Method and apparatus for high-throughput transfection of cells using electroporation

Abstract

An apparatus and method for high-throughput transfection of cells using electroporation is disclosed. A flow channel contains two fluids. The second fluid has one or more cells for transfection. The second fluid may be the same as the first fluid. At least one electrode is attached to the flow channel. The electrode applies an electric field to the cells in the flow channel. A control mechanism controls the electrode to apply one or more specific shape, strength, duration, frequency and number of electric pulses to the cells to optimally increase the permeability of their membranes. Thereby, the cell membrane is transiently compromised such that cell transfection can be achieved without killing the cells. The treated cells can make a full recovery.

Description

METHOD AND APPARATUS FOR HIGH-THROUGHPUT TRANSFECTION OF CELLSUSING ELECTROPORATION

[0001] The present invention pertains to a method and apparatus for a high-throughput transfection of cells using electroporation.BACKGROUND

[0002] The introduction of exogenous materials, e.g., drugs, proteins, various forms of RNA and DNA, into cells is essential for research in basic biology including biomedical and clinical applications. In fact, the ability to genetically modify primary human immune cells using exogenous DNA, RNA and molecular species with gene editing functions has revolutionized cancer treatment via the generation of Chimeric Antigen Receptor T cells, i.e., CAR-T therapy. A panoply of methods exist that promote the intracellular delivery of small and large molecular species including chemical, biological, and physical means. Chemical and biological approaches usually employ various carriers such as vesicles, nanoparticles, peptides, or viruses. Physical methods generally exert their effects by causing membrane disruption and include such techniques as membrane deformation via cell squeezing, microinjection, laser optoporation, particle bombardment and electroporation. All of these treatments promote intracellular gene delivery and other types of cargo transport across membranes.

[0003] Electroporation (EP) is a technique in which electric fields are applied to tissues or cells in order to increase the permeability of their membranes. Permeabilization of biological membranes using high voltage electric pulses has been studied extensively for several decades. Membrane pores produced by the application of such pulses can persist from seconds to minutes thereby enabling such applications as intracellular delivery of drugs, RNA, plasmid DNA as well as direct ablation of tissues and tumors. Electroporation can produce either “reversible” or “irreversible” phenomena. In the case of reversible EP, the cell membrane barrier is transiently compromised to allow passage of cargo into the cell, i.e., cell transfection, but can subsequently fully recover allowing the cell to survive. As opposed to reversible EP, irreversible EP is used clinically to induce cell death directly via high-voltage electric pulses or by introducing normally impermeable chemotherapeutic drugs into the cell, i.e., electrochemotherapy, resulting in tissue and / or tumor ablation.

[0004] Conventional EP cell transfection methods employ moderate electric field strengths and unipolar pulses of relatively long duration, i.e., microsecond (msec) to millisecond(msec) range, and are generally thought to generate pores exclusively in the outer (plasma) cell membrane. The efficiency of cell transfection can vary widely depending on the conditions and cell types used in these techniques.

[0005] Several of the many factors that impact transfection efficiency include the following:Electric (field) parameters - voltage (amplitude), pulse waveform (e.g., unipolar or bipolar), pulse duration and frequency;Pulse regimen - sequence of pulses, e.g., nsEP followed by msEP or msEP or vice versa;Buffer conditions - low conductivity vs. high conductivity, pH, ion composition; Cargo concentrations - plasmid DNA or other macromolecule(s) concentration; Cargo size and charge - larger size cargo requires larger pore sizes; charge of cargo may influence behavior when the electric field is applied;Cell type and size - adherent vs. suspension; primary vs. cell culture; dividing vs. non-dividing;Device geometries - cuvettes, microfluidics, channel dimensions, flow rates, electrode material(s) and configuration;Electronics - drive circuits (waveform generators) and power supplies to produce desired waveforms; andCell viability vs. transfection efficiency.

[0006] In addition to allowing passage of molecules through membranes, there are also some significant undesirable effects of conventional EP methods which include: 1) Joule heating of the cell media and subsequent cells, thereby inducing potential cellular “heat-shock” responses; 2) damaged membranes caused by peroxidation of membrane lipids and other oxidative electrochemical reactions; 3) membrane leakiness causing excessive loss of ATP (the cell energy supply); 4) disruption to intracellular and extracellular ion balance; 5) dysregulation of gene function, i.e. changes in cellular gene expression patterns, and 6) decrease in overall cell viability as a result of a combination of the aforementioned factors. Due to some of the deleterious effects introduced by the conventional EP pulsing regimens and the selectivity of conventional EP to permeabilize cell membranes, there is room for exploration and improvement of current techniques to allow for higher efficiency of cell transfection, reduction of some untoward cell physiological effects, along with improvements in cell viability.SUMMARY

[0007] The present invention pertains to an apparatus and method for high-throughput transfection of cells using electroporation. A flow channel contains two fluids. The second fluid has one or more cells for transfection. The second fluid may be the same as the first fluid. At least one electrode is attached to the flow channel. The electrode applies an electric field to the cells in the flow channel. A control mechanism controls the electrode to apply one or more specific shape, strength, duration, frequency and number of electric pulses to the cells to optimally increase the permeability of their membranes. Thereby, the cell membrane is transiently compromised such that cell transfection can be achieved without killing the cells. The treated cells can make a full recovery.

