Multi-chamber electroporation cartridge and method of use thereof

Abstract

The present disclosure provides systems, devices, and methods for electroporating at least one cell of a plurality of cells. A cartridge divided into a plurality of separation chambers is provided, wherein each chamber of the plurality of separation chambers is configured to contain a culture medium comprising the at least one cell. Each of the plurality of separation chambers is fluidly connected via a fluid exchange path and functionally separated by a constriction disposed within the fluid exchange path. Each of the plurality of separation chambers includes an electrode for applying a pulsed electric field to the at least one cell to effect electroporation of the cell and transfection of the cell with nucleic acid. The constrictions disposed between each chamber prevent electric field interference between adjacent chambers during an electroporation procedure.

Description

Technical Field

[0001] This disclosure provides systems, apparatus, and methods for electroporating cells. More specifically, a multi-chamber electroporation cartridge is provided. Background Technology

[0002] Electroporation procedures can cause varying degrees of cell death, depending on the cell type, the carrier, and electroporation conditions, such as the buffer system and pulse parameters. Pulse parameters are adjusted for each cell type and carrier, but cell death often occurs as an undesirable side effect, reducing the yield of the desired transfection population.

[0003] Transfection of large volumes of cells often employs a flow-through strategy, which consists of repeated transfection of small, fixed-volume cells until the entire volume is processed. The volume processed in each step needs to be as large as possible to process the entire volume quickly enough, but also small enough to allow for the desired performance. This results in limitations on transfection performance and processing time / efficiency. Furthermore, electroporation and / or transfection efficiency often decreases when scaling up from small volumes (i.e., 10 μL to 100 μL) to large volumes (i.e., 0.5 mL to 20 mL).

[0004] There is a need for a system, apparatus, and method for electroporation that improves cell viability, transfection performance, and processing efficiency. This invention addresses these needs. Summary of the Invention

[0005] In a first embodiment, the present invention provides an apparatus for electroporating at least one cell from a plurality of cells. The apparatus includes a housing divided into a plurality of separation chambers. At least one fluid exchange path fluidly connects adjacent chambers of the plurality of separation chambers. A constriction section is disposed within the at least one fluid exchange path to functionally separate adjacent chambers. At least one high-voltage electrode is disposed in each of the plurality of separation chambers and adjacent to the constriction section of the at least one exchange path. At least one counter electrode is disposed opposite the high-voltage electrode and adjacent to the constriction section of the at least one exchange path. The apparatus also includes an inlet.

[0006] In a second embodiment, the present invention provides a method for electroporating at least one cell from a plurality of cells. The method may include the steps of: injecting a culture medium containing at least one cell through an inlet of a cartridge, wherein the cartridge is divided into a plurality of separation chambers; allowing the culture medium to flow through the plurality of separation chambers of the cartridge, including allowing the culture medium to flow through at least one exchange path fluidly connecting adjacent chambers in the plurality of separation chambers; contracting the culture medium as it flows through the at least one exchange path via a contraction portion disposed within the at least one exchange path, the contraction portion functionally separating adjacent chambers; applying an electric field to the at least one cell via at least one high-voltage electrode disposed in each of the plurality of separation chambers and adjacent to the contraction portion of the at least one exchange path, and via at least one counter electrode disposed adjacent to the contraction portion of the at least one exchange path and opposite to the at least one high-voltage electrode; and electroporating the at least one cell.

[0007] In a third embodiment, the present invention provides a method for transfecting at least one cell from a plurality of cells. The method may include the steps of: injecting a culture medium containing at least one cell and at least one nucleic acid through an inlet of a cartridge, wherein the cartridge is divided into a plurality of separation chambers; flowing the culture medium through the plurality of separation chambers of the cartridge, including flowing the culture medium through at least one exchange path fluidly connecting adjacent chambers in the plurality of separation chambers; contracting the culture medium as it flows through the at least one exchange path via a contraction portion disposed within the at least one exchange path, the contraction portion functionally separating adjacent chambers; applying an electric field to the at least one cell via at least one high-voltage electrode disposed in each of the plurality of separation chambers and adjacent to the contraction portion of the at least one exchange path, and at least one counter electrode disposed adjacent to the contraction portion of the at least one exchange path and opposite to the high-voltage electrode; and transfecting at least one nucleic acid into the at least one cell.

[0008] In a fourth embodiment, the present invention provides a system for electroporating at least one cell from a plurality of cells. The system includes a housing divided into a plurality of separation chambers, each of which is configured to contain at least one cell and is fluidly connected via at least one exchange path. The at least one exchange path has a constriction portion disposed therein. At least one high-voltage electrode is disposed in each of the plurality of separation chambers and adjacent to the constriction portion of the at least one exchange path. At least one counter electrode is disposed opposite the high-voltage electrode and adjacent to the constriction portion of the at least one exchange path. An inlet is disposed at the end of the at least one exchange path. The inlet includes an injection inlet, a dead volume region, an electroactive region, and at least one inlet electrode pair consisting of the high-voltage electrode and the counter electrode. The electroporation device is configured to receive the housing and generate electrical pulses via the high-voltage electrode and the counter electrode. Attached Figure Description

[0009] The following figures form part of this specification and are included to further illustrate exemplary embodiments of certain aspects of the invention.

[0010] Figure 1A A side elevation view of an exemplary embodiment of the multi-chamber electroporation box of the present invention is shown.

[0011] Figure 1B It shows Figure 1A Exemplary dimensions of the electroporation box.

[0012] Figure 1C It shows Figure 1A A side cross-sectional view of the fluid exchange path, chamber, and constriction section of the electroporation box.

[0013] Figure 1D It shows Figure 1C A perspective cross-sectional view of the fluid exchange path, chambers, and contractions.

[0014] Figure 2A A side elevation view of another embodiment of the multi-chamber electroporation box of the present invention is shown.

[0015] Figure 2B It shows Figure 2A Exemplary dimensions of the electroporation cell and electrodes in Figure 2A The arrangement within half of the main body layer of the electroporation box.

[0016] Figure 2C It shows Figure 2A A side cross-sectional view of the fluid exchange path, chamber, and constriction section of the electroporation box.

[0017] Figure 2D It shows Figure 2C A schematic view of the chamber and the contraction section, and the electric field generated by the electrodes disposed in the chamber.

[0018] Figure 2E It shows Figure 2D Another schematic view of the chambers, contraction section and electrodes, and the constraint of the electric field between the chambers caused by the contraction section.

[0019] Figure 3A A side cross-sectional view of the inlet of the multi-chamber electroporation box of the present invention is shown.

[0020] Figure 3B It shows Figure 3A A cross-sectional view of the entrance from another side.

[0021] Figure 4A The overmolded circuit board of the multi-chamber electroporation box of the present invention is shown.

[0022] Figure 4B It shows Figure 4A A side cross-sectional view of the overmolded circuit board.

[0023] Figure 5 The circuit board of the multi-chamber electroporation box of the present invention is shown.

[0024] Figure 6 An exemplary method for electroporating and / or transfecting cells using the apparatus of the present invention is shown. Detailed Implementation

[0025] It should be understood that the various aspects disclosed herein can be combined in combinations different from those specifically presented in the specification and figures. It should also be understood that, depending on the example, certain actions or events of any process or method described herein may be performed in a different sequence, may be added, combined, or may be completely omitted (e.g., not all described actions or events are necessary to perform the technique).

[0026] Unless otherwise defined herein, scientific and technical terms as used herein shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural meanings, and plural terms shall include singular meanings.

[0027] The article “a / an” is used in this text to refer to one or more (i.e., at least one) grammatical objects of the article. As an example, “an element” means one or more elements.

[0028] The use of the term "or" in the claims is intended to mean "and / or" unless it is explicitly stated that it refers only to alternatives or that alternatives are mutually exclusive, although this disclosure supports the definition of referring only to alternatives and referring to "and / or".

[0029] As used herein, the terms “comprising” (and any variations or forms of “comprise” and “comprises”), “having” (and any variations or forms of “have” and “has”), “including” (and any variations or forms of “includes” and “include”), or “containing” (and any variations or forms of “contains” and “contain”) are inclusive or open-ended and do not exclude additional unlisted elements or methodological steps.

[0030] The use of the term "for example" and its corresponding abbreviation "eg" indicates that the specific term referenced is a representative example and embodiment of this disclosure, and unless otherwise expressly stated, the representative examples and embodiments are not intended to be limited to the specific examples referenced or cited.

[0031] As used herein, “about” may mean ±10% of the value provided. Where a range is provided, these include boundary values. “About” may additionally or alternatively mean within 10% of the specified value, or within 5% of the specified value, or in some cases within 2.5% of the specified value; or “about” may mean rounded to the nearest significant digit.

