Efficient delivery of cargo across cell membranes using a continuous flow fluidics system

Abstract

Methods, systems, processes and devices for transcellular membrane delivery are provided herein. In one aspect, a device includes a substrate including a mixing channel, a processing chamber and a dilution channel to perform a process of transporting an effective load across the entire cell membrane. On the other hand, a system includes a reservoir for a cell suspension, a transfer solution reservoir and a stop solution connected to a pump. The system also includes an agitator, a heater, a temperature controller and a controller for operating the system. On the other hand, cells in the suspension are mixed with a transfer solution in a microfluidic mixing chip. The transfer solution contains a permeabilizing agent that causes cell permeabilization, thereby allowing the effective load to be transported from the transfer solution through the cell membrane into the cell.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 533,825, filed on July 18, 2017, the entire contents of which are hereby incorporated by reference as a whole. Technical Field

[0003] The subject matter described here relates to the efficient delivery of cargo across cell membranes. Background Art

[0004] Despite some progress, delivering certain particles and / or molecules into cells remains a challenge. For example, some factors, such as the size or charge of the molecule that needs to be delivered into the cell can limit and / or prevent the delivery of molecules into the cell. Delivery across the cell membrane can be complicated by the molecules and / or membranes of the cell. The plasma membrane or cell membrane is a semi-permeable biological membrane that acts as a selective barrier. The cell membrane regulates the chemical composition inside the cell. As a selective barrier to the cell, the cell membrane only allows certain molecules to enter the cell through passive transmembrane displacement across the cell membrane, such as passive diffusion. Hydrophobic small molecules (such as O2, CO2, and N2) and uncharged polar small 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 + , Na + , K + and CI - ) cannot diffuse passively across the cell membrane. Summary of the invention

[0005] The current subject matter provides a cell engineering method and platform that delivers a compound or mixture of compounds (e.g., payload) across a cell membrane into a cell by contacting the cell with a delivery solution containing an effective load and an agent that can reversely permeate or dissolve the cell membrane. In particular, by continuously supplying cells in a suspension and a delivery solution to a system in which the suspension and solution are mixed, the cell is permeabilized and the effective load is delivered across the permeabilized cell membrane. Methods, systems, processes, and devices for transcellular membrane delivery are provided herein.

[0006] In the prior art, batch processing for cell-to-cell delivery can only process a limited number of cells (from 1 to 10 cells) per batch. 8 or 10 9), but may be difficult to scale up to handle higher cell loads. In contrast, some embodiments of the current subject matter relate to delivery technologies that can facilitate continuous flow delivery of a wide range of payloads into cells. Compared to batch processing, the continuous flow platform can achieve a steady-state process in which the concentrations of cells, payload, and osmotic agent can be maintained at a steady state throughout the duration of the process. In addition, the system can be implemented as a closed system and achieve sterile transfection, which makes the system and method suitable for therapeutic applications. Therefore, in some embodiments, the continuous flow platform can provide better consistency throughout the operation, produce high throughput to cover the range of therapeutic capabilities, and have better scale-up potential.

[0007] In one aspect, a device for performing trans-cellular membrane transfer of effective load is provided herein. The device may include a substrate, the substrate including: a first fluid channel, the first fluid channel including a first inlet configured to receive a cell group in a suspension; a second fluid channel, the second fluid channel including a second inlet configured to receive a transfer effective load in a transfer solution; a mixing channel, the mixing channel connecting the first and second fluid channels. The mixing channel can make the first and second fluid channels fluidly connected, and the suspension and the transfer solution can be mixed. The substrate of the device may further include a treatment chamber connected to the mixing channel, the treatment chamber being configured to retain a mixture of the suspension and the transfer solution for a period of time so that the cell group is exposed to the transfer solution. The substrate also includes a third fluid channel, the third fluid channel including a third inlet to receive a stop solution, and a dilution channel may be provided to connect to the treatment chamber and the third fluid channel. The dilution channel can combine the fluid of the treatment chamber with the stop solution supplied from the third fluid channel and output it. The substrate of the device may further include a separator connected to the dilution channel for separating the transformed cells and the waste stream. In addition, the device may include a fourth fluid channel, the fourth fluid channel including a fourth inlet, the fourth inlet being configured to receive one or more additive flows.

[0008] One or more of the following features may be included in any feasible combination. The processing chamber may include one or more diagnostic ports for measuring temperature and pressure in the processing chamber and / or extracting samples. The substrate may include opaque materials and light-transmitting materials capable of forming an optical window. The optical window is arranged to image cells within at least one of the first channel, the mixing channel, the processing chamber and / or the dilution channel.

[0009] In some embodiments, the size and shape of the processing chamber can be set to control the exposure time of the cell population to the delivery solution. For example, the length of the processing chamber can be selected to control the residence time of the mixture of cells and delivery solution before being diluted by the stop solution.

[0010] In an embodiment, the mixing channel can have a geometry including a herringbone pattern, a cross-finger pattern and / or a double T-shaped pattern for mixing. The processing chamber can have a geometry including a straight channel, a serpentine channel, a circular channel and / or an air-filled chamber. The channel width of the fluid channel of the device can be in the range of 0.01 micron to 100 microns, for example, 0.01 micron, 0.05 micron, 0.1 micron, 0.5 micron, 1 micron, 5 microns, 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns or 100 microns. The channel width of the fluid channel of the device can be in the range of 0.01 microns to 100 microns, for example, 0.01 microns, 0.05 microns, 0.1 microns, 0.5 microns, 1 micron, 5 microns, 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns or 100 microns. The channel width can also be in the range of 100 microns to 1 mm, for example, 100 microns, 200 microns, 300 microns, 400 microns, 500 microns, 600 microns, 700 microns, 800 microns, 900 microns or 1 mm, or, alternatively, the range can also be greater than or equal to 1 mm, for example, 1 mm, 2 mm, 3 mm, 4 mm or 5 mm. The substrate of the device may be made of materials including silicon, silicon oxide, silicon carbide, silicon nitride, silicate glass, borosilicate glass, quartz, sapphire, polydimethylsiloxane (PDMS), polyethylene, polypropylene, polyurethane, polytetrafluoroethylene (PTFE), polyether ether ketone (PEEK) and / or stainless steel, such as a steel alloy having a chromium content of at least 10.5%. The stainless steel may include chromium-manganese-nickel alloy, chromium-nickel alloy, etc. The stainless steel may include, for example, 304 grade stainless steel or 316 grade stainless steel. The first inlet, the second inlet, the third inlet and the downstream end of the dilution channel may be configured to be in closed fluid communication.