[0008] These and other objects and advantages of the various embodiments of the invention will be recognized by those of ordinary skill in the art after reading the following detailed description of the embodiments that are illustrated in the various drawing figures.BRIEF DESCRIPTION OF DRAWINGS

[0009] The accompanying drawings, which are incorporated in and form a part of this specification and in which like numerals depict like elements, illustrate embodiments of the present disclosure and, together with the detailed description, serve to explain the principles of the disclosure.

[0010] Figure 1 shows one embodiment of flow in a channel comprising a first fluid and at least a second fluid containing one or more species.

[0011] Figure 2 shows an embodiment wherein species are arranged to be close to the electrode.

[0012] Figure 3a is an example that shows a more general form of a cross-section of a channel.

[0013] Figure 3b shows the cross-sectional area of three fluids contained therein.

[0014] Figure 4a is illustrates a channel with a rectangular cross-section.

[0015] Figure 4b shows a top view of the channel with two electrodes affixed to side walls.

[0016] Figure 4c shows a bulging electric field line.

[0017] Figure 4d shows the cross-section of the channel at field line.

[0018] Figure 5 shows a poration device comprising a flow channel which includes a section formed in a serpentine pattern.

[0019] Figure 6 shows an embodiment wherein the poration device comprises a fluid channel and the device is positioned substantially adjacent to a spatially-resolved detecting device.

[0020] Figure 7 shows one embodiment comprising a Radio-Frequency (RF) power supply, where the RF power supply is connected to a fluid channel by an RF transformer.

[0021] Figure 8 shows an embodiment wherein the poration device comprises at least one printed-circuit board and at least one insulating substrate.

[0022] Figure 9 shows an embodiment wherein a set of pulses are applied to fluid and one or more different sets of pulses are subsequently applied to the fluid.

[0023] Figure 10 illustrates the basic assessment of cell electroporation.

[0024] Figure 11 illustrates two forms of electroporation.

[0025] Figure 12a shows a chart of the viability (via the Hoechst stain) of cells.

[0026] Figure 12b shows a chart depicting the degree of transfection due to the three different pulsing characteristics.

[0027] Figure 13 shows the waveform of a single electrical pulse as a function of time.

[0028] Figure 14 shows a pair of pulses of the same polarity.

[0029] Figure 15 shows a pair of pulses of opposite polarity.

[0030] Figure 16 shows a pair of bipolar pulses where the second pulse is not simply the negative of the first pulse.

[0031] Figure 17 shows a pair of bipolar pulses where the time interval between the first pulse and the second pulse is substantially minimized.

[0032] Figure 18 shows a pair of bipolar pulses where a single transition goes from the first pulse to the second pulse.DETAILED DESCRIPTION

[0033] Reference will now be made in detail to the various embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. While described in conjunction with these embodiments, it will be understood that they are not intended to limit the disclosure to these embodiments. On the contrary, the disclosure is intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the disclosure as defined by the appended claims. Furthermore, in the following detailed description of the present disclosure, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be understood that the present disclosure may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the present disclosure.

[0034] Embodiments of the present invention pertain to a high-throughput transfection of cells using electroporation. In one embodiment, varying the electrical parameters of the pulses can elicit different cellular effects. While the longer pulses, e.g., msec and msec, permeabilize the outer (plasma) membrane, shorter pulses (nanosecond range) with higher amplitudes penetrate into the cell interior and permeabilize internal membranes of cellular organelles. The shorter the pulses are, the easier they penetrate into the cell interior.

[0035] The mechanism of membrane potential build-up following shorter pulse durations is different from that of longer pulse durations, i.e., dielectric stacking versus Maxwell-Wagner polarization, which allows direct effects of the electric field on intracellular membranous structures. This becomes particularly important when one considers an exogenous cargo such as a DNA plasmid that must traverse both the cell and nuclear membranes to exert its desired effects. Any mechanism / method that improves the permeabilization of the cell membrane along with the nuclear membrane while sparing the cell of the adverse effects is a significant improvement over current EP techniques.

[0036] Combining distinct sequences of pulses, e.g., nsec bursts followed by msec or msec bursts or vice versa, possibly with varying polarities, may also prove effective in intracellular and, potentially, intranuclear delivery of cargo. A current hypothesis suggests initial permeabilization of the nuclear membrane using high voltage pulses followed by longer duration micro- or millisecond pulses may be synergistic in transporting macromolecular cargos (e.g., plasmid DNA) directly into the cell nucleus. It has also been posited that the longer duration pulses may promote electrophoretic movement of large, charged molecules, e.g., DNA and RNA, through the complex gel-sol matrix of the cytoplasm thereby reducing the residence time in this cellular compartment. This may, in turn, decrease the effectiveness of cytosolic nucleases that degrade exogenous DNA and RNA before nuclear entry.

