Cell engineering platform

Abstract

The inventive subject matter provides a diagnostic and clinical application scale cell engineering platform for vector-free or viral delivery of payload / cargo compounds and compositions into non-adherent cells. The platform enables rapid delivery of large numbers of cells into a closed system. Related instruments, systems, techniques, articles, and compositions are also described herein.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 679,715, filed June 1, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The subject matter discussed here refers to cell engineering platforms that do not utilize vector delivery. Background Technology

[0004] Cell transfection efficiency varies among different cell types. Transfection of suspension cells, such as non-adherent cells, has proven to be very difficult using conventional methods, especially at scale. Summary of the Invention

[0005] The present invention provides a cell engineering platform that scales up technologies for carrier-free delivery of payload / cargo compounds and compositions to non-adhesive cells. This platform enables rapid delivery of large numbers of cells. For example, some embodiments of the platform can deliver up to 10 cells at a time for diagnostic and therapeutic purposes. 7 Up to 10 9 One or more cells. The platform can be a closed system capable of sterile transfection, delivering mRNA and RNP (ribonucleoprotein particles) to primary T cells, and is easy to use and repeatable.

[0006] By performing the following steps: providing a population of non-adhesive cells and contacting the cell population with a volume of aqueous solution comprising the payload and an alcohol at a concentration greater than 2% (v / v), the platform of the present invention enables the delivery of the payload across the plasma membrane of non-adhesive cells. For example, the alcohol comprises ethanol, such as greater than 5% ethanol. In some embodiments, the aqueous solution contains 5-30% ethanol, such as 12% or 25% ethanol. Other combinations are also possible.

[0007] In one aspect, a method is disclosed herein comprising filling a chamber of a cell engineering platform with a mixture of cells and a first culture medium, and discharging a first culture medium from the chamber to pass the mixture through a filter, thereby depositing cells on the filter. The cell engineering platform may include a chamber, a cap disposed at a first end of the chamber, a base disposed at a second end of the chamber, and a filter holder disposed within the chamber.

[0008] One or more of the following features can be included in any workable combination. The method can include spraying a delivery solution containing a permeant and a payload onto cells deposited on a filter. The method can include applying a stop solution in the chamber. The method can further include filling the chamber with a second media to resuspend the cells from the filter. The first media expelled can be reused as the second media. The method can include agitating the chamber. The method can include extracting the resuspended cells from the chamber. The filling of the chamber can be done automatically by a pump and a controller. The method can include culturing cells in the chamber. The first media can be expelled from the chamber by providing a positive pressure to the chamber. Alternatively or additionally, the first media can be expelled from the chamber by gravity. Further, a stop solution can be applied to wash the cells. The filling of the chamber with a second media can be performed as a cell washing process, a cell concentration changing process, and / or a cell media changing process.

[0009] In another aspect, a system includes a housing configured to receive a filter plate, the filter plate including wells thereon; a differential pressure applicator configured to apply different pressures to the wells; a delivery solution applicator configured to deliver an atomized delivery solution to the wells; a stop solution applicator configured to deliver a stop solution to the wells; and a media applicator configured to deliver a media to the wells.

[0010] One or more of the following features can be included in any workable combination. The housing can include a chamber, a lid disposed at a first end of the chamber, and a base disposed at a second end of the chamber. The filter plate can be disposed within the chamber. The differential pressure applicator can be connected to the lid of the housing and include a showerhead having a plurality of holes at an end of the showerhead. The stop solution applicator can deliver the stop solution to the wells through a septum disposed in the lid of the housing. The media applicator can deliver the media to the wells through a septum disposed in the lid of the housing or through a port of the chamber. The housing can be configured to tilt the filter plate. The delivery solution applicator can include a robotic arm and a spray head, the robotic arm magnetically coupled to the spray head. The system can include a soft elastomeric barrier surrounding the filter plate and the spray head. The soft elastomeric barrier separates the robotic arm and the spray head. The robotic arm is configured to transport the spray head to a plurality of locations on the filter plate. The stop solution applicator and the media applicator are integrated as ports within the housing, and the housing forms a vessel. The vessel includes a waste media outlet for collecting waste media.

[0011] The system may also include a soft elastomeric barrier that encloses the filter plate and the solution delivery applicator within a housing, the soft elastomeric barrier and the housing forming a bioreactor. The housing may include a filter plate base configured to tilt, rotate, and / or vibrate the filter plate.

[0012] The solution delivery applicator may include a robotic arm and a nozzle, the nozzle being a disposable device. The filter plate size can accommodate 10... 7 The system can automatically supply cells to: a filter plate in culture medium; remove culture medium to form a cell monolayer on top of the filter plate; apply a nebulized delivery solution to the cell monolayer; culture the cells; apply a stop solution to the cultured cell monolayer; supply fresh culture medium to the cell monolayer; and tilt, vibrate, and / or rotate the filter plate to resuspend the cells in fresh culture medium. The system is also configured to repeat the application of the nebulized delivery solution, culturing, and application of the stop solution.

[0013] The transfer solution applicator may include an atomizer. The transfer solution applicator may be configured to deliver 10-300 μL of transfer solution per drive. The system may further include a temperature control system configured to control the temperature of the transfer solution and / or the plate (including the orifice). The transfer solution may include an aqueous solution comprising an alcohol with a payload and concentration greater than 2% (v / v). The alcohol includes ethanol. The aqueous solution may contain greater than 5% ethanol. The aqueous solution may include 5-30% ethanol. The aqueous solution may include 12% or 25% ethanol. The aqueous solution may include 12.5-500 mM KCl (potassium chloride). The aqueous solution may contain 10 6 mM KCl.

[0014] The system may further include a filter plate, and the pores may be configured to contain a large number of non-adhesive cells. The non-adhesive cells may include peripheral blood mononuclear cells. The non-adhesive cells may include immune cells. The non-adhesive cells may include T lymphocytes. The payload may include messenger ribonucleic acid (mRNA). The mRNA may encode a gene-editing composition. The gene-editing composition may reduce the expression of PD-1 (programmed cell death protein 1). The mRNA may encode a chimeric antigen receptor.

[0015] This system can be used to deliver cargo compounds or compositions to mammalian cells. The non-adhesive cell population can be a monolayer.

[0016] On the other hand, a system may include a chamber; a cover disposed at a first end of the chamber; a base disposed at a second end of the chamber; and a filter holder disposed within the chamber.

[0017] One or more of the following features may be included in any feasible combination. The filter holder may include a plurality of pores arranged in a predetermined pattern. The filter holder may include a plurality of targets, wherein the plurality of pores are arranged to allow cell deposition on the filter in areas corresponding to the plurality of pores. The system may also include a gasket disposed between the filter holder and the base. The system may also include a filter holder insert located within the filter holder to receive the filter between the filter holder and the filter holder insert. The filter holder insert may include a concave top surface. The filter holder insert may include a plurality of openings corresponding to the plurality of targets. The filter holder may include three targets. The filter holder may include seven targets. The filter holder may include 19 targets. The plurality of targets may be arranged in a square pattern, a rectangular pattern, a triangular pattern, or a linear pattern.

[0018] The system may also include a controller configured to operate at least one of a pump, valves, heating elements, cooling elements, and a stirring device. The pump may be a peristaltic pump or a positive displacement pump. The cover may also include a pressure port and orifices. The base may include a port for receiving or discharging culture medium. The cover may also include a diaphragm. The pressure port may include a showerhead with multiple orifices at its end. A 0.2-micron filter may be connected to the pressure port. The system may also include a pinch valve configured to open and close the pressure port to atmosphere through the 0.2-micron filter. The system chamber may include a port for extracting processed cells. The system is sized to handle cells larger than 1 × 10⁻⁶. 9 T cells.

[0019] The accompanying drawings and the following detailed description illustrate one or more variations of the subject matter described herein. Other features and advantages of the subject matter described herein will become apparent from the specification, drawings, and claims. Attached Figure Description

[0020] Figure 1 illustrates an exemplary implementation of a process involving cell engineering.

[0021] Figure 2 illustrates an exemplary implementation of a cell engineering platform for carrier-free payload delivery across cell membranes.

[0022] Figure 3 illustrates an exemplary implementation of the cell engineering platform of Figure 2, with a portion of it broken down for illustrative purposes.

[0023] Figures 4A-4C show the cover of an exemplary embodiment of the cell engineering platform.

[0024] Figure 5 shows a substrate according to an exemplary embodiment of the unit engineering platform.

[0025] Figure 6 shows a filter holder in an exemplary embodiment of the cell engineering platform of the present invention.

[0026] Figures 7A-7C show various structures of the filter scaffold plug in exemplary embodiments of the cell engineering platform of the present invention.

[0027] Figures 8A-8C show various structures of the sealing gasket in exemplary embodiments of the cell engineering platform of the present invention.

[0028] Figures 9A-9C show flowcharts of efficient loading across the cell membrane without a carrier.

[0029] Figure 10 shows an exemplary implementation of the cell engineering platform.

[0030] Figures 11A-11C show exemplary embodiments of the atomizer in the spraying process. Figure 11D shows an exemplary image after the cell deposition process, and Figures 11E-11F show schematic diagrams of mass flow rate adjustment for atomizer operation.

[0031] Figure 12 is a computer-aided design (CAD) drawing showing an exemplary implementation with three nozzles installed.

[0032] Figure 13 shows a CAD drawing with only one nozzle.

[0033] Figures 14A-14F show exemplary embodiments of the chamber assembly.

[0034] Figure 15A shows an image of an exemplary implementation of a cell engineering platform with a support system and mounting bracket, and Figure 15B shows an image of the platform after the chamber has been removed.

[0035] Figure 16 is a computer-aided design (CAD) diagram showing an exemplary clinical cell engineering platform for cell therapy.

[0036] Figure 17 is an enlarged view of a portion of the platform.

[0037] Figure 18 is a CAD drawing showing an embodiment of the filter plate and filter.

[0038] Figure 19 is a CAD drawing showing a cross-sectional view of an exemplary platform and a rotating filter plate.

[0039] Figures 20A-20C show a series of images illustrating the various components of an exemplary implementation of the platform.

[0040] Figure 21 is a functional block diagram of an exemplary platform.

[0041] Figure 22 is a schematic diagram showing the disposable and reusable parts of an exemplary platform.

[0042] Figure 23 is a table showing some of the experimental performance capabilities of the exemplary platform.

[0043] Figure 24 is a table showing compatible technologies that can be integrated with the exemplary platform of this invention.

[0044] Figure 25 is a flowchart illustrating exemplary cell engineering processing steps.

[0045] Figure 26 is a flowchart illustrating how this exemplary platform can address cell engineering challenges encountered in the production of adoptive cell therapies.

[0046] Figure 27 is a series of figures showing other exemplary bioreactor designs used in this platform.

[0047] Figure 28 shows an image of Kymriah (Tisagenelecleucel), approved by the U.S. Food and Drug Administration (FDA) for the treatment of acute lymphoblastic leukemia (ALL) in children and young adults, which uses the body's own T cells to fight cancer.

[0048] Figure 29 is a process flowchart showing some sub-processes of the subject matter of this invention.

[0049] Figure 30 shows an exemplary operating cycle of an exemplary platform, similar to that shown in Figure 29.

[0050] Figure 31 shows a comparison of processing time and complexity for isolating and activating one million cells. Peripheral blood mononuclear cells (PBMCs), differentiated 3-cell clusters (CD3+), and Pan T cells were compared.

[0051] Figure 32 shows the comparison results of treatment starting materials (e.g., cells and activating agents) with the number of cycles, cell count, number of freezing flasks, and number of freezing bags.

[0052] Figure 33 shows other exemplary platforms for the subject matter of this invention.

[0053] Figure 34 is a CAD drawing showing an example of a single-use bioreactor.

[0054] Figure 35 shows the filter plate container with the stop solution added.

[0055] Figure 36 shows the filter plate container with the new culture medium added.

[0056] Figure 37 shows the tilting of the bioreactor during operation.

[0057] Figure 38 shows a filter plate container during cell culture "pouring out," in which the container (e.g., a reactor) is tilted, rotated, and vibrated to facilitate the removal of cells from the culture medium.

[0058] Figure 39 shows an embodiment of the membrane stent.

[0059] Figure 40 shows an exemplary system. This is an intermediate system that can accommodate up to 5 x 10 7 Cells. This system is compared with the clinical system (full scale, 1 x 10⁻⁶). 9 (One or more cells) have the same characteristics, but do not include translational nozzle systems. Instead, the system utilizes a single nozzle or a multi-nozzle array.

[0060] In different accompanying drawings, the same serial number indicates the same part. Detailed Implementation Plan

[0061] Despite some progress, the transport of certain particles and / or molecules into cells remains a challenge. Factors such as molecular size or charge can limit and / or prevent molecules from entering cells. In particular, transmembrane transport can be complex due to the cell's molecular and / or membrane structure. The cell membrane, or plasma membrane, is a semi-permeable biological membrane that acts as a selective barrier. The cell membrane regulates the internal chemical composition of the cell. For example, selective molecules can only passively diffuse across the cell membrane. Hydrophobic small molecules (such as O2, CO2, and N2) and small, uncharged polar molecules (such as H2O and glycerol) can passively diffuse across the cell membrane. Larger, uncharged polar molecules (such as amino acids, glucose, and nucleotides) and ions (such as H+) can also diffuse across the cell membrane. + Na + K + and Cl - It cannot passively diffuse across the cell membrane.

[0062] Figure 1 illustrates an exemplary implementation of a treatment / procedure involving cell engineering. Referring to Figure 1, cells can be extracted from a patient, isolated (e.g., concentrated or enriched), and then processed using cell engineering methods. The cell-engineered cells can be amplified and returned to the patient. For transmembrane delivery, viral vector methods can be used. However, viral vector-based methods typically require high cost and complex processes, offer limited accessibility, and produce variable and inconsistent results. Electroporation-based methods can also be used. However, electroporation-based methods generally result in higher cell damage and provide poorer cell recovery and cell function.

[0063] The object of this invention is to provide a carrier-free delivery method to address the cost and complexity challenges of cell engineering. To provide a reliable and consistent cell therapy approach, the subject matter of this invention provides a cell engineering method and platform for delivering a compound or mixture of compounds (e.g., a payload) across the cell membrane by contacting cells with a delivery solution (e.g., a carrier), wherein the delivery solution contains the payload and an agent that reversibly permeates or dissolves the cell membrane. Specifically, a system supplies the cells in a suspension, forms a monolayer of the cells by evacuating the suspension, uses (e.g., sprays) the delivery solution to permeate the cells, and delivers the payload through the permeated cell membrane.

[0064] Using some embodiments of the platform of the present invention, other cell engineering processes can be performed before and / or after the carrier-free payload delivery process, which significantly improves productivity and simplifies the overall process. Furthermore, both non-viral and viral transfection methods can be performed within a single platform. Therefore, exemplary embodiments of this platform can offer better scalability than other methods or systems and can process 10 cells in a single process. 11 Or more cells.