[0032] As used in this article, "between" is a range that includes the end of the range. For example, numbers between x and y explicitly include the numbers x and y as well as any numbers that fall within x and y.

[0033] Electroporation can involve applying controlled electrical pulses (e.g., direct current pulses) to at least one of a plurality of cells for a predetermined duration. These pulses can be introduced into at least one of the plurality of cells via electrodes in different waveforms (e.g., square wave pulses or exponentially decaying wave pulses). Upon application of these controlled electrical pulses, a transmembrane potential is induced, which causes reversible rupture of the cell membrane. The transmembrane potential can be described as the potential difference across the cytoplasmic membrane (i.e., between the inside and outside of a biological cell). Rupture of the cell membrane causes permeation or pore formation. This pore formation allows the introduction of molecules (i.e., molecules such as dyes, nucleic acids, or bioactive molecules, including oligonucleotides or peptides) into the cell, a process also known as transfection. Molecules can be continuously introduced into the cell until the pores close by ending the electroporation procedure (or at some point thereafter, if the pores remain in the cell for a period of time). This process can occur anywhere between milliseconds and minutes.

[0034] During electroporation, external molecules are introduced into the cells from an aqueous solution, which can be a buffer solution specifically adapted for cells or a cell culture medium. Temporary pores formed on the cell membrane by pulsed application allow bioactive molecules to first reach the cytoplasm, where they may already be performing their functions or exerting any therapeutic effect to be examined, and then also reach the nucleus, as required in some applications, such as gene therapy. Electroporation and / or transfection procedures are performed within a cartridge containing an electric field generating component designed to facilitate such procedures.

[0035] exist Figures 1A to 2DAn exemplary embodiment of a device or cartridge 100 / 200 for electroporation and / or transfection of at least one cell from a plurality of cells is illustrated herein. The device or cartridge 100 / 200 (also referred to herein as an electroporation cartridge) may be configured to be inserted into and electrically connected to an electroporation device (not shown), which is configured to supply electrical power to the device or cartridge 100 / 200 and generate electrical pulses / fields further described herein. The cartridge 100 / 200 is generally configured to improve cell viability (i.e., survival rate of electroporated / transfected cells), payload delivery (i.e., successful transfection of cells), transfected cell count (i.e., total count of electroporations after cell transfection), and transfection efficiency during electroporation procedures performed in small and large formats. The device typically includes a cartridge 100 / 200 having a body 102 / 202 divided into a plurality of separation chambers 110 / 210. Each of the separation chambers 110 / 210 is a liquid-impermeable structure for containing an aqueous solution or culture medium comprising at least one cell from a plurality of cells and / or a solution containing external molecules for introduction into at least one cell and for allowing flow therethrough. At least one exchange path 112 / 212 extends through the body 102 / 202 of the device or cartridge 100 / 200 and fluidly connects adjacent chambers 110A / 110B / 210A / 210B of the plurality of separation chambers 110 / 210 to allow a flowing culture medium containing at least one cell from a plurality of cells to traverse through the plurality of separation chambers 110 / 210. At least one exchange path 112 / 212 includes at least one constriction 300 / 400 disposed therein for functionally separating adjacent chambers 110A / 110B / 210A / 210B, but still allowing culture medium to flow from one chamber 110A / 210A to an adjacent chamber 110B / 210B along the exchange path 112 / 212 that fluidly connects the multiple separated chambers 110 / 210. Figure 1A and Figure 2A As shown, multiple constriction sections 300 / 400 are disposed between adjacent chambers 110A / 210A / 110B / 210B, and the constriction sections 300 / 400 are described in more detail herein with respect to their corresponding devices 100 / 200. At least one high-voltage electrode 120 / 220 may be disposed in some (suitably all) of the multiple separation chambers 110 / 210, adjacent to at least one constriction section 300 / 400 of at least one fluid exchange path 112 / 212. At least one counter electrode 130 / 230 may be disposed opposite to the high-voltage electrode 120 / 220, adjacent to at least one constriction section 300 / 400 of at least one exchange path 112 / 212.

[0036] Figures 1A to 1DA first embodiment of the device or cartridge 100 of the present invention is shown. The cartridge 100 includes a body 102 having an X-axis along its length (the X-axis is parallel to the fluid exchange path 112), a Z-axis along its width, and a Y-axis along its height. In embodiments, the body 102 of the cartridge may have a width of approximately 60 mm to 80 mm (suitably 73.5 mm) (i.e., along the Z-axis) and a height of approximately 70 mm to 90 mm (suitably 81.5 mm) (i.e., along the X-axis), such as... Figure 1A As illustrated. In an embodiment, the width of the body 102 of the box 100 may be between 50 mm and 200 mm, and the height of the body 102 of the box 100 may be between 50 mm and 200 mm. The width and height of the body 102 of the box 100 may vary according to end-user / laboratory specifications and should not be construed as limited to those described herein. Figures 1A to 1D The embodiment shown. The body 102 is divided into a plurality of chambers 110 along a fluid exchange path 112 for containing and allowing flow of a culture medium containing at least one cell from a plurality of cells, the fluid exchange path being fluidly connected to adjacent chambers 110A, 110B among the plurality of chambers 110. In this embodiment, the body 102 may be divided into a total of twenty-four chambers 110, which are evenly divided into four rows of six chambers 110 along the Z-axis of the body 102, as shown. Figure 1B As illustrated. Each row of multiple chambers 110 can provide a capacity for containing a culture medium containing at least one cell of a plurality of cells with a total volume of 0.5 ml (i.e., across each of the multiple chambers 110), such that the total volume capacity of the four rows of multiple chambers 110 (i.e., the volume capacity of the box 100) is equal to at least 2 ml. In embodiments, the capacity of each individual chamber of the multiple chambers 110 is at least 20 μL, up to about 100 μL, and preferably about 83 μL. In embodiments, the volume capacity of a single row of chambers 110 can be between 0.25 ml and 2 ml. The total volume capacity of the four rows of chambers 110 can be between 1 ml and 8 ml, depending on the end-user specification. The volume capacity of each individual chamber 110, each row of chambers 110, and / or the overall box 100 should not be construed as limited to what is described herein and Figure 1B The embodiments presented herein.

[0037] Surrounding the plurality of chambers 110 on the body 102 is a periphery 104 having a top peripheral portion 104A and a bottom peripheral portion 104B. The top peripheral portion 104A and the bottom peripheral portion 104B are defined by edges slightly inclined relative to the Z-axis, forming an angle 105 relative to the Z-axis. In embodiments, the angle 105 can be between 5 degrees and 30 degrees. More specifically, the angle 105 can be approximately 20 degrees. This angle 105 of the top and bottom portions 104A / 104B of the periphery 104 is formed in the body 102 of the cartridge 100 to prevent any trapping of culture medium containing at least one cell in the corners of the plurality of chambers 110 adjacent to the periphery, and / or to prevent the formation and trapping of air bubbles in the corners of the plurality of chambers 110. The inclined surface of the angle 105 further facilitates the smooth flow of culture medium through the plurality of chambers 110 and the fluid exchange path 112, and prevents any unwanted stagnation.

[0038] Each of the plurality of chambers 110 may contain a high-voltage electrode 120 and / or a counter electrode 130 configured to release / apply an electric field or pulse to a culture medium containing at least one of a plurality of cells. The high-voltage electrode 120 is configured to output a high-voltage electric field, while the counter electrode 130 is configured to output an electric field with a voltage equal to or less than that of the high-voltage electrode 120 and opposite polarity (i.e., positive and negative). In embodiments, the high-voltage electrode 120 may be configured to output a high-voltage electrode field with a voltage between 1 volt and 1200 volts; between 60 volts and 1200 volts; between 100 volts and 1100 volts; between 200 volts and 1000 volts; between 300 volts and 900 volts; between 400 volts and 800 volts; or between 500 volts and 700 volts. In one embodiment, the plurality of chambers 110 include a plurality of high-voltage electrodes 120 and a plurality of counter electrodes 130, wherein the plurality of high-voltage electrodes 120 and the plurality of counter electrodes 130 are alternately arranged within each of the plurality of separate chambers 110. In one embodiment, a chamber 110 includes two counter electrodes 130 arranged opposite each other along a Y-axis within the chamber 110, wherein a fluid exchange path 112 traverses between the two oppositely arranged electrodes. Alternatively, the chamber 110 may include high-voltage electrodes 120 and counter electrodes 130 arranged opposite each other along a Y-axis within the chamber 110, wherein a fluid exchange path 112 traverses between the two oppositely arranged electrodes. Some of the plurality of chambers 110 may be arranged to include two counter electrodes 130 arranged opposite each other along a Y-axis within the chamber 110, while the remaining chambers 110 may include high-voltage electrodes 120 and counter electrodes 130 arranged opposite each other along a Y-axis within the chamber 110. In the chamber 110 including the high-voltage electrode 120 and the counter electrode 130, the polarities of the high-voltage electrode 120 and the counter electrode 130 can be alternated, such that one of the high-voltage electrode 120 and the counter electrode 130 outputs a positive charge, while the other of the high-voltage electrode 120 and the counter electrode 130 outputs a negative charge.