[0011] On the other hand, a system for performing effective loading across cell membranes is provided herein. The system may include the above-mentioned device, and also include a cell suspension reservoir connected to the first inlet of the first fluid channel, a transfer solution reservoir connected to the second inlet of the second fluid channel, and a stop solution reservoir connected to the third fluid channel. At the inlet of the third fluid channel, at least one pump is configured to supply the fluid in the reservoir to the corresponding inlet, and the controller is connected to the pump and configured to adjust the flow rate of the fluid. The system may further include an agitator configured to vibrate at least one of a mixing channel, a treatment chamber or a dilution channel. The agitator may include a low-frequency pressure pulse generator, an ultrasonic vibrator and / or a mechanical vibrator. The system may further include a heater and a temperature controller, which are configured to maintain or adjust the temperature of at least one of a mixing channel, a treatment chamber or a dilution channel. The heater and the temperature controller may include multiple heating zones.

[0012] The delivery solution may include an isotonic aqueous solution including an effective load and an alcohol at a concentration of 2% to 5% or greater than or equal to 5% by volume. The alcohol includes ethanol. The aqueous solution may contain greater than 10% ethanol. The aqueous solution may contain 20-30% ethanol, such as 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%. The aqueous solution may contain 27% ethanol. The aqueous solution may contain 12.5-500mM KCl (potassium chloride), such as 12.5mM, 25mM, 50mM, 75mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM or 500mM KCl. The aqueous solution may contain 106mM KCl.

[0013] The system may further include a filter plate, the wells may be configured to contain a group of non-adherent cells. Non-adherent cells may include peripheral blood mononuclear cells. Non-adherent cells may include immune cells. Non-adherent cells may include T lymphocytes. Effective loading may include messenger ribonucleic acid (mRNA). The mRNA may encode a gene editing component. The gene editing composition may reduce the expression of PD-1 (programmed cell death protein 1). mRNA may encode a chimeric antigen receptor. The current subject matter may be used to deliver cargo compounds or compositions to mammalian cells.

[0014] In yet another aspect, a method for transcellular membrane delivery is provided, the method comprising mixing a cell population in suspension with a delivery payload delivered in a delivery solution. The delivery solution permeabilizes the cell population, and the delivery payload is delivered across the membrane of the cell population.

[0015] The method can be varied in a variety of ways. For example, the cell population can include T cells, and the delivery payload can include nucleic acids, such as mRNA, proteins, and mixtures or complexes of proteins and nucleic acids. In another embodiment, the delivery solution can include alcohol. The suspension and the delivery solution can undergo a first mixing cascade and a second mixing cascade. In one embodiment, mixing the cell population and the delivery payload can include pumping the cell population and the delivery payload into a microfluidic mixing chip. The diameter of the narrowest channel of the microfluidic mixing chip can be larger than the diameter of the cells in the cell suspension, for example, at least 5 times the diameter of the cell population. In another embodiment, the method can include introducing a stop solution into the cell population and delivering the payload after mixing the cell population and the delivery payload, so that the stop solution stops the cell population from experiencing permeability or reduces the permeability. In terms of permeability. Introducing the stop solution can also include providing a user-determined delay process before introducing the cell population and the delivery payload in the stop solution. In another embodiment, the population of cells and the delivered payload can experience laminar flow. The method can also include mixing at least one additive with the cell population in the suspension and the delivered payload in the delivery solution.

[0016] In another aspect, a method for delivering a payload across a cell membrane is provided, the method comprising introducing a cell population and a delivery solution into a microfluidic mixing chip to mix the cell population and the delivery solution. The interaction between the cell population and the delivery solution results in the delivery payload being transferred from the delivery solution across the membrane of the cell population to the cell population.

[0017] The method can be varied in many ways. For example, the interaction between the cell colony and the transfer solution can cause permeabilization of the cell colony. The transfer solution can include alcohol, the cell colony can include T cells, and the delivery payload can include mRNA. In another example, the microfluidic mixing chip can cause the cell colony, and the transfer solution undergoes a first mixing cascade and a second mixing cascade. In other examples, the method can include introducing a stop solution into the cell colony and introducing the transfer solution after the payload is transferred across the membrane of the cell colony. The stop solution reduces or stops cell membrane permeability, and thus any other transfer of the payload across the membrane of the cell colony can be reduced or prevented. The cell colony and the transfer solution may also undergo laminar flow. In other embodiments, the channels in the microfluidic mixing chip can be configured to optimize the transfer of the payload from the transfer solution to the cell colony. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The subject matter of the present invention may be more fully understood by referring to the description of the specific embodiments and the accompanying drawings, in which:

[0019] Figure 1 is a close-up view of one embodiment of a microfluidic mixing chip;

[0020] Figure 2 yes Figure 1 Schematic diagram of the microfluidic mixing chip;

[0021] Figure 3 It is shown in Figure 1 Diagram of cells in suspension and delivery solution mixed in a microfluidic mixing chip shown;

[0022] Figure 4 Such as Figure 1 A top view of a mixing stage in a microfluidic mixing chip is shown;

[0023] Figure 5 yes Figure 1 Schematic diagram of the microfluidic mixing chip;

[0024] Figure 6 is used Figure 1 Figure 1 shows the process of the microfluidic mixing chip.

[0025] Figure 7 is a schematic diagram of a microfluidic device according to an exemplary embodiment;

[0026] Figure 8 is a schematic diagram of a system for delivering a payload across a cell membrane, the system comprising a microfluidic device according to an exemplary embodiment;

[0027] Fig. 9 Exemplary geometries of mixing channels of microfluidic devices are shown: (a) serpentine channel, (b) herringbone channel, (c) interdigitated split-recombination configuration, (d) herringbone split-recombination configuration, and (e) double-T configuration;

[0028] Fig.10 Exemplary geometries of processing chambers of a microfluidic device are shown: (a) a straight channel shape, (b) a serpentine channel shape, and (c) a circular channel shape;

[0029] Fig.11 Exemplary embodiments of efficient cargo delivery across cell membranes using spray methods including syringes are shown;

[0030] Fig.12 An exemplary embodiment of efficient cargo delivery across a cell membrane using a spray method involving a pump is shown;

[0031] Fig.13 The experimental results of using the spray method for efficient delivery of cargo across cell membranes are shown;

[0032] Fig.14Exemplary embodiments of efficient cargo delivery across cell membranes using a trench-type batch processing chamber are described (a) a simulation of cell capture based on cell deposition to the bottom of a trench structure, and (b) the working principle of a trench-type processing chamber;

[0033] Fig.15 is a bright field image showing cells captured in a grooved processing chamber during an experiment of an exemplary embodiment of using the grooved processing chamber for efficient cargo delivery across a cell membrane;

[0034] Fig.16 and Fig.17 shows experimental results obtained in an exemplary embodiment of efficient cargo delivery across cell membranes using a grooved processing chamber, showing the effect of processing time;

[0035] Fig.18 and Fig.19 shows experimental results obtained in an exemplary embodiment of efficient cargo delivery across cell membranes using a grooved processing chamber, showing the effect of ethanol concentration; and

[0036] Fig. 20 and 21 Experimental results obtained in an exemplary embodiment of payload delivery across a cell membrane using a trench-type processing chamber are shown, illustrating the effect of DAPI (4',6-diamidino-2-phenylindole) concentration. Specific implementation plan

[0037] Certain exemplary embodiments will now be described to provide an overall understanding of the principles of structure, function, manufacture and use of the apparatus and methods disclosed herein. One or more embodiments of these embodiments are shown in the accompanying drawings. It will be understood by those skilled in the art that the apparatus and methods specifically described herein and shown in the accompanying drawings are non-limiting exemplary embodiments, and the scope of the present disclosure is limited only by the claims. The features shown or described in conjunction with an exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present disclosure.

