A method for mechanical and hydrodynamic microfluidic transfection and apparatus therefor

Abstract

The invention provides a method for introducing an exogenous substance into a cell, the method comprising exposing the cell to a transient pressure reduction in the presence of the exogenous substance. Apparatus for performing the method of the invention is also provided.

Description

Invention Field

[0001] This invention relates to a method for introducing an exogenous substance into cells, the method comprising exposing the cells to a transient pressure reduction in the presence of the exogenous substance. The transient pressure reduction is preferably coupled to an unstable flow of a liquid in which the cells and the exogenous substance are present. Specifically, this invention relates to the transfection of mammalian cells.

[0002] Related applications

[0003] This application claims priority to Australian Provisional Application No. 2015900021 entitled “Continuous Delivery Technique”, filed on 7 January 2015, the subject of which is incorporated herein by reference in its entirety. Background of the Invention

[0005] Any discussion of the prior art throughout this specification should not be construed as an admission that the prior art is widely known or forms part of common general knowledge in the art.

[0006] Introducing exogenous substances such as small organic molecules, proteins, and nucleic acids into cells, both in vitro and in vivo, is crucial for the research and development of therapies and for advancements in therapeutic delivery strategies.

[0007] For example, introducing fluorescently labeled proteins into cells allows for real-time analysis of protein transport throughout the cell, which can also help identify protein interactions during clinically important stages of a disease or in response to specific triggers. Introducing presumed small organic molecule drugs that cannot effectively cross cell membranes during drug development can provide information about the drug's activity before valuable time and effort are devoted to developing delivery vehicles for these drugs.

[0008] Introducing nucleic acids into cells is a key step in cell therapy manufacturing, where expression vectors encoding genes are delivered across the cell membrane into the cytoplasm to effectively engineer living cells that can be used as therapeutic agents. For example, cell therapy can be used to induce an individual's own immune system to attack cancer cells or evade viruses such as HIV. Given the prevalence of cancer and HIV in the population—with an estimated 35 million people living with HIV in 2013 and an estimated 14 million new cancer cases in 2012—cancer and HIV are specifically correlated from a global health perspective. Utilizing cell-derived gene therapies as part of a global health strategy requires a cell therapy manufacturing method capable of reproducibly producing sufficient quantities of product to potentially treat tens of millions of patients annually at an appropriate price point and under current good manufacturing practices in accordance with regulatory standards.

[0009] Therefore, the ability to introduce exogenous substances, specifically nucleic acids, into cells in a rapid and efficient manner is both a valuable research tool and a useful component of therapeutic strategies.

[0010] Several methods exist for introducing reagents into cells, and the choice of method is usually determined by the cell type, the required level of efficiency, the size of the molecule to be introduced, and the number of cells available.

[0011] Although the terms are used interchangeably, the introduction of reagents such as nucleic acids into eukaryotic cells is generally referred to as "transfection," while the introduction of nucleic acids into prokaryotic cells is generally referred to as "transformation." Transfection and transformation methods can be conveniently classified into three categories: chemical methods, physical methods, and virus-based methods.

[0012] Chemical transfection methods employ reagents such as cationic lipids, calcium phosphate, cationic polymers, and dendritic polymer molecules to essentially package nucleic acids for delivery into cells. However, many of these methods are not suitable for all cell types. Furthermore, they can be impaired by pH fluctuations or salt / phosphate levels in the cell culture medium. Because some of these methods require packaging nucleic acids, the size of the nucleic acid molecules that can be accommodated can be limited. Additionally, chemical transfection methods may require the use of high concentrations of reagents that are expensive and / or toxic to cells, and / or the methods may only achieve low / inconsistent transfection efficiencies.

[0013] Conventional physical methods for transfecting eukaryotic cells include the use of magnetic nanoparticles, electroporation, bioprojectile delivery, and microinjection. However, these methods tend to be quite demanding on cells, often resulting in high mortality rates. These methods may also require immobilized cells, expensive equipment, and / or a greater degree of technical skill for the personnel performing the methods. For example, in some electroporation methods, suspended cells are first permeated, followed by the application of an electric field to facilitate the active delivery of charged exogenous substances. Therefore, these techniques require specialized equipment and consumables to permeate the cell membrane and apply the electric field.

[0014] Virus-based transfection methods rely on viral vectors, including lentiviral, adenoviral, and retroviral vectors for delivering nucleic acids into cells, where the nucleic acids are expressed at high levels via viral promoters. These virus-based methods are expected to prove effective in treating lymphoma and hematopoietic system cancers and are suitable for HIV therapeutics. However, due to variable transfection efficiencies, the cost of manufacturing viral vectors for these types of therapeutics is approximately several thousand dollars per patient. Furthermore, if the methods are not automated, they can be both labor-intensive and prone to manufacturing problems.

