Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
  • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/16—Microfluidic devices; Capillary tubes

Definitions

• the first electrical field is configured to detect presence of a cell between the first pair of electrodes.
• the second electrical field is configured to electroporate the cell between the second pair of electrodes.
• FIGs. 6B - 6E illustrates Green Fluorescent Protein (GFP) expression in HEK293 cells following electroporation
• Electroporation is a means to access the cytoplasm of a cell for delivery of molecules.
• an electric field which can be applied in vitro or in vivo, transiently permeabilizes the cell membrane through which biologically active molecules can enter the cell, such as DNA, RNA, and amino acids.
• the current disclosure describes a system and method for a flow-based, automated cell detection-and-electroporation signal system to detect and electroporate cells.
• the current disclosure describes a device that includes at least two sets of electrodes, where a first set of electrodes is configured to sense the presence of a cell between the electrodes while a second set of electrodes is configured to permeabilize the cells passing between (i.e., separate the sensing region and the electropulsing region to create spatial control of permeabilization) in order to achieve high electroporation efficiency and intracellular delivery while avoiding solution electrolysis.
• Providing electrode set(s) specific for detection and pulsing eliminates the need for a restrictive celltransit time between the electrodes that was determined by the sensing -triggering-pul sing time duration in the prior art devices that forced the cells to have a relatively low speed.
• the device 100 may be designed and fabricated using microfabrication techniques known to those skilled in the art.
• the microfluidic channel 101 (and/or the electrodes) may be patterned on glass slides using techniques such as lithography (e.g., photolithography).
• the microfluidic channels are fabricated using standard soft lithography, where photolithography is used to pattern a negative photoresist on silicon wafers to act as polydimethylsiloxane (PDMS) master molds for replica molding of the microchannels and Ti/Pt electrodes on glass substrates.
• PDMS polydimethylsiloxane
• a PDMS solution is poured over the master mold and baked at 60°C. to produce a hardened negative relief.
• electrodes are fabricated using liftoff techniques on clean glass (e.g., silicon dioxide) slides.
• the electrodes are patterned using a lithographically defined positive photoresist masking layer (EVG620 Exposure system) to define the electrode areas, followed by sputtering a 1000 A thick Ti/Pt layer (Kurt J. Lesker PVD75) and photoresist removal in acetone solution.
• Wires may be soldered on special connective pads patterned on the glass substrates to allow connection with external electronics.
• Alignment marks may be designed and added in order to be able to align the microfluidic channel in parallel with the electrode sets. Methanol can be used as a lubricant between the PDMS and the glass surfaces to help with the alignment.
• the microfluidic channel 101 may be adapted to receive a flow of a plurality of biological cells in a buffer solution from a plurality of inlets 150(a) - (n).
• the microfluidic channel 101 may be designed to hydrodynamically-focus single cells for delivery between the electrodes in the pulsing region 104 via the sensing region 102.
• the inlets are designed to accommodate hydrodynamic flow focusing of the cells in the middle of the channel in order for the cells to flow in a single file line.
• the pulse duration may be about 5 ps to about 20 ms for an electric field strength of about 0.2 - 2.0 kV/cm. In such embodiments, the number of pulses may be about 1-250. In various other embodiments, the pulse duration may be about 50 - 1000 ns for an electric field strength of about 1 - 20 kV/cm. In such embodiments, the number of pulses may be about 1-2000.
• FIGs. 3 A and 3B illustrate the effect of increasing the number of pulses on eTE and SR.
• Survival rate was evaluated through the use of resazurin-based cell viability reagent (Presto Blue HS). As shown in FIGs. 3A and 3B, eTE increased with an increase in number of pulses while the SR decreased.
• the eTE may also be improved by applying a first high field (“HV”) signal (i.e., a permeabilization signal) followed by a second low field (“LV”) signal (i.e., a delivery signal) using two spatially distant pair of electrodes (114(a)-(b) and 114(c)-(d) in the pulsing regions 104(a) and 104(b)) as shown in FIG. IB.
• HV high field
• LV low field
• the two-signal electroporation system may be uniquely designed with the first signal being high amplitude, short duration (using electrodes 114(a)-(b)) which serves to permeabilize the cell membrane without irreversibly damaging the cells, and second signal being longer in duration and lower in amplitude (using electrodes 114(c)-(d)) serving to retain membrane pore opening and to electrophoretically drive molecules into cells.
• the flow rate of the cell as it passes between electrodes 114(a)-(b) (HV signal region) is different from the flow rate of the cell as it passes between electrodes 114(c)-(d) (HV signal region).