[0037] The concepts discussed above are supported by the results from numerical physicsbased models of the electroporation process. Beginning with theoretical treatments described in the literature, techniques such as Finite Element Method (FEM) can be used to model the physical processes of polarization and pore formation and disappearance. Extension of the theory in the reference has yielded models for the exploration of the poration of the nuclear envelope. Exploitation of these models has illustrated several effects, some well-known, and some not well understood broadly. As is well known, the process of pore formation is a threshold effect: below a critical voltage across the cell membrane (or nuclear envelope) pore formation does not occur. Past that threshold, pore formation occurs vigorously. This behavior explains some of the difficultly of electroporation generally: one wants enough voltage, but too much is disastrous: shredding the membrane. Less well understood is that the threshold effect makes permeating the nuclear envelope difficult: the voltage from a single pulse tends to appear across the cell membrane and is only transiently (and not with full amplitude) applied across the nuclear envelope. The use of short pulses with large amplitudesand opposing polarities provides a way around this problem, as shown in the models. These same lessons apply to organelles besides the nucleus.

[0038] Methods and protocols that lead to increased cell transfection and viability performance include the following target areas of parameter space: unipolar and bipolar waveforms; electric field parameters, such as amplitude (voltages in the 5-25kV rang; short pulse lengths in the range of 100-700 nanoseconds; pulse frequencies in the range of 25kHz-l MHz range; and the use of novel formulations of buffers to inhibit electrolytic bubble formation, Joule heating and pH-change related effects as well as promote cell survival.

[0039] Devices that enable high throughput EP are used for genetically modifying cells that may be used in certain cell therapies where very high cell numbers are currently required to produce the desired clinical outcome, e.g. 100-500 million cells for single dose of CAR-T therapy. Several embodiments of high throughput devices include cuvette-like chambers, microfluidic devices that contain a very circumscribed region of electric field application and other microfluidic devices that can have extended regions for E-field interaction.

[0040] The present invention comprises several processes / protocols and high throughput devices for the efficient delivery of plasmid DNA into suspension cells. Basically, electroporation is a process whereby an electric field is induced in a volume containing cells. (A more general term for the objects to which the electric field is applied are “species”, which include cells, bubbles, plastic or glass spheres, lipid vesicles, or any other small particle-like objects.) The electric field so established causes changes in the species, for example the creation of holes (or pores) in the membranes of cells. These changes, and particularly the creation of pores in a cell membrane usually has a threshold behavior whereby if the field is strong enough the change (pores in the membrane) occurs and if it the field is not strong enough, the change does not occur. Thus, a critical goal in the construction of apparatus to perform these tasks is the creation of volumes of substantially uniformly strong electric field.

[0041] In one embodiment, those species are in a fluid which is typically a solution of chemicals in water. Such chemicals are similar to those chemicals used in solutions to keep cells alive. But in the case of poration, the specific chemicals and their concentrations are typically modified to accomplish additionally the effect of poration of the species. The most basic such modification of chemistry is to modify the conductivity of the solution by, forexample adding more or less salt, to promote the application of the field to the species. But many other modifications are conceivable, for example, the addition of proteins, or enzymes, or nutrients, or any combination of the above.

[0042] The electric field is typically generated by conductive electrodes in contact with the solution containing the cells. Conductive electrodes typically have two effects on the fluid and hence on the species being electroporated. First, and desirably, the electrodes cause an electric field to be established within the fluid near to where the electrodes contact the fluid. Second, the electrodes may interact electrochemically with molecules in the fluid to produce one or more gasses, such as hydrogen or oxygen, or the decomposition into subunits of chemicals in the solution, or changes in pH. These second effects are typically undesirable because they modify the chemistry of the solution toward conditions that may be harmful to the species contained in the fluid. For example, a change in pH may cause cells to become distressed or even to die.

[0043] The electric field may also be generated by a time-dependent magnetic field suitably arranged with respect to the fluid. Sometimes, such a suitable arrangement involves fields or currents wrapping around fluids, or fluids wrapping around fields, but a great many arrangements are possible whereby a time-dependent magnetic field causes an electric field in the fluid.

[0044] The fluid may modify the shape or strength of an electric field, compared to what would have existed without the presences of the fluid. Further, multiple different fluids each with different electrical properties, such as conductivity may interact to cause the shape or strength of the field to be modified.

[0045] In the embodiments described below, the combination of electric field, pulse duration, pulse frequency, and number of pulses may be adjusted to cause a desired effect on one or more of the species. For example, short pulses with high electric fields may preferentially cause poration of the nuclear envelope within cells. In another example, longer pulses with lower electric field may cause poration primarily to the cell membrane while not impacting the nuclear envelope. The embodiments below describe various means of controlling or modifying the parameters above. It is an advantage of the inventions described here that these parameters, and hence their effect on the species, can be controlled or modified.

[0046] Figure 1 shows one embodiment of a flow in a channel 101 comprises a first fluid 102 and at least a second fluid 103 containing one or more species 104. As shown in the figure, the species 104, being confined to the second fluid 103 are positioned directly adjacent to the electrode 105 by virtue of the proximity of the second fluid 103 to the electrode.