[0039] Between adjacent chambers 110A, 110B in the plurality of chambers 110 is at least one constriction section 300 disposed within the fluid exchange path 112, for example, such as Figure 1A and Figure 1D As shown in the circled portion. At least one constriction 300 includes a non-conductive wall 304 that extends into the fluid exchange path 112 in a direction parallel to the planar surface of the body 102, or as... Figures 1A to 1D The arrangement is shown along the Z-axis. In this way, the height H (i.e., along the Y-axis) of the plurality of chambers 110 and fluid exchange paths 112 is consistent along the X-axis of the housing 100. In an embodiment, the height H of at least one chamber 110 and / or fluid exchange path 112 is at least 1.5 mm, such as... Figure 1CAs illustrated. In embodiments, the height H of at least one chamber 110 and / or fluid exchange path 112 can be between 0.5 mm and 2 mm, depending on end-user specifications. At least one constriction 300 further includes a rounded protrusion 302 (see...). Figure 1A and Figure 1D The rounded protrusion 302 extends from the non-conductive wall 304 and into the switching path 112, such that the rounded protrusion 302 defines a channel C of the switching path 112. In an embodiment, the rounded protrusion has a radius of at least 0.5 mm. Figures 1A to 1D As shown, the multiple contraction sections 300 are arranged to be staggered between each of the multiple chambers 110 so that each chamber is functionally separated.

[0040] By incorporating the contraction section 300 into the exchange path 112 and between each chamber 110, electric and pressure interferences between adjacent chambers 110A / 110B of the plurality of chambers 110 are constrained or limited by the rounded protrusions 302 of the contraction section 300. More specifically, since the contraction section 300 is placed within the fluid exchange path 112 fluidly connecting the chambers 110A / 110B, a direct electric field applied in the first chamber 110A via electrodes 120 / 130 will not develop in adjacent chambers 110B. This functional separation of the chambers 110 allows for the possibility of partially filling the device or chamber 100 with a culture medium containing at least one cell relative to the full capacity of the chamber 100. Alternatively, functional separation of chambers 110 allows for the identification of an optimal electrical pulse applied via electrodes 120 / 130 to a single isolated chamber 110, which can then be applied to the remaining chambers 110, resulting in higher electroporation and / or transfection efficiency in large-volume applications. As described herein, separating each chamber 110 with the contraction portion 300 further results in enhanced transfection performance.

[0041] Figures 2A to 2C A second embodiment of the device or cartridge 200 of the present invention is shown. The cartridge 200 includes a body 202 having an X-axis along its length (the X-axis is parallel to the fluid exchange path 212), a Z-axis along its width, and a Y-axis along its height. In embodiments, the body 202 of the cartridge may have a width of approximately 60 mm to 80 mm (suitably 73.5 mm) (i.e., along the Z-axis) and a height of approximately 70 mm to 90 mm (suitably 81.5 mm) (i.e., along the X-axis), as... Figure 2AAs illustrated, the width and height of the body 202 of box 200 can vary according to end-user / laboratory specifications. In an embodiment, the width of the body 202 of box 200 can be between 50 mm and 200 mm, and the height of the body 202 of box 200 can be between 50 mm and 200 mm. The width and height of the body 202 of box 200 can vary according to end-user / laboratory specifications and should not be construed as limited to those described herein. Figures 2A to 2C The embodiment shown. The body 202 is divided into multiple chambers 210 along a fluid exchange path 212 for containing and allowing flow of a culture medium containing at least one cell from a plurality of cells, the fluid exchange path fluidly connecting adjacent chambers 210A, 210B among the multiple chambers 210. In this embodiment, the body 202 may be divided into a total of twenty-four chambers 210, which are evenly divided into eight rows of three chambers 210 along the Z-axis of the body 202, as shown. Figure 2A As illustrated. Each row of multiple chambers 210 can provide a capacity for containing a culture medium containing at least one cell of multiple cells with a volume of about 0.25 ml in each of the multiple chambers, such that the total volume capacity of eight rows of multiple chambers 210 (i.e., the volume capacity of the box 200) is equal to at least 2 ml. In an embodiment, the capacity of each separate chamber in the multiple chambers 210 is at least 20 μL, up to and preferably about 83 μL. In an embodiment, the volume capacity of a single row of chambers 210 can be between 0.25 ml and 2 ml. The total volume capacity of four rows of chambers 210 can be between 1 ml and 8 ml, depending on the end-user specification. The volume capacity of each individual chamber 210, each row of chambers 210 and / or the overall box 200 should not be construed as limited to what is described herein and Figure 2A The embodiments presented herein.

[0042] Surrounding the plurality of chambers 210 on the body 202 is a periphery 204 having a top peripheral portion 204A and a bottom peripheral portion 204B. The top peripheral portion 204A and the bottom peripheral portion 204B are defined by edges slightly inclined relative to the Z-axis, forming an angle 205 relative to the Z-axis. In embodiments, the angle 205 can be between 5 degrees and 30 degrees. More specifically, the angle 205 can be approximately 20 degrees. This angle 205 of the top and bottom portions 204A / 204B of the periphery 204 is formed in the body 202 of the cartridge 200 to prevent any trapping of culture medium containing at least one cell in the corners of the plurality of chambers 210 adjacent to the periphery, and / or to prevent the formation and trapping of air bubbles in the corners of the plurality of chambers 210. The inclined surface of the angle 205 further facilitates the smooth flow of culture medium through the plurality of chambers 210 and the fluid exchange path 212, and prevents any unwanted stagnation.

[0043] Each of the multiple chambers 210 may contain a high-voltage (HV) electrode 220 and / or a counter electrode (CE) 230 configured to release / apply an electric field or pulse 240 to a culture medium containing at least one of the multiple cells, such as... Figure 2D and Figure 2E The diagram best illustrates this. High-voltage electrode 220 is configured to output a high-voltage electric field 240, while counter electrode 230 is configured to output an electric field with a voltage equal to or less than that of high-voltage electrode 220 and opposite polarity (i.e., positive and negative). In embodiments, high-voltage electrode 220 may be configured to output a high-voltage electrode field with a voltage between 1 volt and 1200 volts; between 60 volts and 1200 volts; between 100 volts and 1100 volts; between 200 volts and 1000 volts; between 300 volts and 900 volts; between 400 volts and 800 volts; or between 500 volts and 700 volts. In embodiments, multiple chambers 210 include multiple high-voltage electrodes 220 and multiple counter electrodes 230, wherein the multiple high-voltage electrodes 220 and multiple counter electrodes 230 are alternately arranged within each of the multiple separate chambers 210. In one embodiment, chamber 210 includes two counter electrodes 230 arranged opposite each other along the Y-axis within chamber 210, wherein a fluid exchange path 212 traverses between the two oppositely arranged electrodes. Chamber 210 may alternatively include a high-voltage electrode 220 and a counter electrode 230 arranged opposite each other along the Y-axis within chamber 210, wherein a fluid exchange path 212 traverses between the two oppositely arranged electrodes. Some of the plurality of chambers 210 may be arranged to include two counter electrodes 230 arranged opposite each other along the Y-axis within chamber 210, while the remaining chambers 210 may include the high-voltage electrode 220 and the counter electrode 230 arranged opposite each other along the Y-axis within chamber 210.

[0044] Figure 2BThe arrangement of electrodes 220 / 230 within half of the body 202 of the device or housing 200 is shown as if the body 202 were divided across its planar surface (i.e., the Z-axis). Here, the electrodes 220 / 230 are arranged such that the middle rows of the plurality of chambers 210 (i.e., rows two through seven) comprise alternating rows of all high-voltage electrodes 220 and all counter electrodes 230; the top row of chambers 210 (i.e., the first row) comprises two counter electrodes 230 on the outer chambers 210, interleaved with the high-voltage electrodes 220 on the middle chambers 210; and the bottom row of chambers 210 (i.e., the eighth row) comprises two high-voltage electrodes 220 on the outer chambers 210, interleaved with the counter electrodes 230 on the middle chambers 210. From top to bottom (i.e., three columns of eight chambers 210 formed along the X-axis), each chamber 210 alternates between high-voltage electrodes 220 and counter electrodes 230 along the column. The alternating arrangement of the high-voltage electrodes 220 and counter electrodes 230 allows for the same electrode arrangement to be provided on the second half of the body 202 of the device or housing 200 (as opposed to...). Figure 2B (The second half is identical to the first half depicted in the original text), such that by flipping the second half on the rotation axis R, the body 202 of the chamber 200 can be formed, wherein each high-voltage electrode 220 is opposed to the counter electrode 230 along the Y-axis in each chamber 210. In the chamber 210 including the high-voltage electrode 220 and the counter electrode 230, such as in... Figure 2D and Figure 2E In the chamber illustrated, the polarities of the high-voltage electrode 220 and the counter electrode 230 can be alternated, such that one of the high-voltage electrode 220 and the counter electrode 230 outputs a positive charge, while the other of the high-voltage electrode 220 and the counter electrode 230 outputs a negative charge.