[0038] In addition, in the present disclosure, components with the same name shown in the embodiments generally have similar features, and therefore, in a particular embodiment, it is not necessary to fully elaborate on every feature of each component with the same name. In addition, to the extent that linear or circular dimensions are used in the description of the disclosed systems, devices, and methods, such dimensions are not intended to limit the types of shapes that can be used in conjunction with such systems, devices, and methods. Those skilled in the art will recognize that dimensions equivalent to such linear and circular dimensions can be readily determined for any geometric shape. The size and shape of the systems and devices and their components can depend on at least the anatomy of the object in which the systems and devices will be used, the size and shape of the components in which the systems and devices will be used, and the methods and processes in which the systems and devices will be used.

[0039] For a wide range of applications, it is necessary to deliver molecules into living cells. In general, the types of molecules involved can be classified according to the mass of the molecule: (i) small chemical molecules generally have an average molecular weight of: <1,000 Da; (ii) peptides generally have an average molecular weight of: -5,000 Da; (iii) small RNA molecules generally have an average molecular weight of: -15,000 Da; (iv) antibodies generally have an average molecular weight of: -150,000 Da; and (v) nucleic acids, such as DNA, generally have an average molecular weight of: -5,000,000 Da. Further details can be found in International Application No. PCT / US2015 / 057247 filed on October 23, 2015, and expressly incorporated herein.

[0040] There are a variety of methods that can be used to deliver molecules across the plasma membrane and into cells, each depending on the size and chemical nature of the molecule to be delivered. Organic solvents such as dimethyl sulfoxide (DMSO) have been used to deliver small chemical molecules. Although the molecular basis of DMSO's effects on the plasma membrane remains unclear, it is known that DMSO exhibits three different modes of action, each within a different concentration range. At low concentrations, DMSO causes thinning of the plasma membrane and increases the fluidity of the hydrophobic core of the plasma membrane. At higher concentrations, DMSO induces transient water pores in the plasma membrane. At still higher concentrations, single lipid molecules are irreversibly desorbed from the plasma membrane, and the bilayer structure of the plasma membrane then undergoes a detrimental breakdown.

[0041] The introduction of larger biomolecules, such as oligopeptides, polypeptides or proteins, and nucleic acids such as plasmid DNA, oligonucleotides and siRNA is called "transfection". The use of traditional delivery compositions such as DMSO is ineffective for the delivery of these larger molecules. siRNA molecules are usually delivered by liposome-mediated transfection (lipofection). Plasmid DNA is usually delivered using biological (viral), chemical (lipid or chemical polymer-based) or physical (electroporation, magnetofection, injection) methods. However, many cell types, such as primary cells and stem cells, remain "difficult to transfect".

[0042] Many methods are available to chemically "permeabilize" cells and tissues. Many of these methods do not attempt "reversible permeabilization" or focus on delivery into living cells. Instead, these methods aim for "irreversible permeabilization" to deliver a "label" that will attach to a molecule or structure within the cell or tissue for the purpose of visualization or quantification (e.g., immunofluorescence). In these cases, cells and tissues are not viable following permeabilization. Chemicals commonly used in these methods include: alcohols (which dissolve lipids in the plasma membrane), detergents (which form pores in the plasma membrane), and enzymes (which digest proteins and form pores in the plasma membrane).

[0043] Provided herein are methods, systems and devices related to transcellular membrane delivery, especially transcellular membrane delivery to keep cells alive. Effective loading can be delivered to cells across the membrane in a variety of ways, such as using a microfluidic chip to mix cells and solutions containing any effective loading. Compared with various other processes currently used, the processes, devices and systems provided herein have multiple benefits. For example and provided herein as a non-limiting example, the process discussed herein can achieve uniform delivery of various effective loadings within a cell population. Delivery to cells in a suspension can also be completed. The process can allow users to control various transmission process parameters, such as temperature (for example, using a controlled hot plate substrate), humidity, the number of cells to which the effective loading can be transferred, so that microscopic visualization and machine vision unit counting and / or flow control can be performed, and / or termination solution is introduced by a downstream mixing element. Generally, the process herein converts a process that may be difficult to control due to rapid and / or dynamics into a process that may be more controllable. For example, users can obtain control over osmotic pressure and diffusion rate. The process provided herein is also a closed process, which provides better and consistent transfection efficiency, viability and cell function. Thus, this approach can generally address reversible membrane permeabilization through fluid contact and / or mixing and provide control over delivery across cell membranes.

[0044] In one embodiment, by making cell and containing one or more compounds to be delivered and / or composition (that is, effectively loaded) and reversibly permeate and / or reversibly dissolve cell membrane material solution contact, compound or compound mixture (i.e. composition) is delivered to the cytoplasm of eukaryotic cell.In some embodiments, solution can be mixed with cell in the channel of microfluidic chip, for example, by multiple passive or active mixing elements in the channel of microfluidic chip and cell mixing.In addition, cell can be mixed with the solution containing compound with controlled flow velocity and controlled time in temperature and humidity controlled environment.Exemplary reagents for permeating or dissolving eukaryotic cell membrane include alcohol (such as ethanol) and detergent (such as Triton X-100). Other exemplary detergents (e.g., surfactants) can include polysorbate 20 (e.g., Tween 20), 3-[(3-cholamidopropyl)dimethylammonium-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonium]-2-hydroxy-1-propane sulfonate (CHAPSO), sodium dodecyl sulfate (SDS), and octyl glucoside.