[0047] In an alternate embodiment shown in Figure 2, the second fluid is introduced to the channel on the opposite side of the channel 201 from the electrode 205. Thus, in this case, the species 204 ends up relatively far from the electrode 205. A channel 201 can be constructed with a width (201) that may be between 10 micrometer and 5 millimeters. Preferentially, the channel may be constructed with width (201) between 50 micrometer and 2 millimeters. In the first embodiment shown in Figure 1, the species 104 passes by the electrode 105 substantially no further away from the electrode than a small fraction of the width (101), a value that may range from 1 micrometer to 0.5 millimeter, and preferentially from 5 micrometer to 0.2 millimeter. In this sense, the species is arranged to be close to the electrode. In the second embodiment shown in Figure 2, because the second fluid 203 is introduced on the side of the channel 101 that is opposite the electrode 205, the species 204 passes no closer to the electrode that a large fraction of the channel width (201), a value that may range from 9 micrometer to 4.5 millimeter, and preferentially from 45 micrometer and 1.8 millimeter. In this sense, the species 204 is arranged to be far from the electrode.

[0048] FIG. 3a shows a more general form of a cross-section of a channel 301, in the sense that the cross-section is not a simple shape like a rectangle. In this example, electrodes (305 and 307) are positioned substantially on opposite sides of the channel. In this example, three fluids are arranged to flow in a layer-wise fashion such that the first fluid 302 flows adjacent to the second fluid 303 and the second fluid flows adjacent to the third fluid 309 and where the boundary between the first fluid and the second fluid 310 and the boundary between the second fluid and the third fluid 311 do not intersect each other, but rather, terminate on the channel boundary 301 between the two electrodes (305 and 307). In this case, current from one electrode 305 to the other electrode 307 must flow through each of the fluids sequentially. In this way, each of the fluids can be thought of a resistor, with the understanding that a resistor is a so-called lumped element and the fluids are spatially distributed volumes. Nonetheless, thinking of the fluids as effective resistors allows us to reason qualitatively about the voltages and thus electric fields across each of the fluids.

[0049] The conductivities of each of the fluids may be modified, for example by adding more or less salt, such that the strength of the electric field 318 is modified. A specific example is shown in FIG 3c. In this example, the conductivity of the first fluid 302 and the third fluid 309 are increased. Due to this increase, the effective resistance of the first and third fluid (302 and 309) are reduced. Per the well-known analysis of voltage dividers, a larger fraction of the voltage appears across the high-valued resistors, which in this example is the role played by the second fluid 303. Assuming no change to the geometry of the fluids, then the greater voltage across the middle fluid will result in a greater electric field 318 within the second fluid, and the electric fields in the first and third fluids would be reduced. As described above, allowing for the control in this manner of the electric field in the second fluid could allow that field to be optimized to achieve a desired effect such as effective poration of the species 304 in the second fluid. Additionally, the adjusted electric field in the second fluid may allow for reduced cell death.

[0050] In the example above the assumption is made of no change in the geometry of the fluids. Control of that geometry can be achieved by controlling the volume flow of the fluids. In FIG 3b, we again illustrate three fluids 302, 303, and 309. The figure shows the cross- sectional area of the three fluids to be different. But, importantly, especially for small fluidic devices, say, less than 5 mm in any cross-sectional dimension, the fluids all flow at the same linear velocity due to the viscous drag that they exert on each other if not flowing at the same linear velocity. Assuming that the velocities are all the same, then the cross-sectional area of each fluid is proportional to the volume flow rate of that fluid, with the constraint that the total cross-sectional area of all of the fluids must add up to the cross-sectional area of the channel. Therefore, we may control the cross-sectional area of each fluid by controlling the volume flow rate of that fluid, while the fluid velocity is given by the cross-sectional area of the channel combined with the total of the volume flow rates of the fluids. Furthermore, because the effective resistance of the fluids is related to their cross-sectional dimensions, modifying the cross-sectional area of a fluid will affect the voltage appearing across that fluid and hence the electric field within that fluid, and so forth for the other fluids.