[0045] Between adjacent chambers 210A and 210B in the plurality of chambers 210 is at least one constriction section 400 disposed within the fluid exchange path 212, such as Figure 2A The circled part is shown in Figure 2C Further details are provided in the cross-section. For example... Figure 2C As shown, multiple contraction sections 400 are disposed between each of the multiple chambers 210 along the fluid exchange path 212. At least one contraction section 400 extends into the fluid exchange path 212 in a direction perpendicular to the planar surface of the body 202, or is formed along the Y-axis, as shown. Figure 2C The exhibits and in Figures 2D to 2E As illustrated in the diagram. In other words, at least one contraction 400 is orthogonal to the flow extension of the culture medium between electrodes 220 / 230. At least one contraction 400 may include a width W1 formed between a first endpoint 414 of the first edge 410 and a second endpoint 424 of the second edge 420 (see Figure 1). Figure 2EIn one embodiment, the width W1 of at least one contraction portion 400 may be at least 1.5 mm. A second contraction portion 400 is disposed along the Y-axis within the fluid exchange path 212 and opposite to the first contraction portion 400. In this arrangement, the separation distance W2 formed between the tip of the first contraction portion (i.e., at the intersection of the first edge 410 and the second edge 420) and the tip of the second contraction portion is at least 0.5 mm (see [link to original text]). Figure 2C ).

[0046] In one embodiment, at least one chamber 210 has a height H of at least 1.5 mm between each constriction 300. In another embodiment, the height H of at least one chamber 210 and / or the fluid exchange path 212 can be between 0.5 mm and 2 mm, depending on the end-user specification. Each constriction 400 includes a first edge 410 and a second edge 420, wherein the first edge 410 defines a chamber width W3, and the second edge 420 defines a channel C of the fluid exchange path 212, such as... Figure 2E As shown. The first angle 412 formed by the first edge 410 and the adjacent high-voltage electrode 220 or counter electrode 230 is approximately 60 to 120 degrees, 70 to 110 degrees, 80 to 100 degrees, or approximately 90 degrees. The second angle 422 formed by the second edge 420 and the adjacent high-voltage electrode 220 or counter electrode 230 is approximately 60 to 120 degrees, 70 to 110 degrees, 80 to 100 degrees, or approximately 90 degrees. The first edge 410 of the contraction portion 400 is formed and inclined in such a way as to constrain electric field interference between adjacent chambers 210A / 210B during the electroporation procedure, such as Figure 2D As shown, the higher voltage is depicted as being confined within a separate chamber 210, as illustrated by equipotential lines 250 (the voltage range of which is given by...). Figure 2D (See the illustration provided). The second edge 420 of the contraction 400 is formed and inclined in such a way as to prevent turbulence and / or cell stress during the flow of culture medium containing at least one cell through the fluid exchange path 212 (and through each contraction 400 and the associated chamber 210).

[0047] By incorporating the contraction section 400 into the exchange path 212 between each chamber 210, electric and pressure interferences between adjacent chambers 210A / 210B of the plurality of chambers 210 are constrained by the edges 410 / 420 of the contraction section 400. More specifically, since the contraction section 400 is placed within the fluid exchange path 212 fluidly connecting the chambers 210A / 210B, a direct electric field applied in the first chamber 210A via electrodes 220 / 230 will not develop in the adjacent chamber 210B. This functional separation of the chambers 210 allows for the possibility of partially filling the device or cassette 200 with a culture medium containing at least one cell relative to its full capacity. Alternatively, the functional separation of chambers 210 allows for the identification of an optimal electrical pulse applied via electrodes 220 / 230 to a single isolated chamber 210, which can then be applied to the remaining chambers 210, resulting in higher electroporation and / or transfection efficiency in large-volume applications. As described herein, separating each chamber 210 with the contraction portion 400 further results in enhanced transfection performance.

[0048] like Figure 3A and Figure 3B As shown, the device or cassette 100 / 200 may further include an inlet 500 disposed at the inflow end of at least one fluid exchange path 112 / 212. In embodiments of the device or cassette 100 / 200, the fluid exchange paths 112 / 212 intersect at the inlet 500, which serves as a single dedicated port for supplying culture medium containing at least one cell as described herein. A corresponding outlet (not shown) may be disposed at the outflow end of at least one fluid exchange path 112 / 212. In embodiments of the device or cassette 100 / 200, the fluid exchange paths 112 / 212 intersect at an outlet (not shown), which serves as a single outlet for culture medium containing electroporated and / or transfected cells.

[0049] Inlet 500 is configured to minimize dead volume of culture medium / cell suspension not reached by the electric field output of electrodes 120 / 130 / 220 / 230 during the electroporation procedure. Inlet 500 includes an injection inlet 502, a dead volume region 504, an electroactive region 506, and at least one inlet electrode pair 508A / 508B / 509A / 509B. More specifically, injection inlet 502 may be a pre-cut inlet including an insertion guide 512 disposed within the body 102 / 202 of cartridge 100 / 200 near the inlet of injection port 502 for receiving dispenser 510 therein and for facilitating insertion of dispenser 510 into inlet 500. As used herein, “dispenser” refers to any suitable device or structure for dispensing culture medium / cell suspension, including, for example, needles, syringes, pipettes, micropipettes, various tubing, etc. The pre-cut slit configuration of the injection port 502 allows the material constituting the injection port 502 (e.g., rubber) to diffuse outward, make way, or extend into the dispenser 510 as it is inserted into the port 500. The pre-cut slit configuration can further assist in guiding the dispenser 510 through the port 500. The injection port 502 is further presented with an oversized triple-seal design, which ensures a tight seal in the area inside the housing 100 / 200. This oversized setting of the injection port 502 further ensures that the pre-cut portion remains compressed and closed when not interacting with the dispenser 510. In embodiments, the dispenser 510 may be a pipette tip, needle, tube, or any other suitable device for delivering culture medium containing at least one cell of a plurality of cells. The culture medium / cell suspension introduced into the injection port 502 flows into a dead volume region 504, which serves as a narrow passage between the injection port 502 and the electroactive region 506 for the culture medium containing at least one cell of a plurality of cells to flow through. An end stop 514 is disposed at the end of the insertion guide 512 and adjacent to the dead volume region 504, and is configured to receive the end of the dispenser 510 during the procedure of inserting the dispenser 510 into the inlet 500.

[0050] Distributed on either side of and surrounding the electroactive region 506 are pairs of inlet electrodes 508A / 508B / 509A / 509B. These pairs of inlet electrodes are arranged such that the first pair of inlet electrodes 508A / 508B faces the electroactive region relative to the vertical axis L of the inlet 500, and the second pair of inlet electrodes 509A / 509B faces the electroactive region relative to the vertical axis L of the inlet. In an embodiment, the pairs of inlet electrodes 508A / 508B / 509A / 509B include a high-voltage electrode and a counter electrode. More specifically, one of the first pair of inlet electrodes 508A / 508B may be a high-voltage electrode, while the other may be a counter electrode. Similarly, one of the second pair of inlet electrodes 509A / 509B may be a high-voltage electrode, while the other may be a counter electrode. The injection inlet 502, dead volume region 504, electroactive region 506, insertion guide 512, and at least one inlet electrode pair 508A / 508B / 509A / 509B of the inlet 500 are formed within the body 102 / 202 of the device or cassette 100 / 200, such that the inlet 500 is symmetrical about its vertical axis L. This symmetrical orientation of the inlet 500 allows for simplified fabrication of the inlet 500 within the device or cassette 100 / 200 described herein, such as... Figure 3B As shown, the injection port 500 can be held in place by thermoforming the edge 516 above the injection port 502, which ensures that the elastic material of the injection port 502 remains within the injection port 500 and is not accidentally removed when the dispenser 510 is removed.