[0015] Introducing exogenous substances, specifically nucleic acids, into prokaryotic cells is also an important aspect of the manufacture of biologics during therapeutic drug development and, in practice (usually), research. Transformation of bacterial cell lines with exogenous nucleic acids can be achieved through various methods, including chemical transformation and electroporation, for the recombinant production of valuable molecules such as biologic-based drugs (so-called biopharmaceuticals). However, these methods may require cells to be in a “competent” state prior to transformation (e.g., by inducing high cell density and / or nutrient restriction to activate a set of genes), they may not be suitable for all cell types, and / or they may lead to high levels of cell death.

[0016] Therefore, there is a need for a rapid and efficient method for introducing exogenous substances into a range of cell types, overcoming one or more difficulties of known methods. Preferably, the method will deliver acceptable levels of cell viability, and the method will be cost-effective.

[0017] The purpose of this invention is to overcome or improve at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0018] It should be understood that references to "preferred" or "preferred" in this document are intended only as examples. Invention Overview

[0020] Limitations associated with introducing exogenous substances into cells are often related to the toxicity and / or cost of reagents and devices used in physical, viral, and chemical transfection and transformation methods. Furthermore, many transfection methods are not suitable for high-throughput applications, partly because they require significant human intervention throughout the process and large numbers of cells to compensate for low transfection efficiency and / or cell viability. Indeed, human intervention is often a source of inconsistency associated with transfection efficiency in methods heavily reliant on technicians. Even low levels of human-introduced imprecision can have a significant adverse effect on delivery efficiency, cell viability, and / or reproducibility. See, for example, Mitsuyasu et al. (Mitsuyasu RT, et al. (2009). Phase 2 genetherapy trial of an anti-HIV ribozyme in autologous CD34+ cells. Nature Medicine, 15(3):285-292), where a viral vector was used to transfect CD34+ hematopoietic progenitor cells, resulting in a delivery efficiency of 54% ± 17% (mean ± standard deviation) in 38 patients (n = 38).

[0021] The inventors have unexpectedly discovered that cells readily take up exogenous substances when exposed to a sudden decrease in pressure.

[0022] To avoid being bound by theory, a sudden drop in pressure is most likely to permeate the cell membrane without dissolving the cell. A relatively sudden and temporary pressure drop across the cell membrane (resulting in intracellular pressure being greater than extracellular pressure) may cause temporary pores to form in the membrane, allowing the introduction of exogenous substances.

[0023] Therefore, in a first aspect of the invention, a method for introducing an exogenous substance into cells is provided, the method comprising exposing the cells to a transient pressure reduction in the presence of the exogenous substance to thereby introduce the exogenous substance into the cells. The transient pressure reduction does not cause cell lysis, but in some embodiments the cells may become non-viable. Those skilled in the art will understand that when the invention is applied to a cell population, some cells in the population may be dissolved.

[0024] Preferably, the cells are viable after exposure to a transient decrease in pressure.

[0025] Preferably, the cells are selected from the group consisting of: bacterial cells, mammalian cells, yeast cells, plant cells, and insect cells.

[0026] In some preferred embodiments, the cell is a mammalian cell. In other preferred embodiments, the cell is a bacterial cell. In still other preferred embodiments, the cell is a yeast cell. In a further preferred embodiment, the cell is an insect cell. In yet another embodiment, the cell is a plant cell.

[0027] Preferably, the exogenous substance is selected from the group consisting of: small organic molecules, nucleic acids, nucleotides, proteins, peptides, amino acids, lipids, polysaccharides, viruses, quantum dots, carbon nanotubes, radionuclides, magnetic beads, nanoparticles, gold particles, monosaccharides, vitamins, and steroids.

[0028] Preferably, the nucleic acid is selected from the group consisting of: PNA, DNA, RNA, mRNA, miRNA, and siRNA.

[0029] Preferably, the DNA is a plasmid.

[0030] Preferably, the plasmid is an expression vector.

[0031] Preferably, the expression vector expresses PNA, DNA, RNA, miRNA, siRNA, or protein.

[0032] Preferably, the expression vector is a viral vector.

[0033] Preferably, the viral vector is a lentiviral vector or a retroviral vector.

[0034] Preferably, the expression vector is a bacterial artificial chromosome (BAC) or a yeast artificial chromosome (YAC).

[0035] Preferably, the exogenous substance is introduced into the cytoplasm of the cell.

[0036] Preferably, the exogenous substance is introduced into the cell nucleus. In these preferred embodiments, the cell is a mammalian cell, yeast cell, gamete (e.g., sperm cell or egg cell), or insect cell. More preferably, the cell is a mammalian cell.