• the HV signal is designed with the permeabilization signal high in amplitude (e.g., >1 kV/cm) but short in duration (e.g., ⁇ 1 ms) to permeabilize the cell membrane; and the delivery signal lower in amplitude ( ⁇ 0.6 kV/cm) but longer in duration which serves to retain the opening of the pores from the first signal and electrophoretically transport molecules into the cell.
• the permeabilization signal high in amplitude (e.g., >1 kV/cm) but short in duration (e.g., ⁇ 1 ms) to permeabilize the cell membrane
• the delivery signal lower in amplitude ( ⁇ 0.6 kV/cm) but longer in duration which serves to retain the opening of the pores from the first signal and electrophoretically transport molecules into the cell.
• other parameters that may be controlled to improve eTE without causing cell death and electrolysis may include, without limitation, voltage, frequency, duration, pulse amplitude, duty cycle, pulse type, pulse width,
• a DC pulse train there are several interrelated parameters that can potentially affect the delivery efficiency during electroporation, which may be altered to achieve the desired permeabilization: electric field amplitude, pulse duration, pulse train frequency, duty cycle and number of cycles.
• electric field amplitude For example, a 50 kHz pulse train has a 20 ps pulse period, and with a 50% duty cycle, each pulse is 10 ps long. To obtain a 10 ms total pulse application, 1000 cycles are applied for 20 ms at 50 kHz frequency. The duty cycle controls the amount of rest period following each pulse. Any of the above parameters may be altered to achieve the desired premebilization level without causing electrolysis of the cell.
• the duty cycle can be tuned to increase the pulse width improving delivery time at the cost of electrolysis with the pulse train becoming more like a single DC pulse, or decreased to reduce delivery time while increasing the number of pulses to minimize electrolysis.
• the HV and/or the LV signals may be a DC pulse waveform, a series of DC pulse waveforms, an AC pulse waveform (e.g., sine waves), or combinations thereof.
• the electrode pair(s) in the pulsing region 104 may be designed to further improve eTE by, for example, changing the geometry.
• interdigitated electrodes (IDEs), triangle electrodes, or the like may be used to generate the electroporation signal(s). For example, as shown in FIGs.
• the electrode trace in a pulsing region may include a terminal region (adjacent the microfluidic channel) that is triangular in shape in order to improve efficiency and/or to prevent breakage of a thinner cross section electrical trace.
• a terminal region adjacent the microfluidic channel
• Other shapes to increase the cross-section are within the scope of this disclosure.
• interdigitated electrodes may be used.
• Interdigitated electrodes are fabricated through the process of combining two separately addressable electrode arrays, such that the resulting electrode structure is infused in a zipper-like or combshaped arrangement.
• An example device 170 including interdigitated electrodes 174(a) and 174(b) in the pulsing region 174 is illustrated in FIG. 1C.
• the sensing regions 172 and/or 176 may also include interdigitated electrodes.
• Use of IDEs may improve throughput of the device because IDEs span across a longer length region of the microfluidic channel, obviating the need to hydrodynamically focusing the cells through the pulsing region between a pair of pulsing electrodes. For example, a throughput of electroporating about 1000 - 100,000 cells/second may be achieved (i.e., throughput required to address manufacturing needs for cell therapy applications or other applications).
• the spacing may be controlled in such IDEs to allow high field strength pulses (by decreasing the spacing) at lower voltages to avoid solution electrolysis.
• the spacing or gap between the electrodes may be decreased such that less voltage will be required to stimulate equal electric field amplitudes - this may be done using IDE electrodes allowing for lower voltages for the same eTE ( ⁇ 1 ,2V).
• IDEs with 20 pm electrode width - 20 pm spacing and 1 Volt DC as stimulation induces an electric field with 50 kV/m magnitude, whereas 15 um electrode width - 15 pm spacing and just 1 Volt DC as stimulation induces an electric field with 66 kV/m magnitude.
• Table 3 illustrates the voltages at which electrolysis is observed for different voltages and buffers for the IDEs 20 pm electrode width - 20 pm spacing and 15 pm electrode width - 15 pm spacing:
• a second sensing region 106 (using a pair of electrodes across the microfluidic channel 101 and/or three electrodes, as described below) may be included downstream of the pulsing region 104 in order to monitor the permeabilization state of the detected cell via impedance monitoring.
• the cell type may also be determined based on the change in impedance in this sensing region. However, once permeabilized 220, the cell becomes more conductive, and the impedance drops 204 (and the electric current increases). The change in impedance may be detected in, for example, the second sensing region 106 to provide a signature of the permeabilization state of the cell as shown in FIGs.
• various parameters of the pulsing region may be dynamically adjusted to switch to a well-tolerated customized field for the specific cell type and size to maximize delivery, as described herein (e.g., via a controller).