[0051] Figure 4A and Figure 4B An overmolded circuit board 600 integrated with the device or housing 100 as previously described herein is shown, while Figure 5 The exposed circuit board 700 (i.e., before overmolding) is shown in more detail below. Figure 4A As shown, the ultraviolet (UV) light cutout 602 is spatially positioned in a region surrounding or adjacent to the periphery 104 (which, as previously described herein, surrounds a plurality of chambers 110). When forming the device or housing 100, the mold 604 (as...) Figure 4B (as shown in the cross-section) is formed on the circuit board 700 (the circuit board 700 will be Figure 5A conductive copper substrate 702 (described in further detail below) is formed over or around a layer and fixed to a circuit board 700 via a UV-activated adhesive to form an overmolded circuit board 600. Once the overmolded circuit board 600 is formed, a UV light notch 602 thus allows UV light to properly enter for curing the UV-activated adhesive. The implementation of the UV light notch 602 further reduces deformation and mechanical stress on the circuit board 700 and its associated components. In embodiments, the mold can be a cover layer of plastic, carbon, glass, polymer, or any other suitable material. Similar overmolded circuit boards can be formed for integration with the device or housing 200 described herein.

[0052] Figure 5 The circuit board 700 is shown in a side elevation view. The circuit board 700 includes an electrical interface 650 disposed adjacent to an edge of the circuit board 700 and multiple electrical paths 652A / 652B for establishing redundant connections with electrodes 120 / 130 / 220 / 230, wherein the electrical interface 650 is in electrical communication with the multiple electrical paths 652A / 652B. In some embodiments, the circuit board 700 may be a multilayer printed circuit board (PCB), such as... Figure 5 As shown. In embodiments, circuit board 700 can be a single-sided PCB, a double-sided PCB, a rigid PCB, or a flexible PCB. By establishing redundant connections with electrodes 120 / 130 / 220 / 230 via multiple electrical paths 652A / 652B, the electrical interface between electrodes 120 / 130 / 220 / 230 and the overmolded circuit board 600 can be measured, and in-process quality checks can be further permitted. This redundant connection further allows the overmolded metal (i.e., conductive material) of circuit board 700 to be designed such that both the electrical interface 650 and the contact points between electrical paths 652A / 652B and electrodes 120 / 130 / 220 / 230 allow the conductivity of electrodes 120 / 130 / 220 / 230 to be measured during the manufacturing process, ensuring robust electrode fabrication and functionality.

[0053] Systems and methods for electroporation and / or transfection of cells using the apparatus or kit 100 / 200 as described herein are further described. Figure 6An exemplary method 1000 for electroporation of at least one cell from a plurality of cells is illustrated. In a first step 1002, a culture medium containing at least one cell is injected into a device or cartridge 100 / 200. Step 1002 may include injecting the culture medium through an inlet 500 of the cartridge 100 / 200. More specifically, step 1002 may include inserting a dispenser 510 into the injection inlet 502, which may be pre-cut to facilitate receiving the dispenser 510. Inserting the dispenser 510 may include guiding the dispenser through the injection inlet 502 via an insertion guide 512 and stopping the dispenser 510 from traversing the injection inlet 502 via an end stop 514. Inserting the dispenser 510 may include outputting an end stop response, such as tactile feedback (i.e., vibration), an audible indicator, or a flashing / blinking light, upon contact of the end stop 514 with the dispenser 510 to indicate that insertion of the dispenser 510 into the inlet 500 has been completed. Once dispenser 510 is inserted, culture medium containing at least one of the multiple cells can be introduced, flowed, or otherwise injected into device or cassette 100 / 200.

[0054] The next step 1004 of method 1000 includes flowing a culture medium containing at least one cell of a plurality of cells through a plurality of separation chambers 110 / 210 of cartridge 100 / 200. More specifically, step 1004 may include flowing the culture medium through at least one exchange path 112 / 212 that fluidly connects adjacent chambers 110A / 110B / 210A / 210B of the plurality of separation chambers 110 / 210. The flow of the culture medium through the plurality of chambers 110 / 210 and / or at least one fluid exchange path 112 / 212 may be generated by a force of dispenser 510 that introduces, flows into, or otherwise injects the culture medium through inlet 500 into the device or cartridge 100 / 200, as previously described herein. In an embodiment, the culture medium can be generated via a refilling procedure through the flow of multiple chambers 110 / 210 and / or at least one fluid exchange path 112 / 212, wherein a dispenser 510 is a tube connected to an inlet 500, and the dispenser 510 continuously pumps the culture medium from an external source (i.e., a reservoir or bag containing culture medium containing at least one cell of a plurality of cells) into the device or cartridge 100 / 200. During the refilling procedure, after each electroporation / transfection cycle, a separate purge pump (not shown) is used to push air into the cartridge 100 / 200 to ensure that the culture medium containing post-transfected cells is discharged through the outlet and the cartridge 100 / 200 is effectively emptied. The dispenser 510 can then automatically refill the cartridge 100 / 200 with fresh culture medium containing at least one cell for electroporation and / or transfection.

[0055] The next step 1006 of method 1000 includes: contracting the culture medium as it flows through the plurality of chambers 110 / 210 and the at least one exchange path 112 / 212 via at least one constriction 300 / 400 disposed within at least one exchange path 112 / 212. The flow of the culture medium through the plurality of chambers 110 / 210 and / or the at least one fluid exchange path 112 / 212 can be generated by the force of the dispenser 510 that introduces, flows into, or otherwise injects the culture medium through the inlet 500 into the device or cartridge 100 / 200, as previously described herein. The flow of the culture medium through the plurality of chambers 110 / 210 and / or the at least one fluid exchange path 112 / 212 is not affected by the at least one constriction 300 / 400 disposed within the fluid exchange path 112 / 212 due to the slow-moving nature of the culture medium as it flows through it. Step 1006 may include preventing turbulence and / or cell stress during the flow of a culture medium containing at least one cell through the fluid exchange path 112 / 212 and through each contraction 300 / 400 and associated chamber 110 / 210 by allowing the culture medium to flow through the rounded protrusion 302 or the first / second edge 410 / 420 of the contraction 300 / 400, respectively.

[0056] The next step 1008 of method 1000 includes applying an electric field to at least one cell in a culture medium via at least one high-voltage electrode 120 / 220 disposed in each of a plurality of separation chambers 110 / 210 and via at least one counter electrode 130 / 230 disposed opposite the at least one high-voltage electrode 120 / 220, as previously described herein. Applying an electric field to at least one cell causes electroporation of the at least one cell, wherein pores in the cell membrane are opened, and transfection is subsequently achieved. More specifically, the electric field applied to at least one cell may be between 1 volt and 1200 volts. In embodiments, the high-voltage electrode 120 / 220 may be configured to output a high-voltage electrode field with a voltage between 60 volts and 1200 volts; between 100 volts and 1100 volts; between 200 volts and 1000 volts; between 300 volts and 900 volts; between 400 volts and 800 volts; or between 500 volts and 700 volts. The electric field applied to at least one cell can be implemented in a multi-pulse manner, wherein multiple electric field pulses are applied to at least one cell. Step 1008 may include applying an electric field of opposite polarity to at least one cell via at least one counter electrode 130 / 230 disposed within chambers 110 / 210. In an embodiment, a resistance measurement may be obtained prior to step 1008 of applying the electric field, wherein the resistance measurement may be used to help detect incomplete filling of chambers 110 / 210. Incomplete filling of chambers 110 / 210 may favor the generation of arc discharge, or a discharge of relatively high current at a relatively low voltage, especially if the measured output is above 800 volts. Arc discharge may result in potentially incomplete delivery of the electric field from electrodes 120 / 130 / 220 / 230, which may potentially increase damage to at least one cell to be electroporated and / or transfected. The risk of such arc discharge can be mitigated by clipping the voltage of the applied electric field (i.e., limiting the voltage output to ensure it does not exceed a predetermined threshold).

[0057] In an embodiment, method 1000 further includes transfecting cells with one or more molecules (i.e., molecules such as dyes, nucleic acids, or bioactive molecules, including oligonucleotides or peptides) to allow the molecules to enter the cells. Suitably, cells are transfected with nucleic acids that enter the cells via electroporation and are then delivered to the cell nucleus. Transfection with one or more molecules, preferably bioactive molecules, typically does not require any additional treatment, delivery constructs, or encapsulation of the molecules. However, in an embodiment, a viral vector carrying the desired nucleic acid may be used, which can then be transfected into the cells. In other embodiments, lipid-based or polymer-based carriers (e.g., liposomes, micelles, polymer spheres, dendritic structures, or other transfection reagents) may also be used to introduce molecules such as proteins or nucleic acids. In other embodiments, free nucleic acids may be used, or in an embodiment, chemically modified nucleic acids with increased stability (e.g., methylation, etc.) may be used.

[0058] The foregoing description has been presented for illustrative and implementation purposes and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. In view of the above teachings, many modifications and variations are possible. Embodiments and examples have been chosen and described to best explain the principles of the invention, and thereby enable others skilled in the art to best utilize the invention in various embodiments and various modifications as suitable for the particular uses covered. The appended claims are intended to be construed as including other alternative embodiments of the invention.

[0059] The embodiments disclosed herein include the following examples.