[0051] An additional embodiment is shown in FIG 4. In FIG 4a is illustrated a channel 401 with a rectangular cross-section. In this example, one or more conductive fluids are flowing through the channel. In FIG 4a, we illustrate that the flow of the fluid is along the channel and away from the observer. We define this flow direction to be the down-stream direction412. Conversely, the direction opposite to the flow we define to be the up-stream direction413. In FIG 4b is shown a top view of the channel with two electrodes 415 and 417 affixed tothe side walls. Also illustrated are three examples of lines of the electric field 418a, 418b, and 418c with three examples of a species 414 flowing in the fluid. The mirror images of field lines 418b and 418c are shown to illustrate the symmetry of the situation. Field line 418a exists at the center-line of the two electrodes. As a result, it is straight and, in this view, vertical. Field line 18c arises from left edge of one electrode 405) and terminates at the left edge of the other electrode 407. Because of the presence of a volume of conductive fluid to the left of the edge of the electrodes, the field line tends to bulge outward into that volume. Field line 18b is an intermediate case showing bulging as it has partially developed. Necessarily field line 418c (and partially, field line 418b) will, in its bulged portion, be weaker (lower magnitude of electric field) that will field line 418a. As described above, the threshold nature of the change in the species implies that the weaker field strengths of field line 418c may be insufficient to cause the change. It would be desirable to cause field line 418c not to be presented to the species. In FIG 4c is shown an oblique view of the channel 401 with the electrodes 405 and 407 fixed to the side walls. In addition, two conductive areas 414 and 416 are shown affixed to the bottom and top of the channel, upstream of the electrodes 405 and 407. In this view (FIG 4c) the bulging electric field line (not illustrated) is partially bulging toward the viewer. FIG 4d shows the cross-section of the channel at field line 18a. In this case, the field passes through the middle fluid 403 as usual. FIG 4e, however, is the cross-section through the conductors up-stream of the electrodes. In this case, instead of field line 418c passing through the middle fluid 403 containing a species 404, the field line 418c preferentially terminates at the conductor 414 or 416 in the first fluid 402 and reemerges into the third fluid 409 due to the fact that the resistance of the conductive area 414 or 416 can be made lower than the resistance of the second fluid 403. That is to say that the conductors 414 and 416 short-out the second fluid 403. The current passing through fluid 402 passes through the conductor 414 or 416 then flows back into the third fluid 409. This behavior effectively removes the bulging field line 418c from the second fluid containing the species 404.

[0052] In some embodiments, controlling the length of the fluid channel that is exposed to electrodes may be desirable. For example, because the species are flowing at some velocity through the fluid channel, if it is desirable that species be exposed to many pulses from the electrodes, it is then desirable to increase the length of the fluid channel that is exposed to the fluid. FIG 5 shows a poration device comprising a flow channel 520 which includes a section formed in a serpentine pattern 521. An electrode 522 is shown and is patterned 523 andwhere the pattern allows the electrode to be exposed to a substantially large fraction of the portion of the flow channel formed in a serpentine pattern.

[0053] In general, it is desirable to know where cells or other species are located within the poration device. FIG 6 shows one embodiment wherein the poration device 630 comprises a fluid channel 631 and the device is positioned substantially adjacent to a spatially-resolved detecting device 632. Each detecting element of the detecting device is sensitive to a region of the flow channel thus providing information about the location of species 633 within that region. The aggregate of the elements of the detecting device can provide information about the motion of species through the device. Because the fluid channel is positioned substantially adjacent to the detecting device, the sensitivity of individual detecting element so regions of the flow channel is accomplished without the use of lenses or other bulky optics.

[0054] An example of a spatially-resolved detecting device is a pixelated imaging sensor and the detecting elements are the pixels. With a pixelated imaging sensor, different pixels can be made sensitive to different optical characteristics of the species in the fluid channel. For example, some pixels may be sensitive to blue light, red light, or infrared light, in which case, the pixels are sensitive to the scattering of the species at, respectively, blue, red, or infrared. Another example is where some pixels are sensitive to vertical polarization, and some are sensitive to horizontal polarization, in which case the pixels are sensitive to the scattering of the species in, respectively, vertical or horizontal polarization. Because imaging sensors can have very high density of pixels, a multi-channel image of the species in the channel can be made where each channel is made from the subset of pixels corresponding to each optical characteristic that is monitored. If the optical characteristics are chosen appropriately, subcategories of species (e.g. subcategories of cells) may be distinguishable, which information can be used to trigger different poration processing or other downstream processing for each different subcategory.

[0055] An alternative example of a spatially-resolved detecting device comprises an array of electrodes where pairs of electrodes can be used to measure electrical properties of the connection between them and where the measured electrical properties convey information about the location of species within the fluid channel. Additionally, different subcategories of species within the fluid may give rise to different measured electrical properties and the different measured electrical properties may allow for modified processing of each of the subcategories.

[0056] The generation of pulsed electric fields using electrodes requires a generator circuit that applies voltage pulses to the electrodes. Especially when the desired voltage pulses arevery short or very frequent, or are at very high voltages, the circuitry to implement the generator can be difficult to design. In some embodiments, the circuit includes high-voltage transistors that rapidly switch at least one of the electrodes between voltages supplied by conventional high-voltage power supplies. An additional transistor may switch the electrode to ground, thereby removing voltage from the electrode and eliminating the electric field. The duration of individual pulses can be controlled by controlling the switching behavior of the transistors. With modern silicon-carbide transistors, very high voltages and very rapid switching may be had in a single design. The individual voltages applied can be adjusted by adjusting the conventional power supplies. In this way, the switching transistors do not have to simultaneously switch rapidly and control the voltage. One useful alternative embodiment is when the conventional power supplies are chosen to be electrophoresis supplies, which are adjustable over a useful range and are widely available.

[0057] One useful case of the above arrangement is where the voltages consist of at least one positive voltage, at least one negative voltage, and ground or zero voltage. Use of pulses of opposite voltages, rapidly switched, can optimize poration of the nuclear envelope.