[0065] In some implementations, the platform for transcellular payload delivery can be used for carrier-free payload delivery, viral payload delivery, or a combination of carrier-free and viral payload delivery. For example, viral payload delivery can be performed on the platform by collecting cells onto a filter substrate by draining cell-containing culture medium through a filter, followed by delivery of a solution containing a viral payload to the collected cells. Alternatively or additionally, carrier-free viral payload delivery can be performed on the same cells within a single device, which can reduce processing steps, time, and / or costs, increase cell throughput, viability, and therapeutic efficacy, and reduce cell contamination that may occur when cells are transferred between multiple devices. Other aspects of viral payload delivery are discussed in U.S. Provisional Patent Application No. 62 / 855241, filed May 31, 2019, entitled “Method for Viral Delivery to Cell Populations and Virus Production,” Attorney General’s Case No. 048831-520P01US, the entire contents of which are incorporated herein by reference.

[0066] Furthermore, some embodiments of the subject matter of this invention can provide a cell engineering platform that scales carrier-free delivery technologies for payload / cargo compounds and compositions to non-adhesive cells. This example platform can rapidly achieve delivery to large numbers of cells. For example, some embodiments of the platform can deliver 10 cells in a single use. 7 Up to 10 9One or more cells. The platform can be a closed system capable of aseptic transfection, delivering mRNA and RNP to primary T cells, is easy to use, and allows for repeated transfection. Additionally, the platform described in this invention can be used to process human embryonic kidney (HEK) cells.

[0067] This platform enables the delivery of a payload across the cytoplasmic membrane of non-adhesive cells via the following steps: providing a population of non-adhesive cells and contacting the cell population with a volume of aqueous solution, the aqueous solution comprising the payload and an alcohol at a concentration greater than 2% (v / v). For example, the alcohol comprises ethanol, such as greater than 5% ethanol. In some embodiments, the aqueous solution contains 5-30% ethanol, such as 12% or 25% ethanol. Other components are also possible.

[0068] The present invention also provides a platform that automates the delivery process of carrier-free and / or viral payloads, allowing the process to be performed at various scales. When cells are manually loaded onto and / or manually unloaded from the platform, the system's throughput is limited, posing challenges for clinical / therapeutic applications. Depending on the operator and / or various environmental parameters, contamination and inconsistent process issues may arise. Process automation allows for more consistent execution of carrier-free payload delivery processes, significantly reducing concerns about contamination, and thus enabling easier system scaling. Exemplary embodiments of the platform for performing a carrier-free payload delivery process with automated handling will be described below.

[0069] Example 1

[0070] Figure 2 illustrates an exemplary embodiment of a cell engineering platform 100 for carrier-free payload delivery across cell membranes, and for illustrative purposes, Figure 3 shows partially exploded components of the exemplary embodiment of the cell engineering platform 100 of Figure 2. Referring to Figures 2 and 3, the platform 100 for cell engineering includes a chamber 110, a cover 120 disposed at a first end of the chamber 110, a base 130 disposed at a second end of the chamber 110, a filter holder 140, and a filter holder insert 150. The platform may include a gasket 160 disposed between the base 130 and the filter holder 140. The platform 100 may also include a controller.

[0071] Chamber 110 is surrounded by chamber walls. To provide optical observation capabilities, the chamber walls may comprise (e.g., made of) a transparent plastic material, such as polypropylene, acrylic, polycarbonate, etc. The chamber walls may have an overall cylindrical shell shape with open top and bottom surfaces, and the diameter and height of chamber 110 may be determined based on application and system requirements. For example, the inner diameter of chamber 110 may be 110 mm. Optionally or additionally, chamber 110 may include flat surfaces to facilitate monitoring and / or control of the transfection process using optical imaging equipment. Although chamber 110 is shown as a cylindrical shell, the invention is not limited thereto, and chamber 100 may have various shapes, such as square, rectangular, and triangular, as well as other configurations, such as configurations where cell targets are arranged in a linear arrangement.

[0072] Figures 4A-4C are images showing the cover 120. Figure 4A shows the top surface of the cover 120, Figure 4B shows the bottom surface of the cover 120, and Figure 4C shows the side surface of the cover 120. The cover 120 is positioned on top of the cell engineering platform 100, thereby sealing the chamber 110 of the platform 100. For sealing, the cover 120 may include an O-ring 121. The cover 120 may include a pressure port 122. The pressure port 122 allows the chamber 110 to be connected to a pressure source. The pressure source may provide positive or negative pressure to the chamber 110 of the platform 100. Referring to Figures 4A and 4C, the pressure source may be connected to the top of the pressure port 122, which is located above or outside the cover 120. The bottom of the pressure port 122 (located below or inside the cover 120) may include a showerhead, which includes multiple orifices for distributing fluid across the orifices to supply pressure more evenly to the chamber 110. As shown in Figure 4C, the multiple holes of the shower head can be oriented substantially to the side to prevent airflow (e.g., gas or liquid) delivered through the holes from interfering with (e.g., purging) the cells deposited on the filter.

[0073] Alternatively, pressure port 122 may allow chamber 110 to be connected to (e.g., exposed to) ambient pressure. Furthermore, pressure port 122 may allow chamber 110 to be connected to ambient pressure via a filter. For example, the filter may be a 0.2-micron filter. The filter may also be a nanoparticle filter and / or a high-efficiency particulate air (HEPA) filter. When pressure port 122 is connected to ambient pressure via a filter, chamber 110 may discharge due to gravity rather than a pressure source. To open or close the pressure port, a valve may be located upstream of the filter. For example, the valve may be a pinch valve.

[0074] The cover 120 may include (for example, materials such as stainless steel, SS316, plastic, etc.) a material. The cover 120 may also include at least one hole 123 to receive an atomizer, sensor, etc. The sensor may include one or more of a temperature sensor, a humidity sensor, and a pressure sensor. When the atomizer is mounted in at least one hole 123, the atomizer can be mounted using a directional adjustment device such as a universal joint to allow adjustment of the atomizer's orientation. In some embodiments, the cover 120 may include at least one mounting hole 124 to receive a post 170 to secure the cover 120 to the post 170 and support the platform 100.

[0075] Figure 5 shows the base 130 of platform 100. Base 130 is disposed at the bottom of chamber 110 and houses the membrane retainer 140. Base 130 may include an oil drain port 131. Alternatively or additionally, base 130 may include one or more vacuum ports, a conduit connected to a syringe (for resuspending cells in a medium to provide the opportunity for resuspension at different concentrations or in different media), a heating element, a temperature sensor (e.g., PT100 RTD, thermocouple, etc.), and a vibrating device for agitation during the process. Base 130 may include a hole 132 to receive a column 170 mounted thereon.

[0076] Figure 6 shows a filter holder 140. The filter holder 140 may be made of acetal, aluminum, or the like. The filter holder 140 is disposed at the bottom of chamber 110 and fixed to base 130. The filter holder 140 may accommodate at least one filter on its upper surface. The filter holder 140 may include a target configured to allow cell deposition on the filter. The filter holder 140 may include multiple targets. The filter holder 140 may include three targets. At a location corresponding to each target, the filter holder 140 may include multiple orifices 141 to allow cell suspension culture medium to be passed through the orifices 141. When positive pressure is applied to chamber 110 through pressure port 122 of cap 120, cell suspension culture can be discharged through the multiple orifices 141 and base 130, while cells are collected on the filter surface to form a cell monolayer. Alternatively or additionally, discharge can be performed by gravity discharge of the medium by opening a valve connected to pressure port 122 of the 0.2 microfilter to communicate with ambient pressure. The multiple orifices 141 may be arranged in a predetermined pattern. For example, the orifices 141 may be aligned around the outer diameter of the filter and / or along multiple radial directions of the filter. The filter holder 140 may also include an O-ring 142 to provide a seal between the filter holder 140 and the chamber 110.

[0077] Figures 7A-7C show various configurations of the filter holder insert 150. The filter holder insert 150 is disposed at the bottom of the chamber 110 and on the upper surface of the filter holder 140, thereby securing (e.g., fixing or clamping) the filter between the filter holder 140 and the filter holder insert 150. As shown in Figures 7A-7C, the filter holder insert 150 can have various configurations. For example, the filter holder insert 150 can have a configuration including three targets (Figure 7A), one target (Figure 7B), or an open internal portion (Figure 7C). The filter holder insert 150 with three targets (Figure 7A) promotes cell distribution and cell regeneration only within the filtration area 151. When a smaller number of cells is required, a filter holder insert 150 with one target (Figure 7B) can be used. As shown in Figure 2, the filter holder insert 150 may include a concave upper surface to allow cells to deposit on the filter for more efficient reception of the spray plume. The concave shape also prevents the filter from bulging near the center during filling and discharging and resuspends the cells. The configuration of the targets is not limited to the configuration shown, and there can be more than three targets, for example, seven to nineteen or more targets. The multiple targets can be arranged in a square, rectangular, triangular and / or linear configuration.

[0078] In some embodiments, platform 100 may include a gasket 160, such as a rubber gasket, to provide a seal between the base 130 and the bottom surface of filter holder 140. The seal between the base 130 and filter holder 140 prevents cell culture medium from flowing between the base 130 and filter holder 140, allowing the entire cell culture medium to flow through the filter, thereby improving cell collection efficiency. As shown in Figures 8A-8C, gasket 160 may have various shapes corresponding to different filter configurations. Figure 8C illustrates a configuration of gasket 160 suitable for use with the open internal structure shown in Figure 7C. Figures 8A and 8B show gasket 160 to be used with the three target configurations shown in Figure 7A.

[0079] In some implementations, platform 100 includes a pump such as a peristaltic pump or a positive displacement pump, and valves for automatically controlling fluid flow. The pump may be controlled by a controller. The controller may also control the atomizer, temperature / pressure sensors, heating elements of the base, and vibration devices. For atomizer control, the controller may include a human-machine interface (HMI) (e.g., Omron NB3Q-TW01B 5-inch HMI) and a programmable logic controller (PLC) (e.g., Omron NX1NX1P29024DT1 PLC) hardware platform and a 3-channel rotary controller printed circuit board (PCB). The controller may include multiple modules responsible for controlling various components of the platform. The controller may also include a processor configured to execute program instructions to perform vectorless payload transfer processing, operate input / output devices for the user interface, and operate communication modules to connect the platform to a network.

[0080] Referring to Figure 9A, Figure 9A illustrates an exemplary method for carrier-free payload delivery across the cell membrane. In operation, target cells can be mixed in a culture medium of a specific concentration. For example, approximately 60 million cells can be mixed in approximately 60 mL of culture medium. The prepared cell-containing culture medium can be introduced into the chamber via a disposable tubing set and / or a sterile needle / cannulas (step S11). The cell-containing culture medium can be supplied to the chamber through a port in a septum or cap (e.g., a septum for receiving a plastic welded tube). The addition process can be performed manually or automatically using a pump (e.g., a peristaltic pump or a volumetric pump) and a controller. After the cell-containing culture medium is added to the chamber, the chamber is sealed by a shut-off valve. Similarly, the valve operation can be performed manually or automatically using, for example, a solenoid valve and a controller.

[0081] After the valve is closed and the chamber is sealed, the culture medium is discharged through the filter, thereby depositing target cells (e.g., T cells) on the filter surface (step S12). For example, positive pressure can be supplied to the chamber through the central pressure port of the cap. After the culture medium is discharged from the chamber, vacuum pressure can be applied to the chamber to release any remaining positive pressure in the chamber. It may or may not be necessary to eliminate the remaining positive pressure with vacuum pressure. In some embodiments, positive pressure and vacuum pressure can be applied to the chamber alternately during media discharge to adjust / rearrange cell deposition on the filter. The media can also be discharged by opening a valve (e.g., a pinch valve), exposing the pressure port of the cap to ambient pressure, and discharging the media by gravity. A filter (e.g., a 0.2-micron filter) can be provided at the pressure port to prevent foreign particulate matter from entering the chamber.

[0082] Figure 11D shows an exemplary image of the cell deposition pattern after step S12. Figure 11D is an image taken using colored beads that simulate cell behavior. Since there are multiple pores 141 in the filter holder 140, the cell deposition pattern essentially corresponds to the pattern of the multiple pores 141. For step 12, a filter membrane with an appropriate pore size can be selected for efficient cell deposition. The filter membrane allows for easy filtration of the liquid while retaining cells without affecting cell viability. Pores obtained through track etching techniques have the highest yield (e.g., minimal impact on activity and minimal cell loss in the filtrate). To avoid deformation during filtration and promote uniform cell distribution on the target surface, a drain pan with a membrane thicker than the filter can be disposed below the filter. For example, the filter can be 9 µm to 30 µm thick, and the drain pan can be 80 µm thick. During filtration, when the liquid (e.g., cell culture medium) suspending the cells is drained, the cells are only uniformly distributed in the filtration area corresponding to the target site, with minimal loss in the non-filtration areas.

[0083] Therefore, the cell deposition method can be controlled and limited to a specific area where cells are deposited as a monolayer on the filter. Here, a “monolayer” of cells can refer to cells that are substantially horizontally distributed and form one or more vertical cell layers. A monolayer can include one cell layer, one to two cell layers, one to three cell layers, one to five cell layers, or more. The number of cell layers is not limited thereto, and in some embodiments, a monolayer can refer to any number of cell layers.

[0084] Subsequently, a delivery solution containing a cell permeabilizer and a payload (e.g., cargo) is sprayed through an atomizer (step S13). The controller can control the spray volume and duration. For example, the delivery solution can be sprayed for approximately 300 milliseconds. For spraying the delivery solution, the cargo can be introduced into the nozzle via microfibrils or injected via a resealable injection port.

[0085] After the delivery solution is sprayed, a stop solution is introduced through a disposable tubing set and / or a sterile plastic needle / cannulas (step S14). The stop solution can be supplied to the chamber through a port on the septum or cap. The stop solution can be supplied manually or automatically using a pump and controller. The required amount of stop solution is introduced into the chamber. For example, approximately 10 mL of stop solution can be introduced over approximately 20 seconds. However, step S14 is not limited to the application of stop solution and can also provide a cell washing process depending on the composition of the solution applied.

[0086] After the stop solution is introduced, the cells are resuspended (step S15). For resuspension, approximately 60 mL of culture medium, which can be fresh culture medium or previously used culture medium drained from the incubator, can be introduced via a syringe or pump. The fresh culture medium can be supplied to the chamber through a port on the septum or lid. Alternatively, or additionally, the fresh culture medium can be supplied to the chamber from below the filter through a drain hole. The fresh culture medium can be supplied to the chamber by injecting it into the chamber under positive pressure or by applying vacuum pressure to the chamber (e.g., through the pressure port 122), thereby allowing the chamber to absorb the fresh culture medium. For example, the duration of the resuspension step can be from approximately 3 seconds to approximately 1 minute. In some embodiments, various methods, such as platform tilting, agitation (e.g., platform vibration), etc., can be used during or after the resuspension process to improve the resuspension effect. However, step S15 is not limited to the resuspension step and may also include cell concentration changing procedures, cell washing procedures, and / or cell culture medium changing procedures. The cell concentration change process, cell washing process and / or cell culture medium change process can also be performed in steps S11 and S12 by refilling the chamber after step S12 and repeating the drain / refill process multiple times as needed.

[0087] After resuspending the cells in the culture medium, the engineered cells are collected for further processing (step S16). Alternatively, the engineered cells may be cultured in the chamber prior to collection and / or further processing. The platform may be rinsed or washed after subsequent processing. Alternatively, the entire chamber or a portion thereof may be made into a disposable unit that can be disposed of after use and replaced with a new unit.