[0060] Example 1 includes an apparatus for electroporating at least one cell among a plurality of cells, comprising: a box divided into a plurality of separation chambers; at least one exchange path fluidly connecting adjacent chambers among the plurality of separation chambers, the at least one exchange path having a constriction disposed therein for functionally separating the adjacent chambers; at least one high-voltage electrode disposed in each of the plurality of separation chambers and adjacent to the constriction of the at least one exchange path; at least one counter electrode disposed opposite the high-voltage electrode and adjacent to the constriction of the at least one exchange path; and an inlet.

[0061] Example 2 includes the apparatus according to Example 1, wherein the inlet is located at the end of at least one exchange path, and the inlet has an injection inlet, a dead volume region, an electroactive region, and at least one inlet electrode pair consisting of a high-voltage electrode and a counter electrode disposed therein.

[0062] Example 3 includes the apparatus according to Example 2, wherein the injection inlet further includes an insertion guide for receiving the dispenser into the inlet.

[0063] Example 4 includes the device according to Example 1, wherein a first contraction portion of at least one exchange path is formed along the Y-axis of the box, the first contraction portion having a first edge and a second edge, the first edge defining a chamber width and the second edge defining a channel of the exchange path.

[0064] Example 5 includes the apparatus according to Example 4, wherein electric field interference between adjacent chambers is constrained by a first edge.

[0065] Example 6 includes the apparatus according to Example 4, wherein the first angle formed by the first edge and the high-voltage electrode or the counter electrode is approximately 60 degrees to 120 degrees, and the second angle formed by the second edge and the high-voltage electrode or the counter electrode is approximately 60 degrees to 120 degrees.

[0066] Example 7 includes the device according to Example 4, wherein the width of the first contraction portion from the first endpoint of the first edge to the second endpoint of the second edge is at least 1.5 mm.

[0067] Example 8 includes the device according to Example 4, further including a second contraction portion disposed along the Y-axis within at least one exchange path and opposite to the first contraction portion, such that the channel has a separation distance of at least 0.5 mm between the first contraction portion and the second contraction portion.

[0068] Example 9 includes the device according to Example 4, wherein the size of at least one chamber is at least 1.5 mm on the Y-axis.

[0069] Example 10 includes the device according to Example 1, wherein at least one shrinkage portion of the exchange path is formed along the Z-axis of the box, the shrinkage portion having a rounded protrusion extending into the exchange path and defining a channel of the exchange path, the rounded protrusion having a radius of at least 0.5 mm.

[0070] Example 11 includes the apparatus according to Example 10, wherein electric field interference between adjacent chambers is constrained by rounded protrusions.

[0071] Example 12 includes the apparatus according to Example 10, wherein at least one chamber and at least one exchange path are aligned along the height of the Y-axis along the X-axis of the box.

[0072] Example 13 includes the device according to Example 12, wherein the height of at least one chamber and at least one exchange path is at least 1.5 mm.

[0073] Example 14 includes the apparatus according to Example 1, wherein at least one high-voltage electrode and at least one counter electrode have alternating polarities.

[0074] Example 15 includes the apparatus according to Example 1, further comprising a plurality of high-voltage electrodes and a plurality of counter electrodes, wherein the plurality of high-voltage electrodes and the plurality of counter electrodes are alternately arranged in each of a plurality of separation chambers within a housing.

[0075] Example 16 includes the apparatus according to Example 1, wherein the inlet is symmetrical along a vertical axis.

[0076] Example 17 includes the apparatus according to Example 1, wherein each of the plurality of separation chambers has a capacity of at least 20 microliters.

[0077] Example 18 includes the device according to Example 1, wherein the capacity of the box is at least two (2) milliliters.

[0078] Example 19 includes the device according to Example 3, further including an end stop disposed between the injection inlet and the dead volume region for receiving the end of a pipette tip.

[0079] Example 20 includes the device according to Example 1, wherein the top and bottom peripheries of the device surrounding the plurality of chambers are defined by edges having a slope of at least 20 degrees.

[0080] Example 21 includes a method for electroporating at least one cell from a plurality of cells, comprising: injecting a culture medium containing at least one cell through an inlet of a cartridge, wherein the cartridge is divided into a plurality of separation chambers; allowing the culture medium to flow through the plurality of separation chambers of the cartridge, including allowing the culture medium to flow through at least one exchange path fluidly connecting adjacent chambers in the plurality of separation chambers; causing the culture medium to contract as it flows through the at least one exchange path via a contraction portion disposed within the at least one exchange path, the contraction portion functionally separating adjacent chambers; applying an electric field to at least one cell via at least one high-voltage electrode disposed in each of the plurality of separation chambers and adjacent to the contraction portion of the at least one exchange path, and via at least one counter electrode disposed adjacent to the contraction portion of the at least one exchange path and opposite to the at least one high-voltage electrode; and electroporating at least one cell.

[0081] Example 22 includes the method according to Example 21, further comprising applying an electric field of opposite polarity to at least one cell via at least one counter electrode.

[0082] Example 23 includes the method according to Example 22, wherein a first contraction portion of at least one exchange path is formed along the Y-axis of the box, the first contraction portion further having a first edge and a second edge, the first edge defining a chamber width and the second edge defining a channel of the exchange path for contracting it as the culture medium flows through at least one exchange path.

[0083] Example 24 includes the method according to Example 23, further comprising the step of: constraining electric field interference between adjacent chambers via a first edge of a first contraction portion.

[0084] Example 25 includes the method according to Example 21, further comprising a second contraction portion disposed along the Y-axis within at least one exchange path and opposite to the first contraction portion, such that the channel has a separation distance of at least 0.5 mm provided between the first and second contractions within at least one exchange path.

[0085] Example 26 includes the method according to Example 21, wherein a contraction portion of at least one exchange path is formed along the Z-axis of the box, the contraction portion further having a rounded protrusion that defines a channel of the exchange path for contracting it as the culture medium flows through at least one exchange path, the rounded protrusion having a radius of at least 0.5 mm.

[0086] Example 27 includes the method according to Example 26, further comprising the step of: constraining electric field interference between adjacent chambers via the rounded protrusion of the contraction portion.

[0087] Example 28 includes the method according to Example 21, wherein the inlet is located at the end of at least one exchange path, and the inlet further includes an injection inlet having an insertion guide, a dead volume region, and an electroactive region, and injecting culture medium containing at least one cell through the inlet of the cartridge further includes: inserting a pipette tip into the insertion guide of the injection inlet to facilitate proper delivery of the pipette tip into the inlet; and receiving the end of the pipette tip via an end stop disposed between the injection inlet and the dead volume region.

[0088] Example 29 includes the method according to Example 28, further comprising: providing an end stop response when the step of inserting the pipette tip into the injection port is completed.

[0089] Example 30 includes the method according to Example 21, wherein an electric field applied to at least one cell via at least one high-voltage electrode and / or at least one counter electrode is between 1 volt and 1200 volts.

[0090] Example 31 includes a method for transfecting at least one cell from a plurality of cells, comprising: injecting a culture medium containing at least one cell and at least one nucleic acid through an inlet of a cartridge, wherein the cartridge is divided into a plurality of separation chambers; flowing the culture medium through the plurality of separation chambers of the cartridge, including flowing the culture medium through at least one exchange path fluidly connecting adjacent chambers of the plurality of separation chambers; contracting the culture medium as it flows through the at least one exchange path via a contraction portion disposed within the at least one exchange path, the contraction portion functionally separating adjacent chambers; applying an electric field to the at least one cell via at least one high-voltage electrode disposed in each of the plurality of separation chambers and adjacent to the contraction portion of the at least one exchange path, and at least one counter electrode disposed adjacent to the contraction portion of the at least one exchange path and opposite to the high-voltage electrode; and transfecting at least one nucleic acid into the at least one cell.

[0091] Example 32 includes the method according to Example 31, further comprising applying an electric field of opposite polarity to at least one cell via at least one counter electrode.

[0092] Example 33 includes the method according to Example 32, wherein a first contraction portion of at least one exchange path is formed along the Y-axis of the box, the first contraction portion further having a first edge and a second edge, the first edge defining a chamber width and the second edge defining a channel of the exchange path for contracting it as the culture medium flows through at least one exchange path.

[0093] Example 34 includes the method according to Example 33, further comprising the step of: constraining electric field interference between adjacent chambers via a first edge of a first contraction portion.

[0094] Example 35 includes the method according to Example 31, further comprising a second contraction portion disposed along the Y-axis within at least one exchange path and opposite to the first contraction portion, such that the channel has a separation distance of at least 0.5 mm provided between the first and second contractions within at least one exchange path.

[0095] Example 36 includes the method according to Example 31, wherein a contraction portion of at least one exchange path is formed along the Z-axis of the box, the contraction portion further having a rounded protrusion that defines a channel of the exchange path for contracting it as the culture medium flows through at least one exchange path, the rounded protrusion having a radius of at least 0.5 mm.