[0058] Because of the need to keep different populations of cells from contaminating one another, it is highly desirable to make the portion of a poration system containing the fluid disposable. However, for a large portion of the system, for example the power supplies and the switching transistors described above, it is highly desirable that it be durable. FIG. 7 shows an embodiment that accomplishes both goals. It comprises a Radio-Frequency (RF) power supply 734, where the RF power supply is connected to a fluid channel 735 by an RF transformer 736. An appropriate RF power supply can be programmed to supply a wide variety of waveforms to the fluid in the channel.

[0059] In one alternative embodiment, the RF transformer comprises a primary winding 737 that is contained within the durable piece of equipment 738 and further comprises a secondary winding 739 that is contained within a disposable piece of equipment 740. The efficacy of the transformer depends, in part, on accurate positioning of the primary and the secondary with respect to each other. This can be accomplished by providing mechanical features 741 on the disposable and the durable which together allow the two parts to engage in a highly reproducible way. An example of suitable features is a kinematic mount between the disposable and the durable.

[0060] In the construction of devices incorporating both fluid channels and electrodes, the use of printed-circuit-board technology can be advantageous. FIG 8 shows an embodiment wherein the poration device comprises at least one printed-circuit board 850 and at least oneinsulating substrate 851. A flow channel 852 is formed into the substrate where the flow channel is open to a surface 853 of the substrate. The printed-circuit board can have at least one electrical conductor 854 exposed on the surface. Then printed circuit board can be fixed to the substrate such that the conductor is exposed 855 to the flow channel. Solder mask can be used to prevent some portion of the electrical conductor from contacting fluid even if it nominally exposed to the fluid channel. The use of specialized electrode materials such as gold, or platinum, or black-platinum, or silver-chloride that are benign to cellular life is often desirable. In order to accommodate the surface of the printed circuit board to the exposed surface of the substrate, the printed circuit may be constructed using flexible PC technology.

[0061] To control and thus optimize the poration pulses applied to each cell, a detector may be incorporated. Thus, when the detector detects a cell in its sensitive volume, the electroporation pulses may be timed to occur after a delay when the fluid has flowed from the detector volume to the region of the electrodes. This has the advantage that pulses are only applied when needed, thus limiting the problematic effects such as pH change discussed above. Further, if the detector is sensitive to subcategories of cells in the fluid, different electroporation pulses may be applied which are optimized for each detected type of cells. In some embodiments, the detector sensitive volume and the region of the electrodes coincide. Thus, the delay previously described may be zero.

[0062] As discussed above different poration pulses can be chosen to accomplish different poration tasks, for example, preferential poration of the nuclear envelope or preferential poration of the cellular membrane. FIG 9 shows an embodiment wherein a set of pulses 960 is applied to fluid and one or more different sets of pulses 961 are subsequently applied to the fluid where each set is optimized to accomplish a preferred poration task. The characteristics of the pulses that typically would be optimized may include starting time 962, amplitude 963, and stopping time 964 of the pulses. The amplitude of each pulse may be positive or negative and the ordering of positive and negative pulses is useful to achieve a preferred poration task.

[0063] Evaluating the results of poration is useful in deciding the subsequent treatment of species. Detection of the results of poration may be accomplished by optical or impedance techniques similar to those described above. With detected information some individual cells or other species may be routed to subsequent processing if poration was detected to be effective, or if poration was ineffective, those individuals may be routed back to the input to the poration device to be retreated.

[0064] The efficacy of the inventions is illustrated in FIG. 10, FIG. 11, and FIG. 12. FIG. 10 illustrates the basic assessment of cell electroporation. The two rows of photographs illustrate the outcomes of two tests. In the top row is illustrated an experiment where no electroporation is attempted. The bottom row is the result when electroporation is attempted. The left-had column is a conventional bright-field microscope view of a population of cells. As is typical, the contrast of the cells compared to the background brightness is modest. In the middle column is shown the result of staining the respective cell populations with a Hoechst stain. This stain binds to the DNA of viable (that is, living) cells. Therefore, the presence of significant numbers of Hoechst-stained cells in the upper and the lower images illustrates that the electroporation procedure did not significantly reduce the rate of viability of the cells. That is, the cells in the bottom row survived the electroporation process and continued to live. The right-hand column shows the results of either not porating (top) or porating (bottom) the respective populations of cells. Cells that were successfully porated were able to take in a piece of DNAthat, when expressed, caused a fluorescent molecule (green-fluorescent protein, GFP). Taken together, the images in FIG. 10 illustrate that electroporation can cause the uptake (and eventual expression) of the DNA for GFP, without causing excessive cell death.

[0065] FIG. 11 illustrates two forms of electroporation. In the left-hand drawing, pulses are (relatively) high-voltage, short, and are repeated. These are labeled “ns pulses”. This is as opposed to (in the right-hand drawing) a single pulse that is longer, and of lower voltage. Pulses of this variety are labeled “Baseline ps pulses.”