[0088] Figures 9B and 9C show exemplary process flows with exemplary process parameters. However, the process parameters are not limited to those shown in Figures 9B and 9C, and process parameters such as the amount (volume) of culture medium, cell count, concentration, and duration of each step can vary depending on the application. Referring to Figure 9B, the first step may be mixing 6 × 10⁶ cells in 30 ml of culture medium. 7Cells are transferred (step S21). The culture medium can be removed by applying a positive pressure of 20 mbar (step S22), which takes 40 to 60 seconds. In step S23, 100 µL of delivery solution can be delivered to target 1 via 420 ms, and the nebulizer can then be moved and the delivery solution can be repeated on targets 2 and 3. After delivery, the cells can be cultured for 30 seconds (step S24), and 1 mL of stop solution can be delivered to each target site for 30 seconds of culture (step S25). To resuspend the cells, fresh culture medium can be provided from the pump at the bottom. When using the loop insert, 75 mL of culture medium can be used (step S261), and when using the 3-target insert, 50 mL of culture medium can be used (step S262). In step S27, the resuspended cells can be rinsed 15 times (i.e., 5 times per target). Finally, the engineered cells can be transferred to a T75 flask and stored in a culture vessel (step S28).

[0089] Referring to Figure 9C, 6×10⁷ to 8×10⁷ cells can be mixed in 30 ml of culture medium (step S31). The culture medium can be removed by gravity (step S32), which takes 40 to 60 seconds. In step S33, 60-100 µL of transfer solution can be transferred to target 1 in more than 420 ms, and subsequently, the nebulizer can be moved and the transfer solution can be repeated on targets 2 and 3. After transfer, the cells are cultured for 30 seconds (step S34), and 5 mL of stop solution is delivered to each target for 30 seconds of culture (step S35). To resuspend the cells, culture medium can be provided from the pump at the bottom. When using a loop insert, 75 mL of culture medium can be used (step S36). A 10-minute waiting step can be added (step S37). In step S38, the resuspended cells can be rinsed 15 times (i.e., 5 times per target). Finally, the engineered cells can be transferred to a T75 flask and stored in a culture vessel (step S39).

[0090] Figure 10 illustrates an exemplary embodiment of the cell engineering platform, and Figures 11A-11C illustrate exemplary embodiments of an atomizer 1100 for a spraying process. Referring to Figures 11A-11C, the atomizer 1100 includes a liquid orifice plate 1101 and a gas orifice plate 1102 on its lower surface (Figure 11A). On the upper surface of the atomizer 1100, a liquid inlet 1103 and an air inlet 1104 may be formed (Figure 11B). Accordingly, the liquid orifice plate 1101 is connected to a liquid reservoir via the liquid orifice plate inlet 1103, and the gas orifice plate 1102 is connected to a gas reservoir via the air inlet 1104, as shown in Figure 11C.

[0091] The gas (such as air) flow rate can be controlled by a mass flow controller or a volumetric flow controller. By using a mass / volume flow controller, a constant amount (e.g., constant mass and / or constant volume) of gas can be supplied through the nebulizer 1100, ensuring a constant volume of liquid to be sprayed and consistent droplet size, regardless of variations in pressure, temperature, humidity, on / off timing, etc., within the chamber. Mass / volume flow rate regulation also allows for more precise and repeatable delivery of the nebulized solution to the cells. By actively controlling the mass / volume flow rate of the driving gas, the spray plume is less sensitive to variations in plume density, angle, velocity, vortex formation, droplet size, deposition area, etc., resulting in more repeatable and stable transfection performance.

[0092] Figure 11E shows an embodiment of a pneumatic circuit for regulating the mass / volume flow rate of the driving gas. The driving gas can be supplied from a gas cylinder 1110, passing through a gas supply 1111, a control valve 1112, an orifice plate 1113, and a back pressure regulator 1114. The gas can then be controlled by a mass / volume flow controller 1115 before being delivered to the atomizer 1116 via a gas line 1117. The solution to be sprayed can be supplied by a pump 1118. The pump 1118 can be a screw pump, a positive displacement pump, a pneumatic jet pump, etc. Thus, the solution can be sprayed through the atomizer 1116 and delivered to the chamber 1119. Figure 11F shows an embodiment of the control and flow path for spray operation. Figure 11F shows the path of a nozzle.

[0093] Figure 12 is a computer-aided design (CAD) drawing illustrating an exemplary embodiment with three nozzles 1200 installed. Figure 13 shows a CAD drawing of one of the nozzles 1200. Referring to Figure 13, the nozzle 1200 includes a nozzle 1203, a local liquid reservoir 1201, and a cover for a local liquid reservoir 1206, supported on a cylindrical base plate 1202. A threaded hole 1204 allows the nozzle to be secured to a chamber. The cylindrical base plate 1202 includes a valve 1205, such as a pinch valve, for initiating and / or stopping fluid flow from the reservoir.

[0094] Figures 14A-14F illustrate exemplary embodiments of a disposable chamber assembly. Referring to Figure 14B, an exemplary disposable chamber assembly 1400 may include, for example, a chamber wall 1401 made of polycarbonate, a welded cap module 1402, a base module 1403 with a welded membrane, an nebulizer mounting hole 1404, a cell inlet / outlet 1407, and a three-way valve 1408. For operation of the nebulizer, the payload and delivery solution can be supplied through a culture medium port 1405, and compressed air can be supplied through a pneumatic port 1406. Figure 14C shows a side view of an exemplary disposable chamber assembly, Figure 14D shows a cross-sectional view of an exemplary disposable chamber assembly, and Figure 14E shows a front view of an exemplary disposable chamber assembly. Figure 14F shows another exemplary disposable chamber assembled from five targets and five nozzles.

[0095] Figure 15A shows an exemplary implementation of a cell engineering platform with a support system and mounting bracket, while Figure 15B shows a platform with the chamber removed.

[0096] The exemplary implementation described in Example 1 can reduce the number of cells from approximately 10 in a single transfection. 7 10 cells were transfected to approximately 10 9 Single or multiple cells. This platform enables the continuous delivery of cargo such as mRNA to T cells. The system can be aseptically operated within a biosafety cabinet or used as a closed system in any environment. Platform operation can be manual or automated. For automated operation, the fluid handling system can be automatically controlled via controllers and control software. The platform can be configured as a multi-purpose system that can be reused after cleaning. In some embodiments, the platform can be configured as a single-use system comprising disposable components such as disposable chamber units. In some embodiments, the platform can be used for carrier-free payload delivery across cell membranes and / or viral payload delivery across cell membranes.

[0097] Because of its automated process, this cell engineering platform can more stably perform carrier-free payload delivery processes, minimize contamination, and therefore, the system can be scaled. Thus, the cell engineering platform can provide a reliable carrier-free delivery method, reducing the cost and complexity of cell engineering techniques.

[0098] Example 2

[0099] Figure 16 is a computer-aided design (CAD) drawing illustrating another embodiment of a clinical cell engineering platform for cell therapy, while Figure 17 is an enlarged view of a portion of the platform. Platform 1700 includes a robotic arm 1701 with a nozzle, a flexible dome 1702 surrounding a filter base 1703, and a spray nozzle. The platform also includes ports for culture media, reagents, cargo, and waste, thus achieving a closed system.

[0100] The robotic arm 1701 can carry the nozzle to multiple locations within the elastomeric dome 1702 to spray the solution onto the filter base 1703 to form a monolayer of cells. Additionally, the filter base 1703 can be tilted and rotated to immerse the cells in the reagent.

[0101] Figure 18 is a CAD drawing illustrating an embodiment of the filter plate and filter. To achieve transfer, cell monocells can be formed on the filter plate. Positive pressure is applied to the container. During positive pressure, clamp valves in the inlet and outlet directions can be closed. Depending on the degree of activation, the filter can be tracked for etching and used only once. The filter holder includes a pore pattern designed for complete and rapid removal of the culture medium. During operation, spraying can be initiated immediately after monolayer formation.

[0102] Figure 19 is a CAD drawing illustrating a cross-sectional view of an exemplary platform and the rotation of the filter plate. The platform 1700 is shaped to collect waste culture medium beneath the filter plate. The platform 1700 may include a flexible dome 1702, an annular port 1704 for culture medium and stop solution, a filter base 1705, a waste culture medium chamber 1706, and a tilting / rotating unit 1707. The filter base 1705 may be molded from polysulfone and can form a template when cells are extruded under pressure from the culture medium. During operation, the device can be tilted and rotated to remove cells suspended in fresh culture medium.

[0103] Figures 20A-20C are a series of images showing components of an exemplary embodiment of the platform. Figure 20A shows an embodiment of a robotic arm, Figure 20B shows an embodiment of a safety housing for housing the platform, and Figure 20C shows an embodiment of a filter disc. The filter disc may be a polycarbonate track etched (PCTE) filter with a diameter of 293 mm.

[0104] Figure 21 is a functional block diagram of an exemplary platform. The platform includes modules for applying fresh culture medium, stop solution, cells in the culture medium, and cargo in the transfer solution to a main bioreactor. Additionally, the platform may include a filter ranging from 0.2 micrometers to atmospheric size. The platform can utilize the input and output of waste and engineered cells to execute transfer protocols.

[0105] Figure 22 illustrates the disposable and reusable portions of the platform in this embodiment. The robotic arm can be magnetically coupled to the closed filter cartridge, which may include a disposable cartridge that maintains a localized environment between the nozzle and the filter base. Disposable tubing can carry liquid in and out of the bioreactor. The cargo can be introduced into the transfer solution via syringes or other means.

[0106] Figure 23 is a table illustrating some exemplary functions of the exemplary platform. In some implementations, system inputs may include cells, culture media, molecular cargo, stop solutions, and air and / or gases. When performing carrier-free payload delivery, relevant adjustments and standard operating procedures are followed according to the operating protocol. Relevant standard operating procedures include current Good Automated Manufacturing Practice (cGAMP), ASTM E2500, ISO 14791, 21 CFR § 211.68, and 21 CFR § 1271.160[d]).

[0107] In some embodiments, the cells may include adherent cells or non-adhesive cells. Adhesive cells may include at least one of primary mesenchymal stem cells, fibroblasts, monocytes, macrophages, lung cells, neurons, fibroblasts, human umbilical vein (HUVEC) cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells or immortalized cells (e.g., cell lines). In a preferred embodiment, the cell population includes non-adhesive cells, for example, the percentage of non-adhesive cells in the population is at least 50%, 60%, 75%, 80%, 90%, 95%, 98%, 99%, or 100%. Non-adhesive cells include primary cells and immortalized cells (e.g., cells of cell lines). Exemplary non-adhesive / suspension cells include primary hematopoietic stem cells (HSCs), T cells (e.g., (differentiation group 3) CD3+ cells, (differentiation group 4) CD4+ cells, (differentiation group 8) CD8+ cells), natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells, or cell lines such as the Jurkat T cell line. Non-limiting embodiments of T cells may include CD8+ or CD4+ T cells. In some aspects, a CD8+ subset of CD3+ T cells is used. CD8+ T cells can be purified from a population of PBMCs using a positive separation method using anti-CD8 microspheres.

[0108] In this embodiment, cells are cultured in a standard cell culture medium, such as a complete RPMI (Roswell Park Memorial Institute medium) basal medium, heat-inactivated fetal bovine serum (FBS), and, for example, about 10% v / v penicillin, streptomycin, and L-glutamine. In some embodiments, the standard culture medium may be supplemented with cytokines, such as interleukin-2 (IL-2) (200 U / ml).

[0109] In one embodiment, the concentration of the cytokine may be from about 10 U / ml to about 500 U / ml. In other embodiments, the concentration of the cytokine may be about 50 U / ml, about 100 U / ml, about 200 U / ml, about 300 U / ml, about 400 U / ml, or about 500 U / ml. The concentration of the cytokine may be about 200 U / ml.

[0110] In some embodiments, a cell-compatible culture medium is used in the transport method. For example, Prime XV (Irvine Scientific) and X-Vivo (Lonza) are serum-free and animal-component-free culture media that can be used. In some embodiments, the cell culture medium is supplemented with higher concentrations of cytokines. For example, cytokines may include interleukin-2 (IL-2) to enhance proliferation (Tumeh P, et al., J Immunother 2010. 33(6): 759-768 and Besser MJ, et al., Cytotherapy 2009. 11:206-217). In other embodiments, the culture medium may include Immuculturation. TM -XF expansion medium (StemCell Technologies). Like Prime XV, it is a serum-free, xenogeneic T cell culture medium. In some embodiments, TexMACS (Miltenyi Biotech) can be used as an alternative serum-free medium for T cell culture. CTS-OpTmizer-T cell expansion SFM can also be used as a serum-free medium for T cell culture.

[0111] In implementations, molecular cargo (e.g., payload) may include gene-editing tools, small chemical molecules, peptides or proteins, or nucleic acids. The payload may include messenger ribonucleic acid (mRNA). The mRNA may encode a gene-editing composition. The mRNA may encode a chimeric antigen receptor.

[0112] In other embodiments, the molecular cargo to be delivered (e.g., a payload) may include a composition for editing genomic DNA (i.e., a gene-editing tool). For example, the gene-editing composition may include a compound or complex capable of cutting, notching, splicing, rearranging, translocating, recombinating, or otherwise altering genomic DNA. Alternatively or additionally, the gene-editing composition may include: (i) a gene-editing complex that cuts, notches, splices, rearranges, translocates, recombines, or otherwise alters genomic DNA; or (ii) a compound that may be treated or modified to be contained in a cut, notched gene-editing complex that splices, rearranges, translocates, recombines, or otherwise alters genomic DNA. In various embodiments, the gene-editing composition includes one or more of (a) a gene-editing protein; (b) an RNA molecule; and / or (c) a ribonucleoprotein (RNP).

[0113] In some embodiments, the gene-editing composition includes a gene-editing protein, and the gene-editing protein is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a Cre recombinase, a Hin recombinase, or a Flp recombinase. In other embodiments, the gene-editing protein may be a fusion protein that binds a homing endonuclease to a modular DNA-binding domain of TALENs (megaTAL). For example, megaTAL may be delivered as a protein, or mRNA encoding a megaTAL protein may be delivered into the cell.

[0114] In this implementation, the molecular cargo (e.g., the payload) may include small chemical molecules. These small chemical molecules may be less than 1000 Da. The chemical molecules may include MitoTracker. ® Red CMXRos, disodium iodide, methotrexate and / or DAPI (4',6-diamino-2-phenylindole).

[0115] In the implementation scheme, the molecular cargo may be a peptide. The peptide may be 5000 Da. This peptide may include ecallantide (trade name Kalbitor), a 60-amino acid polypeptide used to treat hereditary angioedema and prevent blood loss during cardiothoracic surgery; liraglutide (trade name Victoza for type II diabetes, and Saxenda for obesity); and icatibant (trade name Firazyner, a peptide-like formulation for treating acute attacks of hereditary angioedema). Small interfering RNA (siRNA) molecules are approximately 20-25 base pairs in length, or approximately 10000-15000 Da. siRNA molecules can reduce the expression of any gene product, for example, by knocking out the expression of clinically relevant target or model genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA, GAPDH siRNA-FITC, cyclophilin B siRNA, and / or lamin siRNA. Protein therapy may include peptides, enzymes, structural proteins, receptors, cellular proteins or circulating proteins or fragments thereof. Proteins or peptides are approximately 100-500,000 Da, for example, 1,000-150,000 Da. The protein may include any therapeutic, diagnostic, or investigational protein or peptide, such as β-lactoglobulin, ovalbumin, bovine serum albumin (BSA), and / or horseradish peroxidase. In other instances, the protein may include cancer-specific apoptosis proteins, such as tumor necrosis factor-associated apoptosis-inducing protein (TRAIL).