[0045] Figure 1-6 One embodiment of a system and method for delivery across a cell membrane is shown. Figure 1 A microfluidic mixing chip 100 that can be used in this article is shown. The chip can take many forms. For example, Figure 1 The microfluidic mixing chip shown is manufactured by Dolomite Microfluidics in the United Kingdom. However, a variety of different chips may be used. The embodiment shown herein allows for laminar mixing at low throughputs and also allows for turbulent mixing at higher throughputs, but a variety of other approaches may be employed. The chip 100 includes multiple mixing stages, further comprising a plurality of mixing stages. Figure 4 As shown in Figure 2 As shown, cells are first introduced into the hybrid chip 100 in suspension at a controlled flow rate. T cells are shown here, but a variety of cells can be used. At about the same time as the cells, a delivery solution and additives are introduced into the hybrid chip 100. The delivery solution includes any of many known permeability enhancers, such as those discussed above that reversibly permeate and / or reversibly dissolve cell membranes, and the delivery solution contains a delivery load, such as messenger ribonucleic acid (mRNA), to be delivered to the cell membrane introduced into the chip 100. One or more additives can be optionally used in the process, and the additives can take a variety of forms. Figure 1The chip 100 shown in has two different mixing circuits, and each mixing circuit has 12 mixing stages or channels, and can mix up to 3 fluid streams. However, various mixing circuits, mixing stages and / or streams can be used. Therefore, the mixing of cells can include the ratio of fluid volume to cell volume. Alternatively, mixing can include the ratio of fluid volume to exposed cell area, and the exposed cell area can affect the exposed cell membrane area of ​​the mixing effect when the cells are suspended in the channel of the mixing chip (e.g., chip 100) and / or the fluid tube that affects the effect.

[0046] During the 12 mixing stages, as the cells and the delivery solution are mixed in the mixing chip 100, the cells are permeabilized by controlled contact with the delivery solution. The cells and the delivery solution experience laminar flow in the mixing chip 100, but the laminar flow in the mixing chip 100 is disrupted in the illustrated embodiment by the internal fluid structure in the chip 100, which permeabilizes the cell surface by contrasting contact. There are many ways to permeabilize the membrane of a cell population, such as contacting cells in a suspension with a controlled volume that is effectively loaded with the delivery solution at certain time intervals. The user can control many aspects of the mixing process. For example, the user can control the speed, temperature, and humidity of the process in a visualization of the process.

[0047] like Figure 3 As shown, the embodiments described herein generate multiple laminar flows, and the suspended cells are contacted with a delivery solution that contains a delivery payload. For example, Figure 4 FIG. 1 shows mixing activity during one of the multiple mixing stages of chip 100, showing contact and mixing of the laminar flow with the fluid flow (at Figure 4 , using base, acid, and potassium permanganate indicators). When the cells and transfer solution are mixed through the chip 100, the chip 100 outputs permeabilized cells after the first mixing cascade. The permeabilized cells representing the output of the chip 100 after the first mixing cascade can be fed into the input of the chip 100 of the second mixing cascade, as shown. Figure 5 shown.

[0048] exist Figure 5 The introduction of a stop solution is also shown. The subsequent introduction of a second solution, the stop solution, to stop the permeabilization provides the user with greater control over the mixing process. The user can further introduce a controllable delay before the stop solution mixes with the permeabilized cells. For example, the length of the feedback tube used to introduce the permeabilized cells into the second mixing cascade can be varied. Figure 5In the second mixing cascade process shown, the permeated cells are mixed with a stop solution to stop the permeation of the cells. The subsequent output of the second mixing cascade and the entire process is a cell that has been effectively loaded with transformation and / or transfection by the delivery of the delivery solution. The transformed cells produced by the methods provided herein have enhanced characteristics than cells produced by other methods. Figure 6 Schematic representation of the delivery of payload to cells through the overall process described herein.

[0049] In operation, refer to Figure 6 , a method for delivering an effective load across a cell membrane in a continuous flow platform may include introducing a cell population and a delivery solution into a microfluidic mixing chip (step S100), allowing the cell population and the delivery solution to mix in a first mixing cascade (step S200), thereby allowing interaction between the cell population and the delivery solution to transfer the delivered effective load from the delivery solution to the cell population across the cell membrane (step S300), and after transferring the effective load to the cell membrane of the cell population, further introducing a stop solution into the cell population and the delivery solution, so that the stop solution can prevent any additional transfer across the cell membrane of the cell population (step S400).

[0050] exist Figure 1-6 In the embodiment shown, the narrowest channel of chip 100 is approximately 5 to 10 times the diameter of the T cell. However, various sizes and ratios between channel size and pore diameter can be used, particularly configured to allow an optimal ratio between pore size and / or diameter and channel size and / or width, such that the ratio optimizes mixing and / or controlled contact. In various embodiments, the smallest size of the channel in the mixing chip will be larger than the size and / or diameter of the pore. A clinically relevant therapeutic dose of cells can be 10x10 10 or more, and the number of mixing cascades and / or process flow rates on one or more mixing chips can be optimized for the therapeutic dose, particularly to convert clinically relevant cell samples into acceptable processing times.

[0051] Figure 7 and 8 Other embodiments of microfluidic devices and systems for delivering payloads across cell membranes are shown. Figure 7 and Figure 8, the microfluidic device 200 can be fabricated on a substrate including three microchannel sections: a mixing channel 210, a processing chamber 220, and a dilution channel 230. The mixing channel 210 can receive a reactant stream, such as a suspension including a cell population, a delivery solution including an effective load to be delivered, and various additives, through a plurality of inlets 211. The reactant streams supplied through the inlets 201 can be combined and mixed in the mixing channel 210. To enhance mixing, the mixing channel 210 can include microchannels arranged in, for example, a herringbone, an interdigitated, and / or a double T shape.

[0052] Fig. 9 Non-limiting examples of microchannel geometries for mixing channel 210 are shown. Fig. 9 (a) shows an example of a serpentine flow path around a path-defining wall formed in an interdigitated pattern. In this configuration, many recirculation areas can be formed around the tips of the wall and / or at the corners, and secondary flows can also form as the flow turns and bends. Recirculation and secondary flows can enhance mixing of the cell suspension and the delivery solution.

[0053] Fig. 9 (b) shows an example of a mixing channel wall formed in a herringbone pattern, which can provide a similar effect to the interdigitated pattern. Fig. 9 (c) shows an exemplary embodiment of a mixing channel formed in an interdigitated split-recombination configuration. The three streams entering through each inlet can be merged into a middle channel, then split into upper and lower channels through multiple proximal sub-channels, and then recombined into the middle channel through multiple distal sub-channels. This arrangement can be repeated multiple times to achieve the desired level of mixing. Through multiple split and recombination processes, even laminar flow can be effectively mixed. Figure 3 and 4 A schematic diagram of separation and reconstitution mixing is also shown.