[0066] FIG 12 (parts a and b) show quantitative assessment of three different uses of “ns pulses” and “Baseline ps pulses” shown in Fig. 11. FIG 12a gives the viability (via the Hoechst stain) of cells. FIG 12b shows the degree of transfection due to the three different pulsing characteristics. The left bars in FIG 12a and FIG 12b correspond to only having received ns pulses. The middle bars represent cells that received first a burst of ns pulses followed by a single baseline ps pulse. The right bars represent cells that received first a single baseline ps pulse, followed by a burst of ns pulses.

[0067] The heights of the bars in FIG 12a illustrate that combining ps and ns pulses (in either order) gives approximately equivalent viability, except at the highest voltage where the nspulses alone are inferior. On the other hand, FIG 12b illustrates that combinations of ns and ps pulses, in either order, provides somewhat better transfection compared with ns pulses alone.

[0068] In Figure 13 is illustrated a single pulse as might be applied to cells according to the present invention, where the pulse is illustrated with respect to time. The pulse is characterized by several properties. The amplitude 1301 corresponds to the voltage applied to the processing device. Alternatively, the amplitude 1301 may correspond to the electric field applied to each cell. For any particular design of processing device there will be a well- understood relationship between the voltage applied to the processing device and the electric field applied to the cells. For example, if the processing device comprises two parallel plates where the gap between them is much smaller than the extent of the plates, then the electricfield would be related to the voltage applied approximately according to: £ = where isthe voltage applied, d is the gap between the electrodes, and E is the electric field applied to the cells, where the approximation is accurate when the electric field is examined away from the edges of the parallel plates. I the remainder of this description, amplitude will be referred to in the more generalized sense that encompasses either electric field or voltage.

[0069] Additional properties of the single pulse in Figure 13 are the pulse duration 1302, the rise time of the pulse 1303 from zero to a prescribed amplitude 1301, and the fall time of the pulse 1304 from the prescribed amplitude 1301 substantially to zero. In many cases, the rise time 1303 or the fall time 1304 or both may be much smaller than the pulse duration 1302. In such a case, although an illustration of such a pulse may not show an obvious rise time 1303 or fall time 1304, careful examination of the physical realization of the pulse would show finite, if small, rise time 1303 and fall time 1304. Such a case is to be understood to be included in the description given herein.

[0070] Figure 14 shows a pair of pulses of substantially the same polarity (unipolar pulses). In addition to the properties of the first pulse discussed above, the pair of pulses is characterized by the interval 1411 between the start of the first pulse and the start of the second pulse. In addition, the second pulse is characterized by its rise time 1406, duration 1407, and fall time 1408, and amplitude 1409). It is to be understood that each of the properties of the second pulse (rise time 1406), duration 1407, fall time 1408, and amplitude 1409 may be different from that of the first pulse, or may be substantially equivalent. Finally,the pulse delay 1405 provides an alternate to the pulse interval 1411 for specifying the time between pulses. It is sometimes convenient to refer to the delay 1405 in preference to the interval 1411, where either description can be prescribed to yield substantially the same pair of pulses.

[0071] Figure 15 shows a pair of pulses of substantially opposite polarity (bipolar pulses). The amplitude of the second pulse 1510 may be given either by a negative or a positive number. If a positive number is specified, it is to be understood given the context of the figure that a positive number indicates a pulse of amplitude 1510 substantially opposite to the amplitude 1501 of the first pulse. Whether to use one specification or the other is a matter of convenience where the bipolar nature of the pulses is conveyed by the accompanying drawing. For example, even though the second pulse is negative, we still speak of the “rise” time 1506 of the pulse, where the rise is from substantially zero to the (positive) amplitude of the pulse, even though the voltage (say) is negative.

[0072] Figure 16 illustrates the case where the second pulse has characteristics that are substantially different than those of the first pulse. In the figure, particularly the amplitude 1610 is not the negative of the amplitude 1601 of the first pulse and the pulse duration 1607 of the second pulse is substantially different than the duration 1602 of the first pulse.

[0073] Figure 17 illustrates a pair of bipolar pulses where the pulse delay 1705 between the first pulse and the second pulse is substantially minimized.

[0074] Figure 18 illustrates a pair of bipolar pulses where the fall time of the first pulse 1804, the pulse delay 1805, and the rise time of the second pulse 1806 are replaced by a single transition from the first pulse to the second pulse where the transition is characterized by a transition time 1812.

[0075] In one embodiment, the specific shape, strength, duration, frequency, and number of electrical pulses are controlled to optimally electroporate the cells for a given application to treat the cell while keeping the cell alive. One application may have one set of unique electrical pulses, whereas a different application may have another set of unique electrical pulses. By controlling the electrical pulses in the manners described above, the most optimalelectroporation profile can be achieved depending on the particular type of cells, applications, and / or treatments. The applications and cells vary; hence, the electrical pulse characteristics need to adapt to provide the optimal electroporation under various circumstances. Moreover, the electrical pulse characteristics can be fine tuned using the various embodiments described herein to zero in on the most optimal electroporation profile / plan. Furthermore, the adjustments to the electrical pulses can be made dynamically during the electroporation process.