[0116] The molecular weight of the antibody is typically approximately 150,000 Da. This antibody may include anti-actin antibodies, anti-GAPDH antibodies, anti-Src antibodies, anti-Myc antibodies, and / or anti-Raf antibodies. The antibody may include green fluorescent protein (GFP) plasmids and GLuc plasmids. DNA molecules may be larger than 5,000,000 Da. In some embodiments, the antibody may be a murine monoclonal antibody, such as ibritumomab-tiuxetin, tolmumab-CD3, tositumamab, a human antibody, or a humanized mouse (or other species of origin) antibody. In other embodiments, the antibody may be a chimeric monoclonal antibody, such as abciximab, basciximab, cetuximab, infliximab, or rituximab. In other embodiments, the antibody may be a humanized monoclonal antibody, such as aruntumazumab, bevacizumab, pegylated certolizumab-pegol, daclizumab, gentuzumab-ozogamicin, trastuzumab, tocilizumab, ipilimumab, or panitumab. The antibody may comprise an antibody fragment, such as abatisip, africept, alexicept, or etanercept. This invention includes not only complete monoclonal antibodies but also immunologically active antibody fragments, such as Fab or (Fab)2 fragments; engineered single-chain antibody molecules; or chimeric molecules, such as antibodies containing binding specificity to one antibody, such as mouse-derived antibodies, and the remainder of another antibody, such as human-derived antibodies.

[0117] Molecular cargo (e.g., payload) may include therapeutic agents. The term "therapeutic agent, such as a drug or active agent" refers to any compound intended for therapeutic or diagnostic purposes; this term can be understood to mean any compound administered to a patient for the treatment of a disease. Therefore, therapeutic agents may include proteins, peptides, antibodies, antibody fragments, and small molecules. A therapeutic agent described in U.S. Patent No. 7,667,004 (incorporated herein by reference) entitled "Humanized Anti-vascular Endothelial Growth Factor Antibody" may be used in the methods described herein. Therapeutic agents may include cisplatin, aspirin, statins (e.g., pittavastatin, etc.). The payload may include at least one of torvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promethazine hydrochloride (HCl), chlorpromazine hydrochloride, thioridazine hydrochloride, polymyxin B sulfate, chloramphenicol, phenylfluorouracil hydrochloride, and phenazopyridine hydrochloride, and fluoxetine. The payload may include a diagnostic agent. The diagnostic agent may include at least one detectable marker or label, such as methylene blue, Patent Blue V, and indocyanine green. The payload may include a fluorescent molecule. The payload may include detectable nanoparticles. The nanoparticles may include a quantum dot.

[0118] In some implementations, the cargo may include linear DNA or DNA plasmids.

[0119] In some embodiments, the cells are cultured after transfer and before the addition of a stop solution. In one embodiment, the stop solution comprises phosphate-buffered saline (PBS). The concentration of the phosphate-buffered saline may be approximately 0.5X PBS.

[0120] In some implementations, the delivery solution may be formulated by a GMP (Good Manufacturing Practice) compliant supplier and provided in 10 mL aluminum foil vials. A separate Drug Master File (DMF) is archived for solution delivery. Stability testing and analysis certificates (C of A) may be provided by suppliers with current Good Manufacturing Practices (cGMPs).

[0121] Figure 24 is a table illustrating compatible technologies that can be integrated with the exemplary platform. For example, various selection / isolation / concentration kits can be used. Furthermore, various activation and stimulation technologies can be used, such as anti-CD3 (differentiation cluster 3) monoclonal antibody (mAb) Muromonab-CD3 (OKT3) and interleukin-2 (IL-2), CD3 / differentiation cluster 28 (CD28) Dynabeads®, Transact TM Microbeads, viral peptides, artificial antigen-presenting cells (AAPC), Expamer TM Or ImmunoCult TM Human CD2 / CD3 / CD28 T cell activator. In the examples, various amplification and culture media (aeration static bags, amplification bags, aeration rapid amplification, Xuri) can be used. TM Cell expansion system W25, Xuri TM W5 Quantum Cell Expansion System ® In the examples, various formulations (COBE) can be used. ® 2991 Cell Expert, Cell Saver ® 5. LOVO automated cell processing system and Sefia), cryopreservation (Mr. Frosty) TM VIA Freeze TM Duo, VIAFreeze TM Qyad, Cryomed TM , and CoolCell ® ) and thawing buffer (VIA ThawCB1000, ViaThawSC2, CellSeal) ®Automatic defrosting system and ThawSTAR ® CFTZ transport and cell thawing system.

[0122] Figure 25 is a process flow diagram illustrating exemplary cell engineering processing steps. The exemplary platform can be used between the DynaMag CTS and Sefia / COBE2991. In this example, Dynabead isolation and activation takes approximately two days. After activation, the sample can be added to a WAVE bioreactor / G-rex (gas permeation rapid expansion) to generate immune cells, and then further processed using the DynaMag CTS (ThermoFisher catalog number 12102), for example, for T cell isolation, Solupore Clinical, Sefia / COBE2991, and the output can be measured using flow cytometry.

[0123] Figure 26 is a process flowchart illustrating how this exemplary platform can address cell engineering issues in the production of adoptive cell therapies.

[0124] Example 3

[0125] Figure 27 is a series of images illustrating another exemplary bioreactor design used on the platform. The exemplary bioreactor processes 2 x 10⁻⁶ cells / years. 7 This allows for the delivery of T cells with impressive efficiency, viability, and function. All uptake occurs within an area of ​​approximately 30 mm in diameter. In this configuration, a single spray can achieve an efficiency greater than approximately 60%. In some embodiments, the filter may be a polycarbonate track-etched (PCTE) filter or a polyester track-etched (PETE) filter with a nominal pore size of approximately 1 µm to 3 µm. 1 µm to 3 µm filters are suitable for T cell applications. In some embodiments, the nominal pore size of the filter may be approximately 5 µm to approximately 10 µm or larger. 5 µm to 10 µm filters are suitable for HEK cells, which typically range in size from 11 µm to 15 µm. The filter material and pore size are not limited to these and can be selected based on the type, size, and concentration of target cells.

[0126] Figure 28 is an image of Kymriah (Tisagenelecleucel), approved by the U.S. Food and Drug Administration (FDA) for the treatment of children and adolescents with acute lymphoblastic leukemia (ALL). This product utilizes the body's own T cells to fight cancer. In this embodiment, the basic unit of cell therapy is a single dose, which can be from 10... 6 10 cells 9The number of cells varied. In Figure 28, the dose of CAR-T (chimeric antigen receptor T cell) therapy for Kimriah was as high as 2.5 × 10⁻⁶ cells. 8 CAR+T cells. In other embodiments, the Lonza NuclearFector LV closed / sterile system transfected 10 CAR+T cells in approximately 10 minutes. 9 Cells. In this embodiment, the entire processing time may be less than 1 hour and less than about 20 steps.

[0127] Figure 29 is a process flow diagram illustrating certain aspects of the sub-processes based on the content of this topic. In step 1, T cells are loaded into the culture medium via a peristaltic pump and ¼-inch PVC tubing. This system can be filled with reagents, cells, and gases. In step 2, the culture medium is removed by positive pressure. The culture is stopped, and fresh culture medium is added. In some embodiments, step 2 may be repeated multiple times to improve delivery efficiency. In step 3, the bioreactor is gently tilted to one side to “pour out” the cells and culture medium. In step 4, the bioreactor is gently shaken and rotated to vibrate the filter membrane, assisting in the resuspension of the cells and their exit from the filter bioreactor. In step 5, the bioreactor is returned to an upright position, and the process restarts, for example, returning to step 2 for additional processing, or continuing to pump engineered cells in fresh culture medium into the next unit process.

[0128] Figure 30 illustrates an exemplary operation cycle of the exemplary platform, similar to that shown in Figure 29. The example subprocess execution time is illustrated with a total processing time of approximately 3.5 minutes.

[0129] For some exemplary embodiments, the starting materials may include clusters of differentiated 3 (CD3+) T cells or PBMCs (peripheral blood mononuclear cells). Cells can be activated by various methods, including Dynabeads (e.g., Dynabeads CTS (cell therapy system) microspheres), soluble CD3 / CD28 (differentiation group 28) antibodies, or for T cell activation. T cell TransAct is a polymeric nanomatrix that binds to humanized recombinant CD3 and CD28 agonists to ensure successful activation of quiescent T cells in blood cell populations (e.g., PBMCs or enriched T cell populations) without involving CD4 (differentiation group 4) or CD8 (differentiation group 8).

[0130] Figure 31 illustrates a comparison of the complexity of isolating and activating one million cells. Peripheral blood mononuclear cells (PBMCs), differentiated 3-cell clusters (CD3+), and Pan T cells were compared.

[0131] Figure 32 shows a comparison of various treatment methods for an exemplary number of cells, such as activating reagents, number of cycles, number of cells, number of freezing bottles, and number of freezing bags.

[0132] In some implementations, the system input may include cells, culture medium, molecular cargo, stop solution, and air and / or gas.

[0133] In some embodiments, the cells may include adherent cells or non-adhesive cells. Adhesive cells may include at least one of primary mesenchymal stem cells, fibroblasts, monocytes, macrophages, lung cells, neurons, fibroblasts, human umbilical vein (HUVEC) cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells or immortalized cells (e.g., cell lines). In a preferred embodiment, the cell population includes non-adhesive cells, for example, the percentage of non-adhesive cells in the population is at least 50%, 60%, 75%, 80%, 90%, 95%, 98%, 99%, or 100% non-adhesive cells. Non-adhesive cells include primary cells and immortalized cells (e.g., cells from cell lines). Exemplary non-adhesive / suspension cells include primary hematopoietic stem cells (HSCs), T cells (e.g., (differentiation group 3) CD3+ cells, (differentiation group 4) CD4+ cells, (differentiation group 8) CD8+ cells), natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells, or cell lines such as the Jurkat T cell line. Non-limiting examples of T cells may include CD8+ or CD4+ T cells. In some aspects, a CD8+ subset of CD3+ T cells is used. CD8+ T cells can be purified from PBMC populations using a positive separation method using anti-CD8 microspheres.

[0134] In this embodiment, cells are cultured in a standard cell culture medium, such as a complete RPMI (Roswell Park Memorial Institute medium) basal medium, heat-inactivated fetal bovine serum (FBS), for example about 10% v / v, penicillin, streptomycin, and L-glutamine. In some embodiments, the standard culture medium may be supplemented with cytokines, such as interleukin-2 (IL-2) (200 U / ml).

[0135] In one embodiment, the concentration of the cytokine can be from about 10 U / ml to about 500 U / ml. In other embodiments, the cytokine concentration can be about 50 U / ml, about 100 U / ml, about 200 U / ml, about 300 U / ml, about 400 U / ml, or about 500 U / ml. The cytokine concentration can be about 200 U / ml.

[0136] In some embodiments, a cell-compatible culture medium is used in this delivery method. For example, Prime XV (Irvine Scientific) and X-Vivo (Lonza) are serum-free and animal-free culture media that can be used. In some embodiments, the cell culture medium is supplemented with higher concentrations of cytokines. For example, cytokines may include interleukin-2 (IL-2) to enhance proliferation (Tumeh P, et al., J Immunother 2010. 33(6): 759-768 and Besser MJ, et al., Cytotherapy 2009. 11:206-217). In other embodiments, the culture medium may include Immucult™ XF extended medium (StemCell Technologies). Like Prime XV, it is also a serum-free, xenogeneic T cell culture medium. In some embodiments, TexMACS (Miltenyi Biotech) may be used as an alternative serum-free medium for T cell culture.

[0137] In implementations, molecular cargo (e.g., payload) may include gene-editing tools, small chemical molecules, peptides or proteins, or nucleic acids. The payload may include messenger ribonucleic acid (mRNA). The mRNA may encode gene-editing components. The mRNA may encode chimeric antigen receptors.

[0138] In other embodiments, the molecular cargo to be delivered (e.g., a payload) may include a composition for editing genomic DNA (i.e., a gene-editing tool). For example, the gene-editing composition may include compounds or complexes that cut, notch, splice, rearrange, translocate, recombine, or otherwise alter genomic DNA. Alternatively or additionally, the gene-editing composition may include compounds that: (i) include gene-editing complexes that cut, notch, splice, rearrange, translocate, recombine, or otherwise alter genomic DNA; or (ii) may be treated or modified to be contained in a cut, notch gene-editing complex, splice, rearrange, translocate, recombine, or otherwise alter genomic DNA. In various embodiments, the gene-editing composition includes one or more of (a) gene-editing proteins; (b) RNA molecules; and / or (c) ribonucleoproteins (RNPs).

[0139] In some embodiments, the gene-editing composition includes a gene-editing protein, and the gene-editing protein is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a Cre recombinase, a Hin recombinase, or a Flp recombinase. In other embodiments, the gene-editing protein may be a fusion protein that binds a homing endonuclease to a modular DNA-binding domain of TALENs (megaTAL). For example, megaTAL may be delivered as a protein, or mRNA encoding a megaTAL protein may be delivered to the cell.

[0140] In this embodiment, the molecular cargo (e.g., payload) may include small chemical molecules. These small chemical molecules may be less than 1000 da. The chemical molecules may include MitoTracker. ® Red CMXRos, disodium iodide, methotrexate and / or DAPI (4',6-diamino-2-phenylindole).

[0141] In the implementation scheme, the molecular cargo may be a peptide. The peptide has a molecular weight of approximately 5000 Da. This peptide may include ecallantide (trade name Kalbitor), a 60-amino acid polypeptide used to treat hereditary angioedema and prevent blood loss during cardiothoracic surgery; liraglutide (trade name Victoza for the treatment of type II diabetes, and Saxenda for the treatment of obesity); and icatibant (trade name Firazyner, a peptide-like formulation for the treatment of acute exacerbations of hereditary angioedema). Small interfering RNA (siRNA) molecules are approximately 20-25 base pairs in length, or approximately 10,000-15,000 Da. siRNA molecules can reduce the expression of any gene product, such as clinically relevant target genes or model genes, for example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA, GAPDH siRNAFITC, cyclophilin B siRNA, and / or lamin siRNA. Protein therapy may include peptides, enzymes, structural proteins, receptors, cellular proteins or circulating proteins or fragments thereof. Proteins or peptides are approximately 100-500,000 Da, for example, 1,000-150,000 Da. The protein may include any therapeutic, diagnostic, or investigational protein or peptide, such as β-lactoglobulin, ovalbumin, bovine serum albumin (BSA), and / or horseradish peroxidase. In other embodiments, the protein may include cancer-specific apoptosis proteins, such as tumor necrosis factor-associated apoptosis-inducing protein (TRAIL).