• the parameters for common cell types may be stored in the system.
• the central control algorithm dependent upon the sensory sweep of the cell membrane impedance information during the application electroporation signal(s) - as discussed above - the central control algorithm retains the ability to modify (e.g., change parameters) the electroporation signal(s) based on the continuous tracking/ sweeping of the cell membrane state in order to preserve cell viability.
• Continuous tracking of the cell membrane state may also provide information regarding the cell viability, and the second signal may be terminated either based on the cell viability close to reaching a point of irreversible damage (threshold determined through precalibration) or saturation of the delivered materials (threshold determined through pre- calibration).
• the electroporation system may be designed to operate at a microscale level, and the electroporation signal(s) may be "chopped" into trains of DC pulses at adjustable frequencies (1 HZ - 1 GHz, 0 - 100% duty cycles) with the appropriate amplitude adjustment to meet the permeabilization requirement, in order to measure the cell membrane permeabilization response during electroporation without the generation of electrolysis.
• a database may be created comprising experimental characterization and/or computations modeling of the electroporation signal based on cell type, structure, buffer characteristics, microfluidic channel characteristics, etc.
• a wide range of pulsing conditions may be tested on a cell population level and are analyzed to determine the optimal pulse features for a particular cell type’s required permeabilization threshold and associated parameters, which may be stored in a database for future use.
• the signal may then be designed based on the detected cell and other properties using the database and/or computations modeling based on the database.
• the dimensions of the device such as, without limitation, the gap between electrode pairs in the first pulsing region (XI) and/or the second pulsing region (X2), distance (Y) between the first pulsing region 174(a) and the second pulsing region 174(b), length (LI) of the electrodes in the first pulsing region (i.e., electrodes 114(c) and 114(d), length (L2) of the electrodes in the second pulsing region (i.e., electrodes 114(a) and 114(b)), or the like; may be optimized to achieve a desired electroporation and throughput of the cells (based on, for example, residence time between electrodes and electric field strength).
• the length LI and/or L2 may be optimized to achieve a desired amount of time the cells will experience a given electric field (which may depend upon cell velocity and the lengths LI and/or L2).
• the distances XI and/or X2 may be optimized based on a desired electric field strength (the distance being inversely proportional to the respective distances).
• the distance Y may be optimized based on experimental or real-time data relating to how the length of time between pulses the first pulsing region 174(a) and the second pulsing region 174(b) affects eTE and cell viability.
• XI and X2 may be about 25 - 200 pm, about 50 - 175 pm, about 75 - 150 pm, about 100 - 125 pm, or the like.
• the distance Y may be about 5 - 1000 pm, about 50 - 900 pm, about 100 - 800 pm, about 150 - 700 pm, about 200 - 600 pm, about 250 - 500 pm, about 300 - 400 pm, or the like.
• LI may be about 20 - 100 pm, about 30 - 90 pm, about 40 - 80 pm, about 50 - 70 pm, or the like.
• L2 may be about 100 - 1000 pm, about 200 - 900 pm, about 300 - 800 pm, about 400 - 700 pm, about 500 - 600 pm or the like.
• the first sensing region and the second sensing region may each include three electrodes (instead of two) to form inline impedance cytometers configured to quantify the impedance change between the upstream (pre-electroporation) and downstream (post electroporation) cell impedance (as shown in FIG. IB).
• each cytometer will be comprised of three electrodes, configured so that the impedance signal between electrode pairs B and C is subtracted from the signal between pairs A and B.
• a lock- in amplifier will be used to inject AC excitation signals into the sensor's center counting electrode.
• Relative impedance will be measured using the Wheatstone bridge circuit, by acquiring output voltages VI and V2 across the resistors, respectively. The output voltages may then be fed to a differential amplifier that cancels the common mode noise in between VI and V2.
• the output voltage of a differential amplifier may be provided to the lock-in amplifier to further remove any unwanted noise.
• Different cell types may be electroporated using the system, as described herein. Examples may include, without limitation, 3T3 fibroblasts, human dermal fibroblasts (HDFs), and lymphoblastoid cells (LCLs).
• Cells may be prepared for electroporation using techniques known to those skilled in the art. For example, the 3T3 fibroblasts may be maintained in complete cell media and cultured to 80% confluency before being harvested for experiments. Prior to electroporation, the cells are trypsinized and resuspended in an electroporation buffer.
• small organic compounds such as drugs and molecular probes
• small strands of RNA that are typically used as interfering RNA (siRNA), mRNA, proteins, and plasmid DNA for direct transfection.
• FIG. 7 provides a method for electroporation of single cells in a continuous flow using the smart electroporation device described above.