[0054] Fig. 9 (d) shows an exemplary embodiment of a mixing channel formed in a herringbone split-recombination configuration, which works in the same way as Figure 8 The interdigital splitting and recombination configuration is similar to that of 9(c). Fig. 9 (e) shows an embodiment of a double T (e.g., cross) type mixing channel. The double T type mixing channel can be used as a stand-alone mixing channel 210 or can be used in combination with other mixing channel configurations by placing it upstream of the other configurations described above. Fig. 9 The mixing channel patterns and configurations disclosed in the foregoing are for illustrative purposes only. The channel arrangement pattern is not limited thereto and may include various other patterns and structures. The mixing channel 210 may include a channel width between 0.01 microns and 100 microns, between 100 microns and 1 millimeter, or greater than or equal to 1 millimeter. The channel may have a uniform width throughout, or a varying width.

[0055] After mixing in the mixing channel 210, the reactant stream may be supplied to the processing chamber 220. In the processing chamber 220, the mixture of the suspension and the transfer solution may be retained for a predetermined residence time and permeated therein. The residence time within the processing chamber 220 may be in the range of 0.01 to 60 minutes, such as 0.01 to 0.1 minutes, 0.1 to 1 minutes, 1 to 5 minutes, 5 to 10 minutes, 10 to 30 minutes, or greater than or equal to 30 minutes.

[0056] The process chamber 220 can be implemented in various geometric shapes. For example, for a continuous flow process, the process chamber 220 can be made into a microchannel having a straight channel shape, a serpentine channel shape, and / or a circular channel shape. Fig.10 Non-limiting examples of process chamber 220 geometries are shown. Fig.10 (a) shows an exemplary embodiment of a process chamber 220 arranged in a straight channel shape. The straight channel shape is easy to manufacture and easy to characterize the residence time of the flow. Fig.10 (b) shows an exemplary embodiment of a process chamber 220 arranged in a serpentine channel shape. With the serpentine channel shape, a long channel can be included in a relatively small area, thereby increasing the area utilization of the substrate.

[0057] Fig.10 (c) shows an exemplary embodiment of a treatment chamber 220 arranged in a circular (or spiral) channel shape. Similar to a serpentine channel shape, a circular channel shape can include a long channel length in a relatively small area. However, the geometry of the treatment chamber 220 is not limited thereto. The cross-sectional area, cross-sectional geometry and length of the microchannel of the treatment chamber 220 can be determined based on the total flow rate of the mixture of the suspension and the transfer solution and the desired residence time. The treatment chamber 220 can include a channel width between 0.01 micron and 100 microns, between 100 microns and 1 mm, or greater than or equal to 1 mm. The channel can have a uniform channel width or have a variable width.

[0058] For a semi-continuous (e.g., continuous batch) process, the process chamber 220 can be made into a plenum chamber with a specific volume. Fig.14 An example of a process chamber 220 implemented as a booster chamber is shown in FIG. Fig.14 A groove-type booster chamber is shown in FIG. 2 , but the process chamber 220 is not limited to a groove-type booster chamber. It can be formed into various other geometric shapes, including but not limited to a cube, a sphere, and a cylinder.

[0059] In an embodiment, a pressurized treatment chamber can also be implemented in a system using a continuous flow process. In these embodiments, a small inlet / outlet and a relatively large gas chamber volume may make it difficult to characterize the residence time due to the formation of a recirculation zone. If it is not desirable to have an uncharacterized residence time, a continuously stirred reactor can be used, wherein an agitator is included in the treatment chamber 220. The agitator can be directly connected to a motor or can be driven magnetically. If necessary, a packed bed reactor can also be used. Typically, a packed bed reactor can straighten the fluid flow and make the flow similar to a plug flow, for which the fluid residence time is easy to characterize.

[0060] The processing chamber 220 may also include at least one diagnostic port, such as a temperature port, a pressure port, and a sample extraction port. The temperature port may accommodate a temperature probe, including but not limited to a thermocouple, a resistance temperature detector (RTD), a thermistor, a semiconductor-based sensor, and / or a non-contact infrared sensor. The temperature measurement may be used to monitor the process temperature and further fed back to a temperature controller for feedback temperature control, which will be described later.

[0061] The pressure port can be connected to a pressure sensor, including but not limited to a strain gauge, a piezoelectric sensor, an electromagnetic sensor, a piezoelectric sensor, and / or an optical sensor. The pressure measurement can be used to monitor the process pressure and further fed to the controller for feedback control of process parameters (such as flow). The process pressure can be controlled between 0.9atm and 1atm, 1atm and 1.5atm, or greater than or equal to 1.5atm to achieve effective transport of the payload across the cell membrane.

[0062] Samples can be extracted through the sample extraction port for online and / or offline analysis. The extracted samples can be analyzed to determine process parameters. The extracted samples can inform the process product acceptance or rejection, can inform process cessation or modification of process parameters. In the manufacturing example of cell therapy, cells engineered by the process can be sampled and tested for viability, functionality, expression efficiency, proliferation ability, presence of genes or toxicity.

[0063] In addition, in order to enhance diagnostic capabilities and provide analytical feedback, the processing chamber 220 may also include an optical window made of an optically transparent material. Optical analysis methods include, but are not limited to, optical still / movie imaging, optical / electron microscopy, spectral techniques such as Raman spectroscopy, absorption spectroscopy, and emission spectroscopy. Diagnostic measurements can be used in real time for feedback control of the process or offline for later analysis. The optical window can be made of yellow glass to prevent ultraviolet light from causing photoactivated reactions. As an exemplary embodiment, an optical window can be made by bonding an optically transparent substrate on top of a substrate including the processing chamber 220. Alternatively, the substrate including the processing chamber 220 may include etched features defining the processing chamber. 220, and sandwiched between two optically transparent substrates at the top and bottom thereof, respectively. Bonding of the optically transparent substrate to the device substrate can be achieved by anodic bonding, melt bonding, and / or using an adhesive. In other embodiments, the optical window may be included in at least one of the mixing channel 210, the dilution channel 230, or the separator 240, which will be further described below.

[0064] Examples of analyses that can be performed through the optical window include the following. An optical still image can be obtained to visualize the process. An optical and / or microscopic video can be obtained to visualize the fluid / cell flow in the processing chamber. The visualization can perform cell counting, flow rate monitoring, etc. The optical analysis can further include more complex methods, such as spectroscopy techniques. The optical analysis results can be further used to control the process through the controller 340. In operation, the controller 340 can adjust the flow rate by controlling the pump 350.

[0065] After the mixture of the suspension and the transfer solution permeates in the treatment chamber 220, it can flow into the dilution channel 230. In the dilution channel 230, the treated fluid flowing out of the treatment chamber 220 can be mixed. The dilution channel 230 can be configured as a simple fluid channel, or include more structures, such as Fig. 9 Various patterns for mixing channels 210 shown to enhance mixing and stop solutions between processed fluids. Stop solutions can reduce or stop the permeabilization of cell membranes, thereby reducing or preventing the transfer of additional payloads across the membranes of cell populations, thereby providing better control of the process. In an embodiment, the stop solution can include phosphate buffered saline (PBS). The concentration of PBS can be about 0.5XPBS.