[0142] The molecular weight of an antibody is typically approximately 150,000 Da. This antibody may include anti-actin antibodies, anti-GAPDH antibodies, anti-Src antibodies, anti-Myc antibodies, and / or anti-Raf antibodies. The antibody may include green fluorescent protein (GFP) plasmids and GLuc plasmids. DNA molecules may be larger than 5,000,000 Da. In some embodiments, the antibody may be a murine monoclonal antibody, such as ibritomomab-tiuxetin, muromomab-CD3, tositumamab, a human antibody, or a humanized mouse (or other species of origin) antibody. In other instances, the antibody may be a chimeric monoclonal antibody, such as abciximab, basciximab, cetuximab, infliximab, or rituximab. In other embodiments, the antibody may be a humanized monoclonal antibody, such as aruntumazumab, bevacizumab, pegylated certolizumab-pegol, daclizumab, gentuzumab-ozogamicin, trastuzumab, tocilizumab, ipilimumab, or panitumab. The antibody may comprise an antibody fragment, such as abatisip, africept, alexicept, or etanercept. This invention includes not only complete monoclonal antibodies but also immunologically active antibody fragments, such as Fab or (Fab)2 fragments; engineered single-chain antibody molecules; or chimeric molecules, such as antibodies containing binding specificity to one antibody, such as mouse-derived antibodies, and the remainder of another antibody, such as human-derived antibodies.

[0143] Molecular cargo (e.g., payload) may include therapeutic agents. "Therapeutic agent, such as a drug or active agent" can mean any compound used for therapeutic or diagnostic purposes, and the term can be understood to mean any compound administered to a patient for the treatment of a disease. Therefore, therapeutic agents may include proteins, peptides, antibodies, antibody fragments, and small molecules. The therapeutic agents described in U.S. Patent No. 7,667,004, entitled "Humanized Antibody Against Vascular Endothelial Growth Factor" (incorporated herein by reference), may be used in the methods described herein. The therapeutic agent may include at least one of cisplatin, aspirin, statins (e.g., pittavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promethazine hydrochloride (HCl), chlorpromazine hydrochloride, thioridazine hydrochloride, polymyxin B sulfate, chloramphenicol, phenylfluorouracil hydrochloride, and phenazopyridine hydrochloride), and fluoxetine. The payload may include a diagnostic reagent. The diagnostic reagent may include at least one detectable label or marker, such as methylene blue, Patent Blue V, and indocyanine green. The payload may include a fluorescent molecule. Detectable payloads may include nanoparticles. Nanoparticles may include a quantum dot.

[0144] In one embodiment, cells are cultured after transfer but before the addition of a stop solution. In this embodiment, the stop solution comprises phosphate-buffered saline (PBS). The concentration of the PBS is approximately 0.5 times that of regular PBS.

[0145] Figure 33 shows another exemplary platform based on the current topic. The closed system 3300 may include a bag or reservoir 3301, a pump 3302, an interface filter 3303, a cargo introduction module 3304, and a tilting bioreactor 3305.

[0146] Figure 34 is a CAD drawing showing an exemplary disposable bioreactor. The bioreactor can be tilted at a normal angle of 15 degrees, can be rotated while titrating, can be gently shaken, and may include a vibrating element to vibrate the filter membrane. Goods can be introduced via SMA01. SMA01 is a nebulizer, and in embodiments, any nebulizer may be included. In embodiments, the values ​​when using a nebulizer relate to the nebulization of a volume between approximately 10-300 µl of cell-permeable solution. Exemplary nebulizers are described in U.S. Patent No. 5,411,208 or U.S. Patent No. 6,634,572, which are incorporated herein by reference in their entirety. Other nebulizers are commercially available, such as the DuraMist™ nebulizer (Sigma-Aldrich GXARG1DM04-1EA), nebulizers, OneNeb, Series 2 inert concentric nebulizers, or those used with ICP-OES (Agilent Technologies G8010-60293). In embodiments, the nebulizer may be an ultrasonic nebulizer or a vibrating mesh nebulizer. The input and output tubes can be soldered or use Hospira spin-fit screw-close connectors. Other atomizers are commercially available, such as the DuraMist™ atomizer (Sigma-Aldrich GXARG1DM04-1EA), atomizers, OneNeb, Series 2 inert concentric atomizers, or those used with ICP-OES (Agilent Technologies G8010-60293). In some implementations, the atomizer can be an ultrasonic atomizer or a vibrating mesh atomizer. The input and output tubes can be soldered or use Hospira spin-fit screw-close connectors.

[0147] Figure 35 illustrates a filter plate container with a stop solution added. The stop solution can be added through a series of orifices on the circumference of the filter plate container (as shown). The internal manifold design of the container ensures that the flow rate is equal at each orifice.

[0148] Figure 36 illustrates the filter plate container after the addition of fresh culture medium. After incubation, cell culture medium can be added to it through the second set of circumferential wells. The addition of culture medium can be done at a second time (for mL volumes).

[0149] Figure 37 shows the tilting of the bioreactor during operation. The entire bioreactor is shown in the left figure, and the filter plate container is shown in the right figure. This system is a closed system. The bioreactor (including the filter plate container) can be tilted 15 degrees from its normal angle. The bioreactor can be rotated while tilting. The bioreactor can be gently rocked and contains a vibrating element to vibrate the filter membrane.

[0150] Figure 38 illustrates a filter plate container during cell culture medium “pouring out,” where the container (such as a reactor) is tilted, rotated, and vibrated to facilitate the removal of cells from the culture medium. When the cell culture medium contains cells suspended within it, the filter plate container can be tilted, and the cells can be poured out from the membrane surface as needed by tilting, rotating, and vibrating. For example, this method can remove a “cake” of cells from a filter. Different vibration modes and offsets can be used.

[0151] Current topics can include many components, such as accelerating product introductions, acoustics, additive manufacturing, adhesives, improved assembly, automation, equipment integration, digital prototyping, dynamic tuning, fluid dynamics, human-machine interfaces, optical communications, optics, power electronics, precision injection molding, precision machinery, printed electronics, sensors, software application design, wireless connectivity, and documentation.

[0152] Figure 39 is an embodiment of a membrane scaffold. This exemplary membrane scaffold can be implanted for use with a first exemplary platform (refer to the description in Figures 16-26) or a second exemplary platform (refer to the description in Figures 27-38). Controlling the cell deposition pattern on the filter membrane can be challenging when using positive pressure to form a monolayer membrane on a filter plate. The ridge-pore structure on the filter plate influences the deposition morphology of suspended cells on the filter membrane. Cells deposit on the filter, but only where the ridges and pores are located on the underlying membrane scaffold (e.g., acting as a template). The location of cell deposition and the deposition pattern within that region can be controlled by the membrane scaffold design. Furthermore, discrete cell monolayers can be created on continuous filter membranes using membrane scaffolds containing discrete filtration regions similar to drains.

[0153] As shown in Figure 39, the membrane scaffold comprises a single filtration zone. In this embodiment, a 44 mm polycarbonate track-etched (PCTE) filter membrane (denoted as "a" and indicated by an outer dashed circle) is placed on the membrane scaffold (b). The membrane scaffold has ridges and pores located within a central diameter of 25 mm (c). To illustrate the function of this exemplary design of the filtration unit, the membrane scaffold is inserted into the base of an Amicon stirred cell pressure filtration unit (not shown here). A Dynabeads suspension is added to the chamber and positive pressure is applied. Dynabeads (previously used as a T cell model) are deposited only at the locations of the ridges and pores (indicated by inner dashed circles).

[0154] Figure 40 illustrates an embodiment of an intermediate system capable of processing up to 5 x 10⁷ units. This system possesses the same characteristics as the clinical system (full-size, e.g., capable of processing more than 5 x 10⁷ units per process). 7 Cells, for example, 1 x 10 8 One, and 10 9 (One or more cells), but the system does not include a translational nozzle mechanism, instead using a single nozzle or a multi-nozzle array.

[0155] While some variations have been described in detail above, other modifications or additions are possible. For example, design variations may include filter bases of different geometries, such as rectangular, square, or elliptical shapes. Furthermore, filter bases with different topography may include convex, concave, and textured surfaces with microscopic or macroscopic features. Additionally, target configurations including circular and annular targets are also considered. In embodiments, these modifications or additions can optimize cell deposition under the spray target.

[0156] The subject matter described in this paper offers numerous technical advantages. For example, single-use eliminates the need for system sterilization, significantly reduces the risk of cross-contamination between patient samples, and simplifies the validation process. Furthermore, single-use allows for controlled temperature and humidity within the bioreactor, resulting in healthier cell populations. Another advantage is its ability to co-deliver via a single nozzle or multiple nozzles, and to deliver various cargoes through this co-delivery process. Moreover, the subject matter described is rapid and simple, and the gentle cell handling process maintains cell health and enables the engineering of natural cell populations. Temperature control of the filter substrate further enhances control over the delivery process and facilitates cell recovery from the system. The combination of membrane scaffold design and nebulizer parameters allows for system optimization, delivering different cargoes to different cell types.

[0157] Implementation Scheme

[0158] This invention is based on a surprising discovery that compounds or mixtures of compounds (compositions) can be delivered into the cytoplasm of eukaryotic cells by contacting the compound to be delivered (e.g., the payload) with a solution containing a reagent that can reversibly penetrate or dissolve the cell membrane. Preferably, the solution is delivered to the cells in the form of a spray (e.g., water particles). (See, for example, PCT / US2015 / 057247 and PCT / IB2016 / 001895, both of which are incorporated herein by reference). For example, the cells are coated with a spray but not soaked or immersed in a solution containing the delivered compound. Typical reagents for penetrating or dissolving eukaryotic cell membranes include alcohols and detergents, such as ethanol and Triton X-100, respectively. Other exemplary cleaning agents, such as surfactants, include polysorbate 20 (e.g., Tween 20), 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid (CHAPS), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxy-1-propanesulfonic acid (CHAPSO), sodium dodecyl sulfate (SDS), and octyl glucoside.

[0159] Examples of conditions for achieving coated cell populations include providing fine-particle spray delivery. For example, these conditions do not involve dripping or pipetting large quantities of solution onto the cells to wet or submerge a large number of cells with a given volume of liquid. Therefore, the mist or spray comprises a ratio of fluid volume to cell volume. Alternatively, the conditions comprise a ratio of the volume of the mist or spray to the area of ​​exposed cells, for example, the area of ​​the cell membrane exposed when cells are present as a confluence layer or a substantially confluence layer on the bottom of a tissue culture vessel (e.g., the pores of a tissue culture plate, such as a microplate).

[0160] "Cargo" or "payload" refers to a compound or composition that passes through the cell membrane and enters the cell via an aqueous solution.

[0161] In one aspect, transferring the payload across the cell membrane involves providing a cell population and contacting it with a volume of aqueous solution. The aqueous solution includes the payload and an alcohol content greater than 2%. The volume of the aqueous solution can be a function of the exposed surface area of ​​the cell population or the number of cells in the population.

[0162] In another aspect, the composition for delivering the payload across the cell membrane comprises an aqueous solution including the payload, an alcohol at a concentration greater than 2%, a salt at a concentration greater than 46 mM, a sugar at a concentration less than 121 mM, and a buffer solution at a concentration less than 19 mM. For example, the concentration of the alcohol (e.g., ethanol) does not exceed 50%.

[0163] One or more of the following characteristics may be included in any feasible combination. The volume of solution to be delivered to the cell is a plurality of units, such as a spray, or multiple droplets on water particles. The volume is described relative to a single cell or relative to a confluence or substantially confluence (e.g., at least 75%, at least 80% confluence, such as 85%, 90%, 95%, 97%, 98%, 100%) of the cell population. For example, the volume may be 6.0 x 10⁻⁶ per cell. -7 Microliters to 7.4 x 10⁻⁶ per cell -4 Within the microliter range. The volume can be 4.9 x 10⁻⁶ cells per cell. -6 Microliters to 2.2 x 10⁻⁶ per cell -3 Within the microliter range. The volume can be 9.3 x 10⁻⁶ cells per cell. -6 Microliters up to 2.8 x 10⁻⁶ cells per cell -5 Within the microliter range. The volume could be approximately 1.9 x 10⁻⁶ cells per cell. -5 Microliters, or within the range of ten percent. The volume can be 6.0 x 10⁻⁶ cells per cell. -7 Microliters to 2.2 x 10⁻⁶ per cell -3 Within the microliter range. The volume can be 2.6 x 10⁻⁶ m² / m² exposure area. -9 Micro-increase to 1.1 x 10⁻⁶ per square micrometer of exposure area. -6 Within the microliter range. The volume can reach 5.3 x 10⁻⁶ per square micrometer of exposed area. -8 Micro-increase to 1.6 x 10⁻⁶ per square micrometer of exposure area. -7 Within the microliter range. The volume can be approximately 1.1 x 10⁻⁶ m² / m² exposure area. -7 A slight increase means approximately within the range of 10%.

[0164] Cell fusion refers to the contact between cells on a surface. It can be expressed as an estimated (or calculated) percentage; for example, 10% fusion means 10% of the surface (e.g., a tissue culture vessel) is covered by cells, and 100% means it is completely covered. For example, adherent cells grow in two dimensions on the surface of a tissue culture well, culture dish, or culture flask. Non-adherent cells can be rotated off, aspirated from the top of the cell population using a vacuum or tissue culture medium, or removed by suction or by removing a vacuum from the bottom of the vessel.

[0165] A gas-driven spray forms an aqueous solution, thereby bringing a cell population into contact with a given volume of aqueous solution. The gas can include nitrogen, ambient air, or an inert gas. The spray can comprise discrete volume units ranging in size from 1 nm to 100 µm, for example, with a diameter of 30-100 µm. In some embodiments, the spray comprises discrete volume units with a diameter of approximately 30-50 µm. A total aqueous solution of 20 µl can be delivered via the spray to a depth of approximately 1.9 cm. 2 The cell-occupying area, such as one well of a 24-well culture plate, is supplied with a total volume of 10 µl of aqueous solution to approximately 0.95 cm² of the cell-occupying area, such as one well of a 48-well culture plate. Typically, the aqueous solution contains a payload to be transferred to the cells across the cell membrane, and the second volume is a buffer or culture medium without the payload. Alternatively, the second volume (buffer or medium) may also contain the payload. In some embodiments, the aqueous solution includes the payload and alcohol, and the second volume does not contain alcohol (and optionally does not contain the payload). The cell population may be contacted with the aqueous solution for 0.1–10 minutes before adding the second volume of buffer or culture medium to immerse or suspend the cell population. The buffer or culture medium may be phosphate-buffered saline (PBS). The cell population may be contacted with the aqueous solution for 2 seconds to 5 minutes before adding the second volume of buffer or culture medium to immerse or suspend the cell population. The cell population may be contacted with the aqueous solution (e.g., containing the payload) for 30 seconds to 2 minutes before adding the second volume of buffer or culture medium (e.g., without the payload) to submerge or suspend the cell population. Before adding a second volume of buffer or culture medium to submerge or suspend the cell population, the cell population can be exposed to the spray for approximately 1–2 minutes. During the time between spraying the cells and adding the buffer or culture medium, the cells retain moisture through stratification of the sprayed water.

[0166] The aqueous solution may contain 5% to 30% ethanol. The aqueous solution may include one or more of 75% to 98% H₂O, 2% to 45% ethanol, 6 to 91 mM sucrose, 2 to 500 mM KCl, 2 to 35 mM ammonium acetate, and 1 to 14 mM (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) (HEPES). For example, the transfer solution contains 10 6 mM KCl and 25% ethanol.