[0096] Example 37 includes the method according to Example 36, further comprising the step of: constraining electric field interference between adjacent chambers via the rounded protrusion of the contraction portion.

[0097] Example 38 includes the method according to Example 31, wherein the inlet is located at the end of at least one exchange path, and the inlet further includes an injection inlet having an insertion guide, a dead volume region, and an electroactive region, and injecting a culture medium containing at least one cell and at least one nucleic acid through the inlet of the cartridge further includes: inserting a pipette tip into the insertion guide of the injection inlet to facilitate proper delivery of the pipette tip into the inlet; and receiving the end of the pipette tip via an end stop disposed between the injection inlet and the dead volume region.

[0098] Example 39 includes the method according to Example 38, further comprising: providing an end stop response when the step of inserting the pipette tip into the injection port is completed.

[0099] Example 40 includes the method according to Example 31, wherein an electric field applied to at least one cell via at least one high-voltage electrode and / or at least one counter electrode is between 1 volt and 1200 volts.

[0100] Example 41 includes a system for electroporating at least one cell from a plurality of cells, comprising: a housing divided into a plurality of separation chambers, each of the plurality of separation chambers being configured to contain at least one cell and fluidly connected via at least one exchange path having a constriction therein; at least one high-voltage electrode disposed in each of the plurality of separation chambers and adjacent to the constriction of the at least one exchange path; at least one counter electrode disposed opposite the high-voltage electrode and adjacent to the constriction of the at least one exchange path; an inlet disposed at the end of the at least one exchange path, the inlet having an injection inlet, a dead volume region, an electroactive region, and at least one inlet electrode pair consisting of the high-voltage electrode and the counter electrode disposed therein; and an electroporation device configured to receive the housing and generate electrical pulses via the high-voltage electrode and the counter electrode.

[0101] Example 42 includes the system according to Example 41, wherein the injection inlet further includes an insertion guide for receiving the dispenser into the inlet.

[0102] Example 43 includes the system according to Example 41, wherein a first contraction of at least one exchange path is formed along the Y-axis of the box, the first contraction having a first edge and a second edge, the first edge defining a chamber width and the second edge defining a channel of the exchange path.

[0103] Example 44 includes the system according to Example 43, wherein electric field interference between adjacent chambers is constrained by a first edge.

[0104] Example 45 includes the system according to Example 43, wherein the first angle formed by the first edge and the high-voltage electrode or counter electrode is approximately 60 to 120 degrees, and the second angle formed by the second edge and the high-voltage electrode or counter electrode is approximately 60 to 120 degrees.

[0105] Example 46 includes the system according to Example 43, wherein the width of the first contraction portion from the first endpoint of the first edge to the second endpoint of the second edge is at least 1.5 mm.

[0106] Example 47 includes the system according to Example 43, further including a second contraction portion disposed along the Y-axis within at least one exchange path and opposite to the first contraction portion, such that the channel has a separation distance of at least 0.5 mm between the first contraction portion and the second contraction portion.

[0107] Example 48 includes the system according to Example 43, wherein at least one chamber has a dimension of at least 1.5 mm along the Y-axis.

[0108] Example 49 includes the system according to Example 41, wherein at least one shrinkage portion of the exchange path is formed along the Z-axis of the box, the shrinkage portion having a rounded protrusion extending into the exchange path and defining a channel of the exchange path, the rounded protrusion having a radius of at least 0.5 mm.

[0109] Example 50 includes the system according to Example 49, wherein electric field interference between adjacent chambers is constrained by rounded protrusions.

[0110] Example 51 includes the system according to Example 49, wherein at least one chamber and at least one exchange path are aligned along the height of the Y-axis along the X-axis of the box.

[0111] Example 52 includes the system according to Example 51, wherein the height of at least one chamber and at least one exchange path is at least 1.5 mm.

[0112] Example 53 includes the system according to Example 41, wherein at least one high-voltage electrode and at least one counter electrode have alternating polarities.

[0113] Example 54 includes the system according to Example 41, further comprising a plurality of high-voltage electrodes and a plurality of counter electrodes, wherein the plurality of high-voltage electrodes and the plurality of counter electrodes are alternately arranged in each of a plurality of separation chambers within a housing.

[0114] Example 55 includes the system according to Example 41, wherein the inlet is symmetrical along a vertical axis.

[0115] Example 56 includes the system according to Example 41, wherein each of the plurality of separate chambers has a capacity of at least 20 microliters per chamber.

[0116] Example 57 includes the system according to Example 41, wherein the capacity of the box is at least two (2) milliliters.

[0117] Example 58 includes the system according to Example 41, further including an end stop disposed between the injection inlet and the dead volume region for receiving the end of a pipette tip.

[0118] Example 59 includes the system according to Example 41, wherein the top and bottom peripheries of the device surrounding a plurality of chambers are defined by edges having a slope of at least 20 degrees.

Claims

1. An apparatus for electroporating at least one cell among a plurality of cells, comprising: A box divided into multiple separate chambers; At least one exchange path is fluidly connected to adjacent chambers in the plurality of separated chambers, the at least one exchange path having a constriction disposed therein that functionally separates the adjacent chambers; At least one high-voltage electrode is disposed in each of the plurality of separation chambers and adjacent to the contraction portion of the at least one exchange path; At least one pair of electrodes is configured to be opposite the high-voltage electrode and adjacent to the contraction portion of the at least one exchange path; and Entrance.

2. The apparatus of claim 1, wherein the inlet is disposed at the end of the at least one exchange path, the inlet having an injection inlet, a dead volume region, an electroactive region, and at least one inlet electrode pair consisting of a high-voltage electrode and a counter electrode disposed therein.

3. The apparatus of claim 2, wherein the injection inlet further comprises an insertion guide for receiving the dispenser into the inlet.

4. The apparatus of claim 1, wherein a first contraction portion of the at least one exchange path is formed along the Y-axis of the housing, the first contraction portion having a first edge and a second edge, the first edge defining a chamber width and the second edge defining a channel of the exchange path.

5. The apparatus of claim 4, wherein the electric field interference between the adjacent chambers is constrained by the first edge.

6. The apparatus of claim 4, wherein the first angle formed by the first edge and the high-voltage electrode or the counter electrode is approximately 60 to 120 degrees, and the second angle formed by the second edge and the high-voltage electrode or the counter electrode is approximately 60 to 120 degrees.

7. The device of claim 4, wherein the width of the first contraction portion from the first endpoint of the first edge to the second endpoint of the second edge is at least 1.5 mm.

8. The apparatus of claim 4, further comprising a second contraction portion disposed along the Y-axis within the at least one exchange path and opposite to the first contraction portion, such that the channel has a separation distance of at least 0.5 mm between the first contraction portion and the second contraction portion.

9. The apparatus of claim 4, wherein the size of the at least one chamber is at least 1.5 mm on the Y-axis.

10. The apparatus of claim 1, wherein the contraction portion of the at least one exchange path is formed along the Z-axis of the box, the contraction portion having a rounded protrusion extending into the exchange path and defining a channel of the exchange path, the rounded protrusion having a radius of at least 0.5 mm.

11. The apparatus of claim 10, wherein the electric field interference between adjacent chambers is constrained by the rounded protrusion.

12. The apparatus of claim 10, wherein the at least one chamber and the at least one exchange path are aligned along the height of the Y-axis along the X-axis of the housing.

13. The apparatus of claim 12, wherein the height of the at least one chamber and the at least one exchange path is at least 1.5 mm.

14. The apparatus of claim 1, wherein the at least one high-voltage electrode and the at least one counter electrode have alternating polarities.

15. The apparatus of claim 1, further comprising a plurality of high-voltage electrodes and a plurality of counter electrodes, wherein the plurality of high-voltage electrodes and the plurality of counter electrodes are alternately arranged in each of the plurality of separation chambers within the housing.

16. The apparatus of claim 1, wherein the inlet is symmetrical about a vertical axis.

17. The apparatus of claim 1, wherein each of the plurality of separation chambers has a capacity of at least 20 microliters per chamber.

18. The device according to claim 1, wherein the capacity of the box is at least two (2) milliliters.

19. The apparatus of claim 3, further comprising an end stop disposed between the injection inlet and the dead volume region for receiving the end of a pipette tip.

20. The device of claim 1, wherein the top and bottom peripheries of the device surrounding the plurality of chambers are defined by edges having a slope of at least 20 degrees.