[0066] In addition, a separator 240 may be further included in the device and connected to the end of the dilution channel 230 to separate the transformed cells from the waste stream. The separator may be implemented in the form of an endless filter or a through-flow filter. The embodiment of the separator is not limited thereto, but may include various other embodiments for separating the treated cells from the waste stream.

[0067] The substrate of the microfluidic device 200 can be made of a material including silicon, silicon oxide, silicon carbide, silicon nitride, silicate glass, borosilicate glass, quartz, sapphire or a polymer such as polydimethylsiloxane (PDMS), polyethylene, polypropylene, polyurethane, polytetrafluoroethylene (PTFE) or polyetheretherketone (PEEK). The substrate can be coated with indium oxide (ITO), or can also be made of stainless steel. Stainless steel can include chromium-manganese-nickel alloy, chromium-nickel alloy, etc. Stainless steel can include, for example, 304 grade stainless steel or 316 grade stainless steel. The substrate can be manufactured by machining techniques and / or various micromachining techniques, including but not limited to wet and dry etching, ultrasonic machining and / or laser machining. The microchannel can have a width suitable for the application. In particular, the width of the microchannel can be in the range of 0.01 μm to 100 μm, 100 μm to 1 mm or more.

[0068] Figure 8 A schematic diagram of a system 300 for performing effective load delivery on a cell membrane 300 including a microfluidic device 200 is shown. The system 300 may also include various components to support the operation of the microfluidic device 200. In an embodiment, the system 300 may be implemented as a closed system in which all fluid channels and connections are sealed so that cells and reagents are not exposed to external (e.g., environmental) elements during effective load delivery. The closed system can achieve sterile transfection, making it suitable for therapeutic use.

[0069] System 300 may include a cell suspension reservoir 301. In some embodiments, cells may include adherent cells or non-adherent cells. Adherent cells may include primary mesenchymal stem cells, fibroblasts, monocytes, macrophages, pneumocytes, neuronal cells, fibroblasts, human umbilical vein (HUVEC) cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells or at least one cell or immortalized cells, such as cell lines. In embodiments, a cell colony may include non-adherent cells, for example, the percentage of non-adherent cells in a colony may be at least 50%, 60%, 75%, 80%, 90%, 95%, 98%, 99% or 100% non-adherent cells. Non-adherent cells may be primary cells as well as immortalized cells (e.g., cells of a cell line). Exemplary non-adherent / suspended cells include primary hematopoietic stem cells (HSCs), T cells (e.g., (differentiation cluster 3) CD3+ cells, (differentiation cluster 4) CD4+ cells, (differentiation cluster 8) CD8+ cells), natural killer (NK) cells, cytokine-induced killer (CIK) cells, human cord blood CD34+ cells, B cells or cell lines (such as Jurkat T cell lines). Non-limiting examples of T cells may include CD8+ or CD4+ T cells. In some aspects, CD3+ can be used. + CD8 T cells +Subpopulations. CD8+ T cells can be purified from the PBMC population by positive isolation using anti-CD8 beads.

[0070] In an embodiment, RPMI basal medium, heat-inactivated fetal bovine serum (FBS) (e.g., about 10% by volume) can be used to culture cells in a standard cell culture medium (e.g., complete RPMI (Roswell Park Memorial Institute culture medium)), penicillin-streptomycin and L-glutamine by volume. In certain embodiments, the standard culture medium can be supplemented with cytokines, such as interleukin 2 (IL-2) (200U / ml). In embodiments, the concentration of the cytokine can be about 10U / ml to about 500U / ml. In other embodiments, the concentration of the cytokine can be about 50U / ml, about 100U / ml, about 200U / ml, about 300U / ml, about 400U / ml or about 500U / ml. The cytokine concentration can be about 200U / ml.

[0071] In addition, system 300 can include a transfer solution reservoir 302. The transfer solution (e.g., including cargo molecules) includes an alcohol, such as ethanol having an alcohol content of 1-30%. The transfer solution can include an isotonic aqueous solution that includes an effective load and an alcohol concentration greater than 5% by volume. In embodiments, the alcohol concentration can be in the range of between 2% and 5% by volume, such as 2%, 3%, 4% or 5%. The alcohol can include ethanol. The aqueous solution can contain greater than 10% ethanol. The aqueous solution can contain 20-30% ethanol. The aqueous solution can contain 27% ethanol. The aqueous solution can contain 12.5-500mM KCl (potassium chloride). The aqueous solution can contain 106mM KCl. In some embodiments, the aqueous solution can contain an ethanol concentration of 5% to 30%. The aqueous solution may contain one or more of 75-98% H2O, 2-45% ethanol, 6-91 mM sucrose, 2-500 mM KCl, 2-35 mM ammonium acetate, and 1 to 14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES). For example, the delivery solution may contain 10 6mMKCl and 27% ethanol. In other embodiments, the effective loading delivery solution may include 25% alcohol by volume. Alternatively, the effective loading delivery solution may include 2% to 8% alcohol by volume, or 2% alcohol by volume. The alcohol may include ethanol, and the effective loading includes 5, 10, 20, 25, 30 and up to 40% or 50% by volume ethanol, such as 27% by volume. The embodiment method may include methanol as an alcohol, and the effective loading may include 5%, 10%, 20%, 25%, 30% or 40% methanol by volume. The effective loading may include 2-45% (volume) methanol, 20-30% (volume) methanol or 25% (volume) methanol. The effective loading may include 20% to 30% methanol. Further alternatively, the alcohol may include butanol, and the effective loading may include butanol with a volume ratio of 2%, 4% or more than 8%.

[0072] The system 300 may include a stop solution reservoir 304. In an embodiment, the stop solution may include phosphate buffered saline (PBS). The concentration of PBS may be about 0.5X PBS. In some embodiments, S buffer may be prepared by a GMP (Good Manufacturing Provider) provider and may be provided in a 10 mL vial with a foil top. Separate DMF (dimethylformamide) may be used as S Buffer for record.

[0073] In addition, the system 300 may include an agitator 310, an ultrasonic vibrator, a mechanical vibrator, etc., and the agitator 310 applies stirring through low-frequency pressure pulses. Vibration or impact force can be selectively applied to at least one of the mixing channel 210, the processing chamber 220, the dilution channel 230 or the separator 240 and / or to the entire device 200. Applying vibration increases the chance of collision between cells and effective loads, so that cells and effective loads maintain continuous and uniform contact, thereby achieving better consistency and control. Agitation can keep the cargo on the cell membrane and assist the diffusion process. Agitation can also promote cellular uptake of effective loads by mechanically stimulating the cell membrane.