[0167] The cell population may include adherent cells or non-adhesive cells. The adherent cells may include at least one of primary mesenchymal stem cells, fibroblasts, monocytes, macrophages, lung cells, nerve cells, fibroblasts, human umbilical vein (HUVEC) cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells or immortalized cells (e.g., cell lines). The cell population may also include cells adhering to a suspension in a culture medium. Cells adhering to a suspension in a culture medium may include human embryonic kidney (HEK) cells or macrophages. In a preferred embodiment, the cell population includes non-adhesive cells, for example, the percentage of non-adhesive cells in the cell population is at least 50%, 60%, 75%, 80%, 90%, 95%, 98%, 99%, or 100%. The non-adhesive cells include primary cells and immortalized cells (e.g., cells from cell lines). Exemplary non-adhesive / suspended cells include primary hematopoietic stem cells (HSCs), T cells (e.g., CD3+ cells, CD4+ cells, CD8+ cells), natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells, or cell lines such as the Jurkat T cell line.

[0168] The payload may include small chemical molecules, peptides, proteins, or nucleic acids. Small chemical molecules may have a molecular weight of less than 1000 Da. Chemical molecules may include MitoTracker® Red CMXRos, propidium iodide, methotrexate, and / or DAPI (4',6-diamino-2-phenylindole). Peptides may have a molecular weight of approximately 5000 Da. The peptides may include ecallantide (trade name Kalbitor), a 60-amino acid polypeptide used to treat hereditary angioedema and prevent blood loss during cardiothoracic surgery; liraglutide (trade name Victoza for type 2 diabetes, and Saxenda for obesity); and Icatibant (trade name Firazyner, a peptide-like formulation for treating acute attacks of hereditary angioedema). Small interfering RNA (siRNA) molecules are approximately 20-25 base pairs in length, or approximately 10,000-15,000 Da. siRNA molecules can reduce the expression of any gene product, such as knocking out the expression of clinically relevant target or model genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA, GAPDH siRNA-FITC, cyclin B siRNA, and / or lamin siRNA. Protein therapy may include peptides, enzymes, structural proteins, receptors, cellular proteins, or circulating proteins or fragments thereof. Proteins or peptides are approximately 100–500,000 Da, for example, 1,000–150,000 Da. The protein may include any therapeutic, diagnostic, or investigational protein or peptide, such as β-lactoglobulin, ovalbumin, bovine serum albumin (BSA), and / or horseradish peroxidase. In other instances, the protein may include cancer-specific apoptosis proteins, such as tumor necrosis factor-associated apoptosis-inducing protein (TRAIL).

[0169] The molecular weight of the antibody is typically approximately 150,000 Da. This antibody may include anti-actin antibodies, anti-GAPDH antibodies, anti-Src antibodies, anti-Myc antibodies, and / or anti-Raf antibodies. The antibody may include green fluorescent protein (GFP) plasmids, GLuc plasmids, and BATEM plasmids. The DNA molecular weight may be greater than 5,000,000 Da. In some embodiments, the antibody may be a murine monoclonal antibody, such as ibritomomab-tiuxetin, muromomab-CD3, tositumamab, a human antibody, or a humanized mouse (or other species of origin) antibody. In other embodiments, the antibody may be a chimeric monoclonal antibody, such as abciximab, basciximab, cetuximab, infliximab, or rituximab. In other embodiments, the antibody may be a humanized monoclonal antibody, such as aruntumazumab, bevacizumab, pegylated certolizumab-pegol, daclizumab, gentuzumab ozogamicin, trastuzumab, tocilizumab, ipilimumab, or panitumab. The antibody may comprise an antibody fragment, such as abatisip, africept, alexicept, or etanercept. This invention includes not only complete monoclonal antibodies but also immunologically active antibody fragments, such as Fab or (Fab)2 fragments; engineered single-chain antibody molecules; or chimeric molecules, such as antibodies containing binding specificity to one antibody, such as mouse-derived antibodies, and the remainder of another antibody, such as human-derived antibodies.

[0170] Molecular cargo (e.g., payload) may include therapeutic agents. A therapeutic agent, for example, “medicine or active agent”, may mean any compound used for therapeutic or diagnostic purposes, and the term can be understood to mean any compound administered to a patient for the treatment of a disease. Therefore, therapeutic agents may include proteins, peptides, antibodies, antibody fragments, and small molecules. The therapeutic agents described in U.S. Patent No. 7,667,004, entitled “Humanized Antibody Against Vascular Endothelial Growth Factor” (incorporated herein by reference), may be used in the methods described herein. Therapeutic agents may include at least one of cisplatin, aspirin, statins (e.g., pittavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promethazine hydrochloride (HCl), chlorpromazine hydrochloride, thioridazine hydrochloride, polymyxin B sulfate, chloramphenicol, phenylfluorouracil hydrochloride, and phenazopyridine hydrochloride), and fluoxetine. The payload may include diagnostic reagents. The diagnostic agent may include a detectable tag or label, such as at least one of methylene blue, Patent Blue V, and indocyanine green. The payload may include a fluorescent molecule. Detectable payloads may include nanoparticles. Nanoparticles may include a quantum dot.

[0171] Non-adhesive cell populations can be substantially confluent, for example, with a confluence greater than 75%. Cell confluence refers to cells coming into contact with each other on a surface. It can be expressed as an estimated (or calculated) percentage; for example, 10% confluence means 10% of the surface (e.g., a tissue culture vessel) is covered by cells, and 100% means it is completely covered. For example, adherent cells grow in two dimensions on the surface of tissue culture wells, culture dishes, or culture flasks. Non-adhesive cells can be rotated downwards, pulled from the top of the cell population by vacuum or aspiration of tissue culture medium, or removed from the bottom of the vessel by aspiration or vacuum. Cell populations can form a monolayer of cells.

[0172] The alcohol may be selected from methanol, ethanol, isopropanol, butanol, and benzyl alcohol. The salt may be selected from NaCl, KCl, Na2HPO4, KH2PO4, and C2H3O2NH. In a preferred embodiment, the salt is KCl. The sugar may include sucrose. The buffer may include 4-2-(hydroxyethyl)-1-piperazine ethanesulfonic acid.

[0173] This invention relates to a method for delivering molecules across the plasma membrane. The invention has applications in intracellular delivery and has been used, for example, to deliver molecular biological and pharmacological therapeutic agents to target sites, such as cells, tissues, or organs. The method comprises introducing the molecule into an aqueous composition to form a matrix; atomizing the matrix into a spray; and contacting the matrix with the plasma membrane.

[0174] This invention relates to a composition for delivering molecules across the plasma membrane. The invention finds practicality in the field of intracellular delivery and is applicable, for example, to delivering molecular biological and pharmacological therapeutic agents to target sites, such as cells, tissues, or organs. The compositions of this invention comprise alcohol, salt, sugar, and / or buffers.

[0175] In some implementations, a permeation technique is shown here that facilitates the intracellular delivery of molecules independent of molecular and cell types. Nanoparticles, small molecules, nucleic acids, proteins, and other molecules can be efficiently delivered in situ to suspended or adherent cells, including primary and stem cells, with low cytotoxicity, and the technique is compatible with high-throughput and cell-based automated analysis.

[0176] The exemplary methods described herein include a payload comprising an alcohol. The term "alcohol" refers to a polyatomic organic compound comprising a hydroxyl (-OH) functional group attached to at least one carbon atom. An alcohol may be a monohydric alcohol and may comprise at least one carbon atom, such as methanol. An alcohol may comprise at least two carbon atoms (e.g., ethanol). In other respects, an alcohol comprises at least three carbon atoms (e.g., isopropanol). An alcohol may comprise at least four carbon atoms (e.g., butanol), or at least seven carbon atoms (e.g., benzyl alcohol). Exemplary payloads may comprise no more than 50% (v / v) of alcohol, more preferably, payloads comprising 2-45% (v / v) of alcohol, 5-40% of alcohol, and 10-40% of alcohol. A payload may comprise 20-30% (v / v) of alcohol.

[0177] Most preferably, the payload delivery solution comprises 25% (v / v) alcohol. Alternatively, the payload may comprise 2-8% (v / v) alcohol, or 2% alcohol. The alcohol may comprise ethanol, and the payload comprises 5, 10, 20, 25, 30, and up to 40% or 50% (v / v) ethanol, such as 27%. In an exemplary method, the alcohol is methanol, and the payload may comprise 5%, 10%, 20%, 25%, 30%, or 40% (v / v) methanol. The payload may comprise 2-45% (v / v) methanol, 20-30% (v / v) methanol, or 25% (v / v) methanol. Preferably, the payload comprises 20-30% (v / v) methanol. Alternatively, the alcohol is butanol, and the payload comprises 2%, 4%, or 8% (v / v) butanol.

[0178] In some aspects of the subject matter of this invention, the payload is in a solution or buffer solution.

[0179] According to the subject matter of the invention, the payload comprises at least one salt. The salt is selected from NaCl, KCl, Na₂HPO₄, C₂H₃O₂NH₄, and KH₂PO₄. For example, the concentration of KCl ranges from 2 mM to 500 mM. In some preferred embodiments, the concentration is greater than 100 mM, for example, 10⁶ mM.

[0180] According to an exemplary method of the present invention, the payload may include sugar (e.g., sucrose or disaccharides). According to the exemplary method, the payload includes sugar of less than 121 mM, 6-91 mM, or 26-39 mM. Furthermore, the payload includes 32 mM sugar (e.g., sucrose). Optionally, the sugar is sucrose and the payload includes 6.4, 12.8, 19.2, 25.6, 32, 64, 76.8, or 89.6 mM of sucrose.

[0181] According to the exemplary method of the present invention, the payload may include a buffer (e.g., a weak acid or a weak base). The buffer may include a zwitterion. According to the exemplary method, the buffer is 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid. The payload may include a buffer of less than 19 mM (e.g., 1-15 mM, 4-6 mM, or 5 mM buffer). According to the exemplary method, the buffer is 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid and the payload comprises 1, 2, 3, 4, 5, 10, 12, or 14 mM of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid. More preferably, the payload comprises 5 mM of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid.

[0182] According to an exemplary method of the present invention, the payload comprises ammonium acetate. The payload may include less than 46 mM of ammonium acetate (e.g., ammonium acetate in the range of 2-35 mM, 10-15 mM, or 25 mM). The payload may include 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or 33.6 mM of ammonium acetate.

[0183] The volume of the aqueous solution is driven by a gas, which may include compressed air (e.g., ambient air), and other embodiments may include inert gases such as helium, neon, and argon.

[0184] In some aspects of this subject matter, cell populations may include adherent cells (e.g., lung, kidney, immune cells, such as macrophages) or non-adhesive cells (e.g., suspended cells).

[0185] In certain aspects of the subject matter of this invention, the cell population may be substantially fused, and said substantially may include greater than 75% fusion. In a preferred embodiment, the cell population may form a single monolayer.

[0186] According to the exemplary method, the payload to be delivered has an average molecular weight of up to 20,000,000 Da. In some embodiments, the average molecular weight of the payload to be delivered can reach 2,000,000 Da. In some embodiments, the payload to be delivered can have an average molecular weight of up to 150,000 Da. In further embodiments, the payload to be delivered has an average molecular weight of up to 15,000 Da, 5,000 Da, or 1,000 Da.

[0187] Payloads delivered via the cell membrane may include small chemical molecules, peptides or proteins, polysaccharides or nucleic acids, or nanoparticles. Small chemical molecules may be less than 1000 Da, peptides may have a molecular weight of about 5000 Da, siRNA may have a molecular weight of about 15000 Da, antibodies may have a molecular weight of about 150,000 Da, and DNA may have a molecular weight greater than or equal to 5,000,000 Da. In a preferred embodiment, the payload includes mRNA.

[0188] According to an exemplary method, the payload comprises molecules to be transferred in the range of 3.0–150.0 µM, more preferably, molecules to be transferred in the range of 6.6–150.0 µM (e.g., 3.0, 3.3, 6.6, or 150.0 µM molecules to be transferred). In some embodiments, the payload to be transferred has an average molecular weight of up to 15,000 Da, and the payload comprises molecules to be transferred in the range of 3.3 µM.

[0189] According to an exemplary method, the payload to be delivered has an average molecular weight of up to 15,000 Da, and the payload comprises 6.6 µM of molecules to be delivered. In some embodiments, the payload to be delivered has an average molecular weight of up to 1,000 Da, and the payload comprises 150.0 µM of molecules to be delivered.

[0190] According to a further aspect of this subject matter, a method for transferring molecules of more than one molecular weight through a plasma membrane is provided; the method includes the steps of: introducing molecules of more than one molecular weight into an aqueous solution; and contacting the aqueous solution with the plasma membrane.

[0191] In some embodiments, the method includes introducing a first molecule having a first molecular weight and a second molecule having a second molecular weight into a payload, wherein the first and second molecules may have different molecular weights, or wherein the first and second molecules may have the same molecular weight. According to an exemplary method, the first and second molecules may be different molecules.

[0192] In some implementations, the payload to be delivered may include therapeutic or diagnostic agents, including, for example, cisplatin, aspirin, various statins (e.g., pittavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, imipramine hydrochloride, chlorpromazine hydrochloride, thioridazine hydrochloride, polymyxin B sulfate, chloramphenicol, diflubenzuron hydrochloride, and oxazolidin hydrochloride), and fluoxetine. Other therapeutic agents include antibacterial agents (aminocyclohexanes (e.g., gentamicin, neomycin, streptomycin), penicillins (e.g., amoxicillin, ampicillin), glycopeptides (e.g., avopalene, vancomycin), macrolides (e.g., erythromycin, tilmicosin, tylosin), quinolones (e.g., sarafloxacin, enrofloxacin), streptomycin (e.g., virginiamycin, quinoplastin, carbapenems, lipopeptides, oxazolidinones, cycloserine, ethambutol, ethoxyamide), etc. Isonicotinamide, para-aminosalicylic acid, and pyrazinamide. In some embodiments, antiviral drugs (e.g., abacavir, acyclovir, enfovirtidine, entecavir, nelfinavir, nevirapine, nevirapine, oseltamivir-retgravir, ritonavir, stavudine, and valacyclovir). This therapy may include protein therapy for treating a variety of diseases, such as cancer, infectious diseases, hemophilia, anemia, multiple sclerosis, and hepatitis B or C.

[0193] Other exemplary effective devices may include detectable markers or labels, such as methylene blue, patent blue V, and indocyanine green.

[0194] The methods described herein may also include a payload comprising a detectable portion or detectable nanoparticles (e.g., quantum dots). The detectable portion may include fluorescent molecules or radioactive reagents (e.g., 125I). The presence of a fluorescent molecule can be detected by fluorescence when it is exposed to light of an appropriate wavelength. The most commonly used fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, terephthalaldehyde, and fluorescein. Such molecules can also be labeled with fluorescent emitting metals (e.g., 152Eu) or other lanthanides. These metals can be attached to the molecule using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Such molecules can also be detected by coupling with a chemiluminescent compound. The presence of the chemiluminescent labeled molecule is then determined by detecting the luminescence that occurs during a chemical reaction. Examples of particularly useful chemiluminescent labeled compounds include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalates.