[0076] Although the subject matter has been described in language specific to structural features and / or methodological acts, it is to be understood that the subject matter defined in this disclosure is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts described above are disclosed as example forms of implementing this disclosure.

[0077] Embodiments according to the invention are thus described. While the present invention has been described in particular embodiments, the invention should not be construed as limited by such embodiments, but rather construed according to the following claims.

Claims

CLAIMSWhat is claimed is:

1. An apparatus for transfection of a particle using electroporation, comprising: a flow channel having a first fluid, wherein at least one particle is in the first fluid; a first electrode coupled to the flow channel for applying an electric field to the particle; a control mechanism for controlling the electrode to cause a shape, strength, duration, frequency, and number of pulses of the electric field to be regulated to optimize the electroporation of the particle.

2. The apparatus of Claim 1, wherein the particle comprises a cell and electroporation of the cell enables passage of a treatment into the cell, allowing the cell to fully survive.

3. The apparatus of Claim 1, wherein the first electrode is positioned adjacent to the first fluid.

4. The apparatus of Claim 1, wherein the first electrode is positioned adjacent to a second fluid.

5. The apparatus of Claim 1 further comprising a third fluid and a second electrode, wherein the first electrode and the second electrode reside on opposite sides of the flow channel and the first fluid flows adjacent to the second fluid which flows adjacent to the third fluid.

6. The apparatus of Claim 5, wherein current from the first electrode flows through the first fluid, the second fluid, and the third fluid to the second electrode.

7. The apparatus of Claim 1, wherein conductivities of the first fluid and the second fluid are controlled.

8. The apparatus of Claim 1, wherein a geometry of the first fluid and the second fluid is controlled by controlling a volume flow of the fluids.

9. The apparatus of Claim 1, wherein a first cross-sectional area of the first fluid is controlled by controlling a first volume flow rate of the first fluid and a second cross-sectional area of the second fluid is controlled by controlling a second volume flow rate of the second liquid.

10. The apparatus of Claim 5 further comprising a first conductive area placed on top of the flow channel and a second conductive area placed at bottom of the flow channel, wherein the first conductive area and the second conductive area are upstream of the first electrode and the second electrode.

11. The apparatus of Claim 1 further comprising a controller for controlling a length of the flow channel.

12. The apparatus of Claim 1, wherein at least one portion of the flow channel comprises a serpentine shape.

13. The apparatus of Claim 1 further comprising a detecting device to detect a location of the particle within the flow channel.

14. The apparatus of Claim 13, wherein the detecting device comprises a pixelated imaging sensor.

15. The apparatus of Claim 13, wherein the detecting device comprises an array of electrodes used to measure electrical properties of connections between pairs of electrodes.

16. The apparatus of Claim 1, wherein the control mechanism generates pulses of at least one positive voltage and at least one negative voltage.

17. The apparatus of Claim 16, wherein the control mechanism generates a ground voltage and switches between the positive voltage and the negative voltage.

18. The apparatus of Claim 1, wherein the control mechanism controls a starting time, stopping time, amplitude, and delay of electrical pulses.

19. The apparatus of Claim 1 further comprising a printed circuit board and a substrate, wherein the flow channel is formed into the substrate and is open to a surface of the substrate and the printed circuit board includes at least one electrical conductor exposed on the surface.

20. The apparatus of Claim 1 further comprising a detector that detects particles in the flow channel in order to apply poration pulses to the particles.

21. A method apparatus for transfection of a cell using electroporation, comprising: suspending the cell in a flow channel having a fluid; applying an electric field to the cell to increase permeability of the cell; controlling the electric field to cause a shape, strength, duration, frequency, and number of pulses of the electric field to be regulated to optimize the electroporation of the cell, wherein an application can be more effective at treating the cell while keeping the cell alive.

22. The method of Claim 21 further comprising controlling an electrical current through a first fluid, a second fluid, and a third fluid to electroporate the cell.

23. The method of Claim 21 further comprising controlling a conductivity of the fluid.

24. The method of Claim 21 further comprising controlling a volume flow of the fluid to define a geometry of the fluid.

25. The method of Claim 21 further comprising controlling a first conductive area placed on top of the flow channel and a second conductive area placed at a bottom of the flow channel, wherein the first conductive area and the second conductive area are upstream of a first electrode and a second electrode used to generate the electric field.

26. The method of Claim 21 further comprising detecting a location of the cell within the flow channel.

27. The method of Claim 21 further comprising switching between a positive voltage and a negative voltage corresponding to the electric field.

28. The method of Claim 21 further comprising controlling a starting time, stopping time, amplitude, and delay of electrical pulses corresponding to the electric field.

29. A system for transfection of cells using electroporation, comprising: a flow channel having cells suspended in a fluid; a plurality of electrodes coupled to the flow channel for applying one or more electric fields to the cells; a control mechanism for adjusting the electrodes to generate a pre-determined shape, strength, duration, frequency, and number of electrical pulses to optimally electroporate the cells for a given application to treat the cell while keeping the cell alive.

30. The system of Claim 29, wherein the electrical pulses are selectively adjusted to optimally electroporate the cells for a plurality of different applications.

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