[0074] The system 300 may also include a heater 320 and a temperature controller 330 to maintain or change the temperature of the entire device or a specific portion of the device. The heater 320 and the temperature controller 330 may include a single heating zone or multiple heating zones. The temperature controller 330 may obtain input from a temperature detector disposed in the processing chamber 220 and generate a feedback control signal to operate the heater 320. The heater 320 may be implemented as a resistive heater and / or an infrared heater. Since the reactivity of the reagent, the biological reaction of the cells and cell membranes, and the flow of the fluid are all affected by the process temperature, controlling the process temperature can enhance the consistency of the process. Using a heater to introduce a thermal gradient between substrate regions can promote cold shock, thereby promoting intercellular transfer.

[0075] The system 300 may include an additive reservoir 303. In the additive reservoir 303, one or more additives may be stored for use in the process, and the additives may take a variety of forms. For example, the additives may stabilize the protein-based payload and / or may enhance the solubility of the payload.

[0076] At least one pump 350 may be further included in the system 300 to continuously supply the fluid in the reservoir to the corresponding inlet. In an embodiment, each reservoir may include a pump. In addition, the system 300 may include a controller 340. The controller 340 may record and / or process data streams input from temperature sensors, pressure sensors, and various other sensors. The controller 340 may also generate control signals to the temperature controller 330 and / or pump 350 to adjust the process temperature and / or flow rate respectively. The controller 340 may further include a computer or similar user interface device to interact with the operator of the system 300.

[0077] Some embodiments of the subject matter described herein provide a cell engineering platform that can scale up the technology of effectively loading and delivering to cells. The platform can achieve rapid delivery to a large number of cells. The platform can be made into a closed system. In some embodiments, the subject matter described herein can achieve aseptic transfection, and mRNA and RNP are delivered to primary T cells and other cells. In addition, the platform is easy to use and can achieve repeatable and continuous delivery.

[0078] In some embodiments, the subject matter described herein relates to delivery technologies that can facilitate delivery of a wide range of payloads to cells. Compared to batch processes, some embodiments of continuous flow platforms that deliver payloads across cell membranes can provide a steady-state process in which cell concentrations, payload concentrations, and osmotic agent concentrations are maintained at a steady state over time. Thus, aspects of continuous flow platforms can provide better continuity throughout the operation, can generate high throughput to cover a range of therapeutic capabilities, and have better scalability.

[0079] Example 1

[0080] In one embodiment of the methods provided herein, an automated syringe pump is loaded with 2×1 mL Hamilton syringes and set to a flow rate of 500 μL per minute. Syringe A contains 500 μL of Avectas delivery solution, which contains 50 μg of propidium iodide (PI; 150 μL). Syringe B contains 500 μL of 2×10 7 Connect the syringes to the Dolomite Hybrid Chip so that Syringe A is connected to Inlet 2 and Syringe B is connected to Inlet 3. Plug Inlet 1. The chip is primed with the cells and delivery solution containing PI. Once the pump is activated to run at 500 μL / min, samples (each containing 50 μL and 5x10 5 cells). The first sample (perfusion mixture) was discarded and the remaining five samples were incubated at room temperature for 1, 1.5, 2, 2.5, and 3 minutes, respectively. Stop solution (0.5X PBS; 150 μl) was added for 30 seconds, followed by complete Jurkat medium (300 μl). The cells were allowed to rest for 5 minutes before preparation for flow cytometry. The cells were centrifuged at 500 × g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 150 μL PBS. The cells were analyzed by flow cytometry for the presence of intracellular PI. Controlled mixing by the Dolomite Mixing Chip induced PI uptake in each sample with a maximum uptake of 11.8% (2 minutes) compared to the control group.

[0081] The devices disclosed herein can also be designed to be discarded after a single use, or they can be designed to be used multiple times. The components of the current subject matter described herein can be processed before use. First, a new or used instrument can be obtained and cleaned if necessary. The instrument can then be sterilized. In one sterilization technique, the instrument is placed in a sealed container, such as a plastic bag or a TYVEK bag. The container and the instrument are then placed in a radiation field that can penetrate the container, such as gamma radiation, X-rays, or high-energy electrons. The radiation kills bacteria on the instrument and in the container. The sterilized instrument can then be stored in a sterilization container. The sealed container keeps the instrument sterile until it is opened in a medical facility. If the device is sterilized, it can be done by a variety of other means known to those skilled in the art, including beta or gamma radiation, ethylene oxide, steam, and liquid baths (e.g., cold soaking). An exemplary embodiment of sterilizing a device including an internal circuit is disclosed in U.S. Patent No. 8,114,345, entitled "System and Method for Sterilizing Implantable Medical Devices," filed on February 8, 2008. It is preferred that if the device is implanted, it is hermetically sealed. This can be accomplished in a number of ways known to those skilled in the art.

[0082] Example 2

[0083] In some embodiments, efficient cargo delivery across cell membranes can be achieved by nebulization methods. Fig.11 and Fig.12 Two exemplary embodiments of the spray method tested are shown. In the exemplary embodiment of the spray method, a suspension containing cells is mixed with a delivery solution containing an effective load, and the mixture is injected through a sprayer, through which the mixture is broken into fine droplets. The atomized droplets are collected on a plate with a plurality of pore structures to accommodate the liquid. After a certain residence time (e.g., incubation time), a stop solution is applied and the treated sample is subsequently analyzed.

[0084] Unlike batch processes using a monolayer approach, a spray approach may not require the formation of a monolayer to deliver an effective load (e.g., nucleic acid) to cells (e.g., human T cells). In addition, a spray approach may not require the use of a filter plate, and cells may be more easily recovered due to the lack of a filter screen. Furthermore, a spray approach may be adapted to include a continuous flow rate.

[0085] In a first exemplary implementation of the spray method using a 3 mL syringe 460, as Fig.11 As shown, cells (2x10 6) was mixed into a delivery solution containing an effective load mixed in a 1:1 ratio in a 3 mL syringe 460 attached to an AriMist nebulizer 410 (Burgener Research, Inc., Mississauga, Ontario, Canada). A first exemplary embodiment of the spraying method may also include a pre-column connector 420, a valve 430, a valve seat 440, and a Luer connector 450. In an embodiment, the valve 430 and / or the valve seat 440 may be omitted. The cells and the delivery solution were discharged through the nebulizer 410. After 30 seconds of incubation, the cells were dropped into a 24-well plate and 150 μl of the delivery solution. After another 30 seconds of incubation, culture medium (350 μl) was added. At the end of the spray test, the cells were cultured overnight in a humidified incubator at 37°C and 5% CO2, and green fluorescent protein (GFP) fluorescence was evaluated by flow cytometry. In 1 donor and 2 experiments, the expression level of GFP was 25% plus or minus 5.0% and the viability was 70% ( Fig.13 ).