[0195] In other embodiments, the payload to be delivered may include a composition for editing genomic DNA (i.e., a gene editing tool). For example, the gene editing composition may include a compound or complex that cuts, notches, splices, rearranges, translocates, recombines, or otherwise alters genomic DNA. Optionally or additionally, the gene editing composition may include: (i) a gene editing complex that may cut, notch, splice, rearranges, translocates, recombines, or otherwise alters genomic DNA; or (ii) a treatable or alterable compound included in a gene editing complex that cuts, notches, splices, rearranges, translocates, recombines, or otherwise alters genomic DNA. In various embodiments, the gene editing composition includes one or more (a) gene editing proteins; (b) RNA molecules; and / or (c) ribonucleoside proteins (RNPs).

[0196] In some embodiments, the gene-editing composition includes a gene-editing protein, and the gene-editing protein is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a Cre recombinase, a Hin recombinase, or a Flp recombinase. In other embodiments, the gene-editing protein may be a fusion protein that binds a homing endonuclease to a modular DNA-binding domain of TALENs (megaTAL). For example, megaTAL may be delivered as a protein, or mRNA encoding a megaTAL protein may be delivered to the cell.

[0197] In various embodiments, the gene editing composition comprises an RNA molecule, and the RNA molecule comprises sgRNA, crRNA, and / or trace RNA.

[0198] In some embodiments, the gene-editing composition comprises an RNP, and the RNP comprises a Cas protein and sgRNA or crRNA and tracrRNA. Some aspects of the subject matter of this invention are particularly useful for controlling the duration and duration of presence of a particular gene-editing compound in cells.

[0199] In various embodiments of the subject matter of this invention, (a) the gene-editing composition can be detected in the cell population or its progeny after approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5-2, 0.5-6, 6-12, or 0.5-72 hours following contact of the cell population with the aqueous solution, or (b) less than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5-2, 0.5-6, 6-12, or 0.5-72 hours following contact of the cell population with the aqueous solution.

[0200] In some implementations, the cellular genome in the cell population or its progeny contains at least one site-specific recombination site of Cre recombinase, Hin recombinase, or Flp recombinase.

[0201] Some aspects of the present invention relate to cell populations comprising a gene-editing compound and the insertion of another gene-editing compound into the cell population. For example, a component of an RNP can be introduced into cells expressing or already containing another component of an RNP. For example, cells in the cell population or their progeny may contain sgRNA, crRNA, and / or tracrRNA. In some embodiments of the cell population, the cell population or its progeny expresses sgRNA, crRNA, and / or tracrRNA. Alternatively or additionally, cells in the cell population or their progeny represent the Cas protein.

[0202] Various embodiments of the subject matter of this invention include Cas proteins. In some embodiments, the Cas protein is the Cas9 protein or a mutant thereof. Exemplary Cas proteins (including non-limiting examples of Cas9 and Cas9 mutants) are described herein.

[0203] In various aspects, the concentration of Cas9 protein ranges from approximately 0.1 to approximately 25 micrograms. For example, the concentration of Cas9 protein can be approximately 1 microgram, approximately 5 micrograms, approximately 10 micrograms, approximately 15 micrograms, or approximately 20 micrograms. Alternatively, the concentration of Cas9 can range from approximately 10 ng / µL to approximately 300 ng / µL, for example, from approximately 10 ng / µL to approximately 200 ng / µL; or from approximately 10 ng / µL to approximately 100 ng / µL; or from approximately 10 ng / µL to approximately 50 ng / µL.

[0204] In some embodiments, the gene editing composition comprises (a) a first sgRNA molecule and a second sgRNA molecule, wherein the nucleic acid sequence of the first sgRNA molecule is different from the nucleic acid sequence of the second sgRNA molecule; (b) a first RNP comprising the first sgRNA and a second RNP comprising the second sgRNA, wherein the nucleic acid sequence of the first sgRNA molecule is different from the nucleic acid sequence of the second sgRNA molecule; (c) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule; (d) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule, and further comprises a tracrRNA molecule; or (e) a first RNP comprising the first crRNA and tracrRNA and a second RNP comprising the second crRNA and tracrRNA, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule.

[0205] In some respects, the ratio of Cas9 protein to guide RNA can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0206] In some implementations, increasing the number of times cells pass through the delivery process (or increasing the number of doses) can increase the percentage of edits; wherein, in some implementations, the number of doses may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

[0207] In various embodiments, the first and second sgRNA or the first and second crRNA molecules collectively comprise a nucleic acid sequence complementary to a side-chain target sequence of a gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence, wherein the gene, exon, intron, or genomic nucleic acid sequence is approximately 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 1-100 kilobases in length, or at least approximately 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 1-100 kilobases in length. In some embodiments, paired RNPs comprising the first and second sgRNA or the first and second crRNA molecules can be used to create polynucleotide molecules comprising a gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence.

[0208] In some embodiments, the target sequence of sgRNA or crRNA has a nucleotide length of about 12 to about 25, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 17-23, or 18-22. In some embodiments, the target sequence length is 20 nucleotides or about 20 nucleotides.

[0209] In various implementations, the first and second sgRNA or first and second crRNA molecules are complementary to an extrachromosomal side-face sequence, which is also contained within the expression vector.

[0210] Various aspects of the subject matter of this invention relate to the delivery of multiple components of a gene-editing complex, wherein the components are not combined together. In some embodiments, the gene-editing composition comprises at least one gene-editing protein and at least one nucleic acid, wherein the gene-editing protein and the nucleic acid are not bound to or combined with each other.

[0211] The subject matter of this invention enables high gene editing efficiency while maintaining high cell viability. In some embodiments, upon contact with an aqueous solution, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99% or more of the cells or their progeny are genetically modified. In various embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99% or more of the cell population or their progeny are viable after contact with the aqueous solution.

[0212] In some embodiments, the gene-editing composition induces single-strand or double-strand breaks in intracellular DNA. In some embodiments, the gene-editing composition further includes a repair template polynucleotide. In various embodiments, the repair template includes (a) a first side chain region comprising a nucleotide sequence complementary to a sequence of about 40 to about 90 base pairs on one side of the single-strand or double-strand break, and a second side chain region comprising a nucleotide sequence complementary to a sequence of about 40 to about 90 base pairs on the other side of the single-strand or double-strand break; or (b) a first side chain region comprising a nucleotide sequence complementary to a sequence of at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on one side of the single-strand or double-strand break, and a second side chain region comprising a nucleotide sequence complementary to a sequence of at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on the other side of the single-strand or double-strand break. Non-restrictive descriptions of gene editing (including repair templates) using the CRISPR-Cas system are presented in Ran et al. (2013) Nat Protoc. 2013 Nov; 8(11): 2281–2308, the entire contents of which are incorporated herein by reference. Implementation schemes involving repair templates are not limited to those involving the CRISPR-Cas system.

[0213] In various embodiments of the subject matter of this invention, the volume of the aqueous solution is delivered to the cell population in the form of a spray. In some embodiments, the volume per cell is 6.0 x 10⁻⁶. -7 Microliters to 7.4 x 10⁻⁶ per cell -4 Between microliters. In some embodiments, the spray contains colloidal or subparticles with a diameter of 10 nm to 100 µm. In various embodiments, the volume is in the range of 2.6 x 10⁻⁶. -9 Exposure surface area per microliter per square micrometer and 1.1 x 10 -6 Between microliters per square micrometer of exposed surface area.

[0214] In some embodiments, the size of the RNP is approximately 100 Å × 100 Å × 50 Å or 10 nm × 10 nm × 5 nm. In various embodiments, the size of the spray particles is adjusted to accommodate at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more RNPs per spray particle.

[0215] For example, a cell population can be brought into contact with a volume of aqueous solution by atomizing an aqueous solution with gas. In some embodiments, the cell population is brought into contact with the aqueous solution for 0.01-10 minutes (e.g., 0.1-10 minutes), and then a second volume of buffer or culture medium is added to submerge or suspend the cell population.

[0216] In various embodiments, the cell population includes at least one of primary or immortalized cells. For example, the cell population may include mesenchymal stem cells, lung cells, neurons, fibroblasts, human umbilical vein (HUVEC) cells and human embryonic kidney (HEK) cells, primary or immortalized hematopoietic stem cells (HSCs), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, and B cells. Non-limiting examples of T cells may include CD8+ or CD4+ T cells. In some aspects, a CD8+ subset of CD3+ T cells is used. CD8+ T cells can be purified from the PBMC population using positive isolation with anti-CD8 microspheres. In some aspects, primary NK cells are isolated from PBMCs, and GFP mRNA can be delivered via a platform delivery technology (i.e., 3% expression rate and 96% survival rate at 24 hours). In other aspects, NK cell lines, such as NK92, may be used.

[0217] Cell types also include previously modified cells, such as T cells, NK cells, and MSCs, to enhance their therapeutic effects. For example: T cells or NK cells expressing chimeric antigen receptors (CAR T cells and CAR NK cells, respectively); T cells expressing modified T cell receptors (TCRs); MSCs modified virally or non-virally to overexpress therapeutic proteins that complement their inherent properties (e.g., Epo delivered using a lentiviral vector or BMP-2 delivered using AAV-6) (described in Park et al, Methods, 2015 Aug; 84-16); MSCs pretreated with non-peptide drugs or magnetic nanoparticles to enhance efficacy and external stability, respectively, modulating targeting (Park et al, 2015); and using partially functionalized MSCs to enhance their homing to therapeutic sites through enzymatic modification (e.g., fucosyltransferase), chemical conjugation (e.g., SLeX on MSCs chemically modified with N-hydroxysuccinimide (NHS)), or non-covalent interactions (e.g., engineering the cell surface with palmitated proteins as hydrophobic anchors for subsequent antibody binding) (Park et al, 2015). For example, T cells modified to express chimeric antigen receptor (CAR T cells) (e.g., primary T cells or T cell lines) can be further treated with gene-edited proteins and / or complexes containing specific guide nucleic acids targeting the CAR coding sequence according to the present invention to edit the gene encoding CAR, thereby reducing or stopping CAR expression in the modified T cells.

[0218] Various aspects of the present invention relate to the delivery of gene-editing compounds and complexes to cells and tissues without expression vectors, such as the delivery of Cas-gRNA ribonucleoproteins for genome editing in primary human T cells, hematopoietic stem cells (HSCs), and mesenchymal stromal cells (MSCs). In some embodiments, mRNA encoding these proteins is delivered into the cells.

[0219] Various aspects of the CRISPR-Cas system are known in the art. Non-limiting aspects of this system include U.S. Patent No. 9,023,649, published May 5, 2015; U.S. Patent No. 9,074,199, published July 7, 2015; U.S. Patent No. 8,697,359, published April 15, 2014; U.S. Patent No. 8,932,814, published January 13, 2015; PCT International Patent Application No. WO 2015 / 071474, published August 27, 2005; Cho et al., (2013) Nature Biotechnology Vol 31 No 3 pp 230-232 (including supplemental information); and Jinek et al., (2012) Science Vol 337 No 6096 pp 816-821, the entire contents of which are incorporated herein by reference.

[0220] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs or modifications thereof. These enzymes are known; for example, the amino acid sequence of the *Streptococcus pyogenes* Cas9 protein can be found under accession number Q99ZW2 in the SwissProt database and accession number Q99ZW2.1 in the NCBI database. UniProt database accession numbers A0A0G4DEU5 and CDJ55032 provide another example of the amino acid sequence of the Cas9 protein. Another non-limiting example is the *Streptococcus thermophilus* Cas9 protein, whose amino acid sequence can be found in the UniProt database under accession number Q03JI6.1. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments, the CRISPR enzyme is Cas9, and may be Cas9 derived from *Streptococcus pyogenes* or *Streptococcus pneumoniae*. In various embodiments, the CRISPR enzyme directs the cleavage of one or both strands at a location on the target sequence (e.g., within the target sequence and / or within complement of the target sequence). In some embodiments, the CRISPR enzyme cleaves one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs. In some embodiments, the vector encodes a CRISPR enzyme mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands containing the target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine in the RuvCI catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a two-strand cleaving nuclease to a nickase (single-strand cleaving). Other examples of mutations that make Cas9 a nickase include, but are not limited to, H840A, N854A, and N863A. In various aspects of the invention, nickases can be used for genome editing via homologous recombination.

[0221] In some implementations, Cas9-nickase can be used in combination with guide sequences, for example, two guide sequences targeting the sense and antisense strands of the DNA target, respectively. This combination allows both strands to be cleaved and used to induce NHEJ.

[0222] As a further embodiment, two or more catalytic domains (RuvC I, RuvC II, and RuvCIII) of Cas9 can be mutated to produce a mutant Cas9 that substantially lacks all DNA cleavage activity. The D10A mutation can be combined with one or more of the H840A, N854A, or N863A mutations to produce a Cas9 enzyme that substantially lacks all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered substantially lacking in DNA cleavage activity when the DNA cleavage activity of the mutant enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or lower relative to its non-mutated form. Other mutations may be useful; mutations in the corresponding amino acids may produce similar effects if Cas9 or other CRISPR enzymes originate from species other than Streptococcus pyogenes.

[0223] In some embodiments, the delivered protein (e.g., a Cas protein or a variant thereof) may include a subcellular localization signal. For example, the Cas protein within an RNP may contain a subcellular localization signal. Depending on the context, a fusion protein containing, for example, Cas9 and a nuclear localization signal may be referred to herein as "Cas9" without specifying that it contains a nuclear localization signal. In some embodiments, the payload (e.g., an RNP) includes a fusion protein that contains a localization signal. For example, the fusion protein may contain a nuclear localization signal, a nucleolar localization signal, or a mitochondrial targeting signal. These signals are known in the art, and non-limiting embodiments are described in the following references: Kalderon et al., (1984) Cell 39 (3 Pt 2): 499–509; Makkerh et al., (1996) Curr Biol. 6 (8):1025–7; Dingwall et al., (1991) Trends in Biochemical Sciences 16 (12): 478–81; Scott et al., (2011) BMCBioinformatics 12:317 (7 pages); Omura T (1998) J Biochem. 123(6):1010-6; Rapaport D (2003) EMBO Rep. 4(10):948-52; and Brocard & Hartig (2006) Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1763(12):1565–1573, each of these documents is incorporated herein by reference in its entirety. In various embodiments, the Cas protein may contain more than one localization signal, such as 2, 3, 4, 5 or more nuclear localization signals. In some embodiments, the localization signal is located at the N-terminus of the Cas protein, while in other embodiments, the localization signal is located at the C-terminus of the Cas protein.

[0224] In some implementations, the enzyme-coding sequence encoding a CRISPR enzyme is codon-optimized for expression in specific cells (e.g., eukaryotic cells). These eukaryotic cells can be cells of a specific organism or cells derived from a specific organism, such as mammalian cells, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to a method of modifying nucleic acid sequences by replacing at least one codon in the natural sequence (e.g., approximately or more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) with a codon that is more frequently used or most frequently used in the host cell's gene, thereby improving expression in the host cell of interest while maintaining the natural amino acid sequence. Different species exhibit specific codon preferences for specific amino acids. Codon preferences (differences in codon use between organisms) are generally associated with the translation efficiency of messenger RNA (mRNA), which in turn depends on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The preference of tRNAs chosen in a cell usually reflects the most commonly used codons in peptide synthesis.