• step 701 cells in a continuous flow system are hydrodynamically focused such that a single cell is introduced into a defined upstream detection region.
• Concepts relating to hydrodynamic focusing are known to those skilled in the art. As discussed above, hydrodynamic focusing may not be required when the pulsing electrodes are IDEs.
• a cell detection signal is applied across the upstream detection area and the impedance is monitored.
• the cell detection signal may be an AC detection waveform obtained either from simulation models or known literature.
• the AC detection waveform may be used to monitor the presence or absence of a cell within the detection area based on a change in impedance. Detection of a cell may trigger the smart electroporation system for signal application.
• an electroporation signal is applied across the pulsing region.
• an electroporation signal may include a single pulsing signal such as a short- duration, high frequency DC pulse for permeabilization followed by a long-duration, low frequency DC signal for delivery into the cell, as described above.
• the electroporation signal can include a HV pulsing signal applied in a first upstream region followed by a second LV pulsing signal applied in a downstream region of the microfluidic channel.
• permeabilization status of the cell following may be determined by applying a sensing signal downstream of the electroporation region (704).
• changes in the cell impedance following electroporation may be detected by monitoring the impedance of a permeabilized cell as it passes through a second sensing region.
• the parameters of the electroporation signal for 703 as well as the flow of cells through the pulsing region may be controlled based on a difference between the impedance detected using the upstream detection signal and the downstream sensing signal (705).
• the electroporation signal(s) in the pulsing region(s) may be dynamically controlled using a feedback control that continuously monitors the impedance changes pre and post electroporation.
• the difference in upstream and downstream impedance (i.e., before and after permeabilization) of the cell may be correlated to experimental data to determine electroporation parameters.
• the experimental data may be received from using the electroporation system on immobilized cells (for a particular cell type), where the experimental data is optimized to determine the optimal electroporation parameters and cell velocity for various cell types.
• the electroporation signal may be experimentally predesigned based on the pulse characteristics of the first and second detection signals (and corresponding impedance differences), cell type, cell size, buffer characteristics, and other properties and desired eTE and/or survival rate (as discussed above).
• FIG. 8 depicts an example of internal hardware that may be used to contain or implement the various computer processes and systems as discussed above.
• the smart electroporation discussed above may include hardware such as that illustrated in FIG. 8.
• An electrical bus 800 serves as an information highway interconnecting the other illustrated components of the hardware.
• CPU 805 is a central processing unit of the system, performing calculations and logic operations required to execute a program.
• CPU 805, alone or in conjunction with one or more of the other elements, is a processing device, computing device or processor as such terms are used within this disclosure.
• a CPU or “processor” is a component of an electronic device that executes programming instructions.
• the term “processor” may refer to either a single processor or to multiple processors that together implement various steps of a process.
• processor includes both the singular and plural embodiments.
• Read only memory (ROM) 810 and random access memory (RAM) 815 constitute examples of memory devices.
• memory device and similar terms include single device embodiments, multiple devices that together store programming or data, or individual sectors of such devices.
• a controller 820 interfaces with one or more optional memory devices 825 that service as date storage facilities to the system bus 800.
• These memory devices 825 may include, for example, an external or internal disk drive, a hard drive, flash memory, a USB drive or another type of device that serves as a data storage facility. As indicated previously, these various drives and controllers are optional devices. Additionally, the memory devices 825 may be configured to include individual files for storing any software modules or instructions, auxiliary data, incident data, common files for storing groups of contingency tables and/or regression models, or one or more databases for storing the information as discussed above.
• Program instructions, software or interactive modules for performing any of the functional steps associated with the processes as described above may be stored in the ROM 810 and/or the RAM 815.
• the program instructions may be stored on a non- transitory, computer readable medium such as a compact disk, a digital disk, flash memory, a memory card, a USB drive, an optical disc storage medium, and/or other recording medium.
• An optional display interface 840 may permit information from the bus 800 to be displayed on the display 845 in audio, visual, graphic or alphanumeric format. Communication with external devices may occur using various communication ports 850.
• a communication port 850 may be attached to a communications network, such as the Internet, a local area network or a cellular telephone data network.
• the hardware may also include an interface 855 which allows for receipt of data from input devices such as an imaging sensor 860 of a scanner or other input device 865 such as a keyboard, a mouse, a joystick, a touchscreen, a remote control, a pointing device, a video input device and/or an audio input device.
• input devices such as an imaging sensor 860 of a scanner or other input device 865 such as a keyboard, a mouse, a joystick, a touchscreen, a remote control, a pointing device, a video input device and/or an audio input device.

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• 2023
  • 2023-10-06 EP EP23875863.5A patent/EP4599040A2/de active Pending
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