21. A method for electroporating at least one cell of a plurality of cells, comprising: Culture medium containing at least one cell is injected through the inlet of a box, wherein the box is divided into multiple separate chambers; Flowing the culture medium through the plurality of separation chambers of the cartridge includes flowing the culture medium through at least one exchange path, the at least one exchange path being fluidly connected to adjacent chambers of the plurality of separation chambers; As the culture medium flows through the at least one exchange path, the culture medium is contracted via a contraction portion disposed within the at least one exchange path, the contraction portion functionally separating the adjacent chambers; An electric field is applied to the at least one cell via at least one high-voltage electrode disposed in each of the plurality of separation chambers and adjacent to the contraction portion of the at least one exchange path, and via at least one counter electrode disposed adjacent to the contraction portion of the at least one exchange path and opposite to the at least one high-voltage electrode. as well as Electroporation is performed on at least one of the cells.

22. The method of claim 21, further comprising applying an electric field of opposite polarity to the at least one cell via the at least one counter electrode.

23. The method of claim 22, wherein a first contraction portion of the at least one exchange path is formed along the Y-axis of the container, the first contraction portion further having a first edge and a second edge, the first edge defining a chamber width, and the second edge defining a channel of the exchange path for contracting as the culture medium flows through the at least one exchange path.

24. The method of claim 23, further comprising the following steps: The electric field interference between the adjacent chambers is constrained via the first edge of the first contraction portion.

25. The method of claim 21, further comprising a second contraction portion disposed along the Y-axis within the at least one exchange path and opposite to the first contraction portion, such that the channel has a separation distance of at least 0.5 mm provided between the first contraction portion and the second contraction portion within the at least one exchange path.

26. The method of claim 21, wherein the contraction portion of the at least one exchange path is formed along the Z-axis of the box, the contraction portion further having a rounded protrusion defining a channel of the exchange path for contracting as the culture medium flows through the at least one exchange path, the rounded protrusion having a radius of at least 0.5 mm.

27. The method of claim 26, further comprising the following steps: The rounded protrusions of the contraction section constrain the electric field interference between the adjacent chambers.

28. The method of claim 21, wherein the inlet is disposed at the end of the at least one exchange path, and the inlet further comprises an injection inlet having an insertion guide, a dead volume region, and an electroactive region, and the inlet of the cartridge for injecting culture medium containing the at least one cell further comprises: Insert the pipette tip into the insertion guide of the injection port to facilitate proper delivery of the pipette tip into the port; as well as The tip of the pipette is received via an end stop located between the injection inlet and the dead volume region.

29. The method of claim 28, further comprising: When the step of inserting the pipette tip into the injection port is completed, an end stop is provided in response.

30. The method of claim 21, wherein the electric field applied to the at least one cell via the at least one high-voltage electrode and / or the at least one counter electrode is between 1 volt and 1200 volts.

31. A method for transfecting at least one cell from a plurality of cells, comprising: A culture medium containing at least one cell and at least one nucleic acid is injected through the inlet of a box, wherein the box is divided into multiple separation chambers; Flowing the culture medium through the plurality of separation chambers of the cartridge includes flowing the culture medium through at least one exchange path, the at least one exchange path being fluidly connected to adjacent chambers of the plurality of separation chambers; As the culture medium flows through the at least one exchange path, the culture medium is contracted via a contraction portion disposed within the at least one exchange path, the contraction portion functionally separating the adjacent chambers; An electric field is applied to the at least one cell via at least one high-voltage electrode disposed in each of the plurality of separation chambers and adjacent to the contraction portion of the at least one exchange path, and at least one counter electrode disposed adjacent to the contraction portion of the at least one exchange path and opposite to the high-voltage electrode. as well as Transfect the at least one nucleic acid into the at least one cell.

32. The method of claim 31, further comprising applying an electric field of opposite polarity to the at least one cell via the at least one counter electrode.

33. The method of claim 32, wherein a first contraction portion of the at least one exchange path is formed along the Y-axis of the container, the first contraction portion further having a first edge and a second edge, the first edge defining a chamber width, and the second edge defining a channel of the exchange path for contracting as the culture medium flows through the at least one exchange path.

34. The method of claim 33, further comprising the following steps: The electric field interference between the adjacent chambers is constrained via the first edge of the first contraction portion.

35. The method of claim 31, further comprising a second contraction portion disposed along the Y-axis within the at least one exchange path and opposite to the first contraction portion, such that the channel has a separation distance of at least 0.5 mm provided between the first contraction portion and the second contraction portion within the at least one exchange path.

36. The method of claim 31, wherein the contraction portion of the at least one exchange path is formed along the Z-axis of the cartridge, the contraction portion further having a rounded protrusion defining a channel of the exchange path for contracting as the culture medium flows through the at least one exchange path, the rounded protrusion having a radius of at least 0.5 mm.

37. The method of claim 36, further comprising the following steps: The rounded protrusions of the contraction section constrain the electric field interference between the adjacent chambers.

38. The method of claim 31, wherein the inlet is disposed at the end of the at least one exchange path, and the inlet further comprises an injection inlet having an insertion guide, a dead volume region, and an electroactive region, and injecting a culture medium containing the at least one cell and at least one nucleic acid through the inlet of the cartridge further comprises: Insert the pipette tip into the insertion guide of the injection port to facilitate proper delivery of the pipette tip into the port; as well as The tip of the pipette is received via an end stop located between the injection inlet and the dead volume region.

39. The method of claim 38, further comprising: When the step of inserting the pipette tip into the injection port is completed, an end stop is provided in response.

40. The method of claim 31, wherein the electric field applied to the at least one cell via the at least one high-voltage electrode and / or the at least one counter electrode is between 1 volt and 1200 volts.

41. A system for electroporating at least one cell among a plurality of cells, comprising: A box divided into multiple separate chambers, each of which is configured to contain at least one cell and is fluidly connected via at least one exchange path having a constriction disposed therein; At least one high-voltage electrode is disposed in each of the plurality of separation chambers and adjacent to the contraction portion of the at least one exchange path; At least one pair of electrodes is configured to be opposite the high-voltage electrode and adjacent to the contraction portion of the at least one exchange path; An inlet is provided at the end of the at least one exchange path, the inlet having an injection inlet, a dead volume region, an electroactive region, and at least one inlet electrode pair consisting of a high-voltage electrode and a counter electrode disposed therein; as well as An electroporation device configured to receive the box and generate electrical pulses via the high-voltage electrode and the counter electrode.

42. The system of claim 41, wherein the injection inlet further includes an insertion guide for receiving the dispenser into the inlet.

43. The system of claim 41, wherein a first contraction of the at least one exchange path is formed along the Y-axis of the box, the first contraction having a first edge and a second edge, the first edge defining a chamber width and the second edge defining a channel of the exchange path.

44. The system of claim 43, wherein the electric field interference between adjacent chambers is constrained by the first edge.

45. The system of claim 43, wherein the first angle formed by the first edge and the high-voltage electrode or the counter electrode is approximately 60 to 120 degrees, and the second angle formed by the second edge and the high-voltage electrode or the counter electrode is approximately 60 to 120 degrees.

46. ​​The system of claim 43, wherein the width of the first contraction portion from the first endpoint of the first edge to the second endpoint of the second edge is at least 1.5 mm.

47. The system of claim 43, further comprising a second contraction portion disposed along the Y-axis within the at least one exchange path and opposite to the first contraction portion, such that the channel has a separation distance of at least 0.5 mm between the first contraction portion and the second contraction portion.

48. The system of claim 43, wherein the dimension of the at least one chamber is at least 1.5 mm along the Y-axis.

49. The system of claim 41, wherein the contraction portion of the at least one exchange path is formed along the Z-axis of the box, the contraction portion having a rounded protrusion extending into the exchange path and defining a channel of the exchange path, the rounded protrusion having a radius of at least 0.5 mm.

50. The system of claim 49, wherein the electric field interference between adjacent chambers is constrained by the rounded protrusion.

51. The system of claim 49, wherein the at least one chamber and the at least one exchange path are aligned along the height of the Y-axis along the X-axis of the housing.

52. The system of claim 51, wherein the height of the at least one chamber and the at least one exchange path is at least 1.5 mm.

53. The system of claim 41, wherein the at least one high-voltage electrode and the at least one counter electrode have alternating polarities.

54. The system of claim 41, further comprising a plurality of high-voltage electrodes and a plurality of counter electrodes, wherein the plurality of high-voltage electrodes and the plurality of counter electrodes are alternately arranged in each of the plurality of separation chambers within the housing.

55. The system of claim 41, wherein the inlet is symmetrical about a vertical axis.

56. The system of claim 41, wherein each of the plurality of separate chambers has a capacity of at least 20 microliters per chamber.

57. The system of claim 41, wherein the capacity of the box is at least two (2) milliliters.

58. The system of claim 41, further comprising an end stop disposed between the injection inlet and the dead volume region for receiving the end of a pipette tip.

59. The system of claim 41, wherein the top and bottom peripheries of the device surrounding the plurality of chambers are defined by edges having a slope of at least 20 degrees.

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