[0225] Therefore, based on codon optimization, genes can be tailored to achieve optimal gene expression in a given organism. Codon usage tables are readily available, for example, in "codon usage databases," and these tables can be adapted in many ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNAsequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms optimize specific codon sequences for expression in specific host cells; for example, GeneForge (Aptagen; Jacobus, Pa.) can be used. In some implementations, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) in the sequence encoding the CRISPR enzyme correspond to the most frequently used codons for a specific amino acid.

[0226] Generally, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target polynucleotide sequence to hybridize with it and directly and sequence-specifically bind the CRISPR complex to the target sequence. In some embodiments, when optimal alignment is achieved using a suitable alignment algorithm, the complementarity between the guide sequence and its corresponding target sequence is approximately or greater than approximately 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or greater. In some embodiments, the complementarity is 100%. Optimal alignment can be determined by using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., the Burrows-Wheeler aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, CA), soap (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides long. In some embodiments, the guide sequence is less than approximately 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides long. The ability of the guide sequence to specifically bind the CRISPR complex to the target sequence can be assessed by any suitable analysis. For example, sufficient to form a CR Components of the CRISPR system in the ISPR complex (including the guide sequence to be tested) can be provided to host cells with the corresponding target sequence, for example, by transfection with a vector encoding the CRISPR sequence, followed by evaluation of preferential cleavage within the target sequence, for example, by Surveyor analysis as described herein. Similarly, cleavage of the target polynucleotide sequence can be evaluated in vitro by providing the target sequence, components of the CRISPR complex (including the guide sequence to be tested and a control guide sequence different from the test guide sequence), and comparing the binding or cleavage rates at the target sequence between the test and control guide sequence reactions.

[0227] CRISPR Cas technology has facilitated the development of genome engineering across multiple cell types. Recent studies have shown that providing Cas9-gRNA editing tools in the form of ribonucleoproteins (RNPs) offers several advantages over providing plasmids encoding Cas9 and gRNAs. These advantages include faster and more efficient editing, less deviation from the target, and less toxicity. RNPs have been delivered via liposome transfection and electroporation, but these delivery methods still have limitations, particularly for certain clinically relevant cell types, including toxicity and inefficiency. Therefore, there is a need for a vector-free (e.g., virus-free) method for delivering biologically relevant payloads (e.g., RNPs) across the plasma membrane and into the cell. "Cargo" or "payload" is a term used to describe a compound or composition that crosses the cell membrane and enters the cell via an aqueous solution.

[0228] The subject matter of this invention relates to a delivery technology that accelerates the delivery of large-scale payloads into cells with low toxicity. Genome editing can be achieved by delivering RNPs to cells using certain aspects of the present subject matter. The levels then decrease until Cas9 is no longer detectable. The delivery technology itself does not adversely affect the activity or function of Jurkat and primary T cells. The subject matter of this invention relates to gene editing of clinically relevant cell types via Cas9-RNPs with minimal toxicity.

[0229] Compared to expression vector-mediated delivery, the transient and direct delivery of CRISPR / Cas components such as Cas and / or gRNA offers advantages. For example, a specific amount of Cas, gRNA, or RNP can be added at more precise timing and within a limited timeframe compared to using an expression vector. Components expressed from a vector may be produced in varying quantities and at different times, making it difficult to achieve consistent gene editing without deviating from the target editing. Furthermore, pre-formed complexes (RNPs) of Cas and gRNAs cannot be delivered along with the expression vector.

[0230] In one aspect, this subject matter describes cells attached to a solid support (e.g., strips, polymers, beads, or nanoparticles). The scaffold or support can be a porous or non-porous solid scaffold. Well-known supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. For the purposes of this subject matter, the support can be partially soluble or insoluble. The scaffold material can have virtually any possible structural configuration. Thus, the scaffold construction can be spherical, for example, in the form of magnetic beads, or conical, on the outer surface of a cylindrical tube, such as the inner surface of a cylindrical tube. Alternatively, the surface can be flat, such as a thin plate or test strip. Preferred scaffolds include polystyrene beads.

[0231] In other respects, the solid carrier comprises a polymer, with cells chemically bound, immobilized, dispersed, or bonded to the polymer. The polymer carrier can be a polymer network and can be in bead-like form (e.g., via suspension polymerization). Cells on such a scaffold can be sprayed together with an effective load containing an aqueous solution of the present invention to deliver the desired compound to the cytoplasm of the scaffold. Exemplary scaffolds include supports and other implantable medical devices or structures.

[0232] Other implementation plans

[0233] In the foregoing specification and claims, phrases such as “at least one” or “one or more” may appear after a list of conjunctions for elements or features. The term “and / or” may also appear in a list of two or more elements or features. Unless implied or explicitly contradicted by the context in which the phrase is used, the phrase means any element or feature listed alone, or any referenced element or feature combined with any other recited element or feature. For example, the phrases “at least one of A and B,” “one or more of A and B,” and “A and / or B” each mean “A alone, B alone, or A and B together.” A similar interpretation may also mean that the list includes three or more items. For example, the phrases “at least one of A, B, and C,” “one or more of A, B, and C,” and “A, B, and / or C” each mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” Furthermore, the use of the term “based on” in the claims means “at least partially based on,” even if such an unreferenced feature or element is permitted.

[0234] The subject matter described herein can be implemented in systems, apparatus, methods, and / or articles according to desired configurations. The embodiments set forth in the foregoing specification do not represent all embodiments consistent with the subject matter described herein. Rather, they are merely examples of aspects consistent with the subject matter described herein. Although some variations have been described in detail above, other modifications or additions may be made. In particular, other features and / or variations may be provided in addition to those features and / or variations set forth herein. For example, the embodiments described above may refer to combinations or sub-combinations of the various features disclosed above and / or combinations and sub-combinations of several further features disclosed above. Furthermore, the logical flows depicted in the drawings and / or described herein do not necessarily require the specific or sequential order shown to achieve the desired results. Other embodiments may be included within the scope of the following claims.

Claims

1. A method for spraying a transfer solution containing a payload and a permeabilizer onto cells deposited on a filter, comprising: A mixture of cells and a first culture medium is introduced into the chamber of the cell engineering platform; The first culture medium is discharged from the chamber through a filter, causing the cells to deposit on the filter. A delivery solution containing payload and penetrant is sprayed onto the cells deposited on the filter; The cell engineering platform includes: Chamber; A cover disposed at the first end of the chamber; A base disposed at the second end of the chamber; and A filter holder is installed inside the chamber.

2. The method of claim 1, further comprising applying a stop solution in the chamber.

3. The method of claim 2, further comprising filling the chamber with a second culture medium to resuspend the cells from the filter.

4. The method of claim 3, wherein the discharged first culture medium is reused as a second culture medium.

5. The method of claim 3, further comprising agitating the chamber.

6. The method of claim 3, further comprising extracting the resuspended cells from the chamber.

7. The method according to claim 3, wherein, The chamber is filled automatically by a pump and a controller.

8. The method of claim 1, further comprising culturing cells within the chamber.

9. The method according to claim 1, wherein, The first culture medium is discharged from the chamber by applying positive pressure to the chamber.

10. The method according to claim 1, wherein, The first culture medium is discharged from the chamber by gravity.

11. The method according to claim 2, wherein, Apply a stop solution to wash the cells.

12. The method according to claim 3, wherein, The process of filling the chamber with a second culture medium is performed as at least one of a cell washing process, a cell concentration change process, and / or a cell culture medium change process.

13. A system for delivering cargo compounds or compositions to mammalian cells, comprising: A housing configured to receive a filter plate, the filter plate including pores including a first culture medium comprising cells; A differential pressure applicator is configured to apply different pressures to the pores to drain the first culture medium from the chamber through the filter of the filter plate, leaving the cells deposited on the filter plate including the pores; A transfer solution applicator configured to deliver an atomized transfer solution into the orifice, the transfer solution applicator including a nozzle; A stop solution applicator configured to deliver a stop solution to the orifice; as well as A culture medium applicator configured to deliver culture medium into the well.

14. The system according to claim 13, wherein, The outer casing includes: chamber; A cover disposed at the first end of the chamber; and A base is disposed at the second end of the chamber; The filter plate is disposed within the chamber.

15. The system according to claim 14, wherein, The differential pressure applicator is connected to the cover of the housing and includes a shower head having multiple holes at its end.

16. The system according to claim 14, wherein, The stop solution applicator delivers the stop solution into the orifice through a diaphragm disposed on the cover of the housing.

17. The system according to claim 14, wherein, The culture medium applicator delivers the culture medium into the orifice through a diaphragm disposed in the cover of the housing or through a port of the chamber.

18. The system according to claim 13, wherein, The housing is configured to tilt the filter plate.

19. The system according to claim 13, wherein, The solution delivery applicator includes a robotic arm and a nozzle, the robotic arm being magnetically coupled to the nozzle.

20. The system of claim 19, further comprising a flexible barrier surrounding the filter plate and the nozzle, the flexible barrier separating the robotic arm and the nozzle.

21. The system according to claim 19, wherein, The robotic arm is configured to deliver the nozzle to multiple locations on the filter plate.

22. The system according to claim 13, wherein, The stop solution applicator and the culture medium applicator are integrated as ports within the housing, and the housing forms a container.

23. The system according to claim 22, wherein, The container includes a waste culture medium outlet for collecting waste culture medium.

24. The system of claim 13, further comprising a soft elastomer barrier that surrounds the filter plate and the solution transfer applicator within a housing, the soft elastomer barrier and the housing forming a bioreactor.

25. The system according to claim 13, wherein, The housing includes a filter plate base configured to tilt, rotate, and / or vibrate the filter plate.

26. The system according to claim 13, wherein, The solution transfer applicator includes a robotic arm and a nozzle, the nozzle being a disposable device.

27. The system according to claim 13, wherein, The filter plate is sized to accommodate 10 7 More than one T cell.

28. The system according to claim 13, wherein, The system is configured to operate automatically. Provide cells to the filter plate in the culture medium; Remove the culture medium to form a cell monolayer on top of the filter plate; Atomized delivery solution is applied to the cell monolayer; Culture the cells; Apply a stop solution to the cultured monolayer cells; as well as Provide fresh culture medium to the cell monolayer.

29. The system according to claim 28, wherein, The system is configured to automatically tilt, vibrate, and / or rotate the filter plate to resuspend the cells in a fresh culture medium.

30. The system according to claim 28, wherein, The system is configured to repeatedly apply the atomized delivery solution, incubate, and apply the stop solution.

31. The system according to claim 13, wherein, The solution delivery applicator includes an atomizer.

32. The system according to claim 31, wherein, The solution delivery applicator further includes a mass flow controller or a volume flow controller to regulate the airflow and control the atomizer.

33. The system according to claim 13, wherein, The transfer solution applicator is configured to deliver 10-300 microliters of transfer solution per drive.

34. The system of claim 13, further comprising a temperature control system configured to control the temperature of the transfer solution and / or the filter plate including the pores.

35. The system according to claim 13, wherein, The delivery solution includes an aqueous solution, which includes a payload and an alcohol with a concentration greater than 2%.

36. The system according to claim 35, wherein, The alcohols include ethanol.

37. The system according to claim 35, wherein, The aqueous solution contains more than 5% ethanol.

38. The system according to claim 35, wherein, The aqueous solution contains 5-30% ethanol.

39. The system according to claim 35, wherein, The aqueous solution includes 12% or 25% ethanol.

40. The system according to claim 35, wherein, The aqueous solution contains 12.5-500 mM KCl.

41. The system according to claim 35, wherein, The aqueous solution contains 106 mM KCl.

42. The system of claim 13, further comprising the filter plate, wherein the pores are configured to contain a large number of non-adhesive cells.

43. The system according to claim 42, wherein, The non-adhesive cells include peripheral blood mononuclear cells.

44. The system according to claim 42, wherein, The non-adhesive cells include immune cells.

45. The system according to claim 42, wherein, The non-adhesive cells include T lymphocytes.

46. ​​The system according to claim 35, wherein, The payload includes messenger ribonucleic acid.

47. The system according to claim 46, wherein, The messenger ribonucleic acid-encoded gene editing composition.

48. The system according to claim 47, wherein, The gene-editing composition reduces PD-1 expression.

49. The system according to claim 46, wherein, The messenger RNA encodes a chimeric antigen receptor.

50. The system of claim 13, for delivering cargo compounds or compositions to mammalian cells.

51. The system according to claim 42, wherein, The non-adhesive cell population is a monolayer.

52. A system for delivering cargo compounds or compositions to mammalian cells, comprising: chamber; A cover located at the first end of the chamber; A base located at the second end of the chamber; A filter holder installed inside the chamber; as well as The nozzles are arranged to apply atomized delivery solution to cells deposited on the filter holder.

53. The system according to claim 52, wherein, The filter holder includes a plurality of holes arranged in a predetermined pattern.

54. The system according to claim 53, wherein, The filter scaffold includes multiple targets, wherein multiple pores are arranged to allow cell deposition on the filter in areas corresponding to the multiple pores.

55. The system of claim 52, further comprising a gasket disposed between the filter holder and the base.

56. The system of claim 54, further comprising a filter holder insert located within the filter holder to accommodate a filter between the filter holder and the filter holder insert.

57. The system according to claim 56, wherein, The filter holder insert includes a concave top surface.

58. The system according to claim 56, wherein, The filter holder insert includes multiple openings corresponding to the plurality of targets.

59. The system according to claim 58, wherein, The filter holder includes three targets.

60. The system according to claim 58, wherein, The filter holder includes seven targets.

61. The system according to claim 58, wherein, The filter holder includes nineteen targets.

62. The system according to claim 56, wherein, The multiple targets are arranged in a square pattern, a rectangular pattern, a triangular pattern, or a linear pattern.

63. The system according to claim 52, wherein, The system further includes a controller configured to operate at least one of a pump, a valve, a heating element, a cooling element, and a stirring device.

64. The system according to claim 63, wherein, The pump is a peristaltic pump or a positive displacement pump.

65. The system according to claim 55, wherein, The cover further includes a pressure port and an orifice.

66. The system according to claim 52, wherein, The base includes a port for receiving or discharging culture medium.

67. The system according to claim 65, wherein, The cover also includes a diaphragm.

68. The system according to claim 67, wherein, The pressure port includes a shower head, which has multiple holes at its end.

69. The system according to claim 65, wherein, A 0.2-micron filter was connected to the pressure port.

70. The system according to claim 69, wherein, The system also includes a pinch valve configured to open and close a pressure port to the atmosphere via a 0.2-micron filter.

71. The system according to claim 52, wherein, The system's chambers include ports for extracting and processing cells.

72. The system according to claim 52, wherein, The system is sized to handle sizes greater than 1×10 9 T cells.

73. A system for delivering cargo compounds or compositions to mammalian cells, comprising: A housing configured to receive a filter plate, the filter plate including pores that contain a first culture medium comprising cells. in The outer casing includes: a chamber, a cover disposed at a first end of the chamber, and a base disposed at a second end of the chamber, wherein the filter plate is disposed within the chamber; A differential pressure applicator is configured to apply different pressures to the pores to drain the first culture medium from the chamber through the filter of the filter plate, leaving the cells deposited on the filter plate including the pores; A transfer solution applicator configured to deliver an atomized transfer solution into the orifice, the transfer solution applicator including a nozzle; A stop solution applicator configured to deliver a stop solution to the orifice; and A culture medium applicator configured to deliver culture medium into the well.

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