[0037] Preferably, the instantaneous pressure reduction is a reduction of at least 10 kPa.

[0038] Preferably, the instantaneous pressure reduction is a reduction of at least 100 kPa.

[0039] Preferably, the instantaneous pressure reduction is a reduction of at least 500 kPa.

[0040] Preferably, the instantaneous pressure reduction is a reduction of at least 1000 kPa.

[0041] Preferably, the cells are exposed to the instantaneous pressure reduction in the presence of the exogenous substance for at least 10 nanoseconds.

[0042] Preferably, the cells are exposed to the instantaneous pressure reduction in the presence of the exogenous substance for at least 100 nanoseconds.

[0043] Preferably, the cells are exposed to the instantaneous pressure reduction in the presence of the exogenous substance for at least 1 microsecond.

[0044] Preferably, the cells are exposed to the instantaneous pressure reduction in the presence of the exogenous substance for no more than 1 millisecond.

[0045] Preferably, when exposed to the instantaneous pressure reduction, the exogenous substance and the cells are in a liquid.

[0046] Preferably, the cells are exposed to the instantaneous pressure reduction within a closed channel, the closed channel having dimensions configured to allow the flow of the fluid containing the exogenous substance and the cells through it.

[0047] Preferably, the flow of the liquid within the channel has a fluctuating velocity.

[0048] Preferably, the flow has a minimum peak velocity of at least 1 m / s.

[0049] Preferably, the flow has a minimum peak velocity of at least 5 m / s.

[0050] Preferably, the flow has a maximum peak velocity of no more than 50 m / s.

[0051] Preferably, the flow has a maximum peak velocity of no more than 100 m / s.

[0052] Preferably, the channel is configured to influence the flow of the liquid such that there are one or more regions within the channel where the flow of the liquid is laminar, and / or one or more regions within the channel where the flow of the liquid is crepitus, and / or one or more regions within the channel where the flow of the liquid is unstable.

[0053] Preferably, the flow of liquid around the diverter in at least one of the regions within the channel has a Reynolds number (Re) of the object. o This is sufficient to induce unstable flow.

[0054] Preferably, the object's Reynolds number (Re o () is at least 40.

[0055] Preferably, the object's Reynolds number (Re o () is no more than 2000.

[0056] Preferably, the flow of the liquid is affected by one or more diverters within the channel.

[0057] Preferably, one or more regions within the channel where the flow of the liquid is unstable are downstream of the distributor.

[0058] Preferably, the instantaneous pressure exposed to the cells downstream of the shunt is reduced.

[0059] Preferably, the diverter is an obstacle placed within the enclosed channel.

[0060] Preferably, the obstacle is a pillar. More preferably, the pillar is cylindrical.

[0061] Preferably, the obstacle is positioned within the channel such that the cell must pass through a gap with a width and height or diameter at least 1.01 times the minimum diameter of the cell as it flows through the channel.

[0062] Preferably, the gap has a width and height or diameter that is at least 1.01 times the minimum diameter of the cell.

[0063] Preferably, the gap has a width and height or diameter that are at least twice the minimum diameter of the cell.

[0064] Preferably, the gap has a width and height or diameter that are at least 10 times the minimum diameter of the cell.

[0065] Preferably, the gap has a width and height or diameter that are at least 100 times the minimum diameter of the cell.

[0066] Preferably, the obstacle has a maximum width of 10 nanometers.

[0067] Preferably, the obstacle has a maximum width of 20 micrometers.

[0068] Preferably, the obstacle has a maximum width of 100 micrometers.

[0069] Preferably, the obstacle has a maximum width of 1 mm.

[0070] There are many advantages to adapting high-throughput methods for introducing exogenous substances into cells to meet the needs of large-scale manufacturing. For example, devices such as microfluidic devices can be suitably designed and operated to expose cells to one or more transient pressure reductions in the presence of exogenous substances. Advantageously, such devices can be manufactured from simple plastics at very low cost, potentially in the range of only a few dollars per device.

[0071] Therefore, in a second aspect of the invention, an apparatus is provided for use in a method for introducing an exogenous substance into cells in a liquid, the apparatus comprising:

[0072] At least partially closed channels, said channels having dimensions configured to allow the flow of said cells and exogenous substances suspended in a liquid through them; and

[0073] One or more splitters within the channel;

[0074] The aforementioned shunt causes at least one region of reduced pressure immediately downstream of the shunt.

[0075] Preferably, the pressure reduction region occurs in at least one unstable flow region immediately downstream of the splitter.

[0076] Preferably, the device is a microfluidic device.

[0077] Preferably, the device is based on Figure 4 To construct.