[0086] A second exemplary embodiment of the spray method was tested using a liquid pump 530 manufactured by ELVEFLOW (Paris, France). Fig.12 ), where equal volumes of cells and transfer solution were mixed in a 1.5 mL Eppendorf tube. The tube was connected to an ELVEFLOW liquid pump via a pinch valve 520 and atomized into a 24-well plate via an AriMist atomizer 510. After 30 seconds of incubation, stop solution was added, followed by culture medium.

[0087] The 3 mL syringe and ELVEFLOW liquid pump configuration was tested using human T cells mixed with the delivery solution prior to dropletization via the AriMist nebulizer. Four cell concentrations (5x10 6 , 2.5x10 6 , 1.25x10 6 and 0.5x10 6 ). The total number of cells used for the 3mL syringe setting is 2x10 6 . The percentage expression of GFP mRNA was measured using a BD Accuri C6 flow cytometer. Viability was assessed using 7-AAD. Cell debris was excluded from whole cells using forward and side scatter parameters. Single cells were selected by excluding doublets with high FSC values, while FSC values ​​between Fig.13 GFP expression was analyzed on gated live cells.

[0088] Example 3

[0089] In one embodiment, efficient cargo transfer across the cell membrane can be achieved using a trench-type processing chamber as a batch process. An example of a trench-type processing chamber is Fig.14 , which is a microfluidic chip developed by Dolomite Center Ltd. With a grooved processing chamber, cells can be captured in the grooves of the complete growth medium. In some embodiments, at least 20 individual cells are captured to analyze each of them. A bright field image of the cells captured in the grooves is shown in Fig.15 After capturing the cells, a background (e.g., reference) image is taken using bright field and fluorescence filters. Subsequently, the complete growth medium is removed from the reservoir and replaced with 50 microliters of a transfer solution containing an effective load. In operation, the medium can be removed from the reservoir using a 200 microliter pipette tip that matches the chip. After a set time (usually less than 3 minutes), the transfer solution is removed from the reservoir and replaced with 50 microliters of a stop solution. The cells are then imaged for uptake of the effective load. In addition, after the cells are exposed to the stop solution for approximately 15 minutes, a live / dead stain is added to the reservoir. The cells are imaged again to take up detectable markers, such as FITC (fluorescein isothiocyanate) and PI (propidium iodide), to determine whether the cell membrane is closed. In addition, in another test, the effective load is added to both the cell culture medium and the stop solution to expand the exposure range and maximize the absorption of the effective load. This method was validated for 2 microliters of DAPI (4',6-diamidino-2-phenylindole) with 35% ethanol.

[0090] Figure 16-21 Results of an efficient loading transfer experiment using an exemplary trench-type processing chamber are shown. In particular, Fig.16 and 17 The effect of delivery time is shown. In this experiment, DAPI at a concentration of 3 μM and ethanol at a concentration of 25% were used while varying the delivery time. The results show that the maximum uptake of effective loading was achieved at a delivery time of 9 minutes. However, reverse permeabilization was not achieved due to the uptake of PI by the cells. No PI was taken up at a delivery time of 3 minutes. Fig.18 and 19 The effect of ethanol concentration is shown. In this experiment, the DAPI concentration was 3 μM, and a 3-minute delivery time was used while varying the ethanol concentration. The results show that the highest DAPI uptake was achieved at 35% ethanol concentration. Figure 20-21 The effect of DAPI concentration is shown. In this experiment, a 35% ethanol concentration and a 3-minute delivery time were used while varying the DAPI concentration. The results show that using a 2 μM DAPI concentration maximizes DAPI efficient loading and reverse permeabilization.

[0091] According to the description of the above-mentioned embodiment, those of ordinary skill in the art should understand other features and advantages of the subject matter of the present invention. Therefore, the present invention is not limited by the content specifically shown and described, but by the content limit indicated by the appended claims. All publications and references cited here are clearly incorporated into this paper by reference in full.

Claims

1. A method for delivering a payload across a cell membrane, comprising: mixing a cell population in suspension with a payload to be delivered in a delivery solution, the mixing being performed using a substrate containing a microfluidic channel, wherein the cell population comprises a non-adherent cell population, wherein the non-adherent cells comprise peripheral blood mononuclear cells (PBMC), immune cells or T lymphocytes, wherein the delivery solution causes the cell population to undergo permeabilization and the payload to be delivered is delivered across the cell membrane of the cell population, wherein the delivery solution comprises an alcohol; and After mixing the cell population and the payload to be delivered, introducing a stop solution into the cell population and the payload to be delivered, such that the stop solution stops the cell population from undergoing permeabilization; wherein at least a portion of the population of cells that cease to undergo permeabilization are viable, and At least a portion of the population of cells that ceased to undergo permeabilization are analyzed and are viable.

2. The method of claim 1, wherein the cell population comprises T cells and the payload to be delivered comprises mRNA.

3. The method according to claim 1, wherein: The suspension and the transfer solution undergo a first mixing cascade and a second mixing cascade.

4. The method according to claim 1, wherein: Mixing the cell population and the payload to be delivered includes pumping the cell population and the payload to be delivered into a microfluidic mixing chip.

5. The method according to claim 4, wherein: The narrowest channel of the microfluidic mixing chip is at least 5 times larger than the diameter of the cell population.

6. The method of claim 1, further comprising introducing a stop solution into the cell population and the payload to be delivered after mixing the cell population and the payload to be delivered, such that the stop solution stops the cell population from undergoing permeabilization.

7. The method according to claim 1, wherein: The cell population and the payload to be delivered are subjected to laminar flow.

8. The method of claim 1, further comprising mixing the cell population in the suspension and the payload to be delivered in the delivery solution with at least one additive.

9. The method according to claim 1, wherein: The delivery solution comprises an isotonic aqueous solution comprising an effective load and an alcohol concentration greater than 5% (v / v).

10. The method according to claim 9, wherein: The alcohol includes ethanol.

11. The method according to claim 9, wherein: The aqueous solution includes greater than 10% ethanol.

12. The method according to claim 9, wherein: The aqueous solution comprises between 20-30% ethanol.

13. The method according to claim 9, wherein: The aqueous solution includes 27% ethanol.

14. The method according to claim 9, wherein: The aqueous solution includes 12.5-500 mM KCl.

15. The method according to claim 9, wherein: The aqueous solution included 106 mM KCl.

16. The method according to claim 9, wherein: The payload includes messenger ribonucleic acid (mRNA).

17. The method according to claim 16, wherein: The mRNA encodes a gene editing composition.

18. The method according to claim 17, wherein: The gene editing composition reduces the expression of PD-1.

19. The method according to claim 16, wherein: The mRNA encodes a chimeric antigen receptor.

20. The method of claim 1 for delivering a cargo compound or composition into a mammalian cell.

21. The method of claim 17, wherein after introduction of the stop solution, 70% of the cells remain viable.

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