[0078] Preferably, the device is based on Figure 5 To construct.

[0079] Preferably, the device is based on Figure 6 To construct.

[0080] Preferably, the device is based on Figure 7 To construct.

[0081] Preferably, the device is based on Figure 8 To construct.

[0082] Preferably, the device is based on Figure 9 To construct.

[0083] Preferably, the device is based on Figure 10 To construct.

[0084] Preferably, the device is used in a method for introducing exogenous substances into cells in a liquid according to any of the foregoing aspects.

[0085] Therefore, in a third aspect of the invention, a cell containing exogenous substances produced according to any of the foregoing aspects is provided.

[0086] Therefore, in a fourth aspect of the present invention, a cell suspension comprising the cells of the fourth aspect is provided.

[0087] Therefore, in a fifth aspect of the invention, a pharmaceutical composition comprising the cells of the third aspect or the cell suspension of the fourth aspect, and a pharmaceutically acceptable diluent, cryoprotectant, carrier, or excipient is provided.

[0088] Therefore, in a sixth aspect of the present invention, a kit comprising the apparatus of the second aspect is provided.

[0089] This invention relates to the introduction of exogenous substances into cells. As used herein, the term "exogenous" means any substance present outside the cell prior to exposing the cell to a transient pressure reduction in the presence of the exogenous substance. It should be understood that the term "exogenous" relates to substances that develop, grow, or originate outside the cell. Exogenous substances can be naturally occurring or synthetic. In the context of this application, the term "naturally occurring" within the scope of the substances involved means any substance present in nature and may include bioactive substances. Naturally occurring substances may be modified in ways that are not naturally occurring in nature and suitably isolated from nature by techniques known in the art. In the context of this application, the term "synthetic" means not naturally occurring but prepared by human technical intervention. In the context of synthetic proteins and nucleic acids, this covers molecules produced by recombinant, chemically synthesized, or combinatorial techniques well known in the art. Synthetic substances may be imitations of naturally occurring substances or may not resemble substances present in nature.

[0090] The exogenous substance may be biologically active in the cells in which it is introduced. Alternatively, the exogenous substance may have no detectable effect on the cells after its introduction.

[0091] The cell can be any cell with a cell membrane or cell wall, which may temporarily permeate when the cell is exposed to a transient decrease in pressure. In the method of the present invention, the cell may or may not be viable before or after exposure to the transient decrease in pressure. The cell may or may not be senescent. Since the method of the present invention can be a passive method of introducing exogenous substances into cells, it should be understood that the method of the present invention is not necessary for viable and / or actively dividing cells. For example, in the case of introducing exogenous substances into cells to identify specific organelles in the cytoplasm, the method of the present invention can be performed on dead cells (cells no longer capable of metabolism). In another instance, if the exogenous substance introduced into the cell is a selective marker of cell death, the method of the present invention can be performed on a mixture of live and dead cells.

[0092] In specific embodiments of the invention, the cells are bacterial cells, mammalian cells, yeast cells, gamete cells (e.g., sperm cells or egg cells), plant cells, or insect cells. It should be understood that the invention also contemplates progenitor cells, and specifically stem cells, and more preferably hematopoietic stem cells or mesenchymal stem cells. Cells may be in culture, extracted from tissue samples, and / or immortalized. It should be understood that in those embodiments contemplated as plant cells, the cell wall is completely or partially removed to form protoplasts prior to treatment according to the method of the invention. Cells may be derived from primary cultures or from continuous (subculture) cultures. Cells may originate from any tissue type. Cells may or may not be terminally differentiated. Appropriately, the cells are isolated cells. “Isolated” means that a substance is substantially or essentially free of components that normally accompany the substance in its natural state, or components present during its production when purified or produced by synthetic means. Therefore, the term “isolated” also includes purified or synthetic substances within its scope.

[0093] As those skilled in the art will understand, the preferred starting cell density may depend on the cell type and / or exogenous material. In a preferred embodiment of the invention, and particularly in a preferred embodiment relating to mammalian cells, the starting cell density is between about 2 million cells / mL and about 10 million cells / mL, and all integers therebetween.

[0094] In the context of "introducing exogenous substances into cells" as described herein, the term "introduction" means the delivery, travel, or transfer of an exogenous substance to at least the outermost barrier of the cell, namely the cell wall or cell membrane. Exogenous substances can travel beyond the outermost barrier of the cell and cross the cell wall or cell membrane to enter the cytoplasm. Exogenous substances can travel to organelles within the cell. Specifically, exogenous substances can travel to the cell nucleus.

[0095] In embodiments of the present invention, the exogenous substance introduced into the cell is selected from the group consisting of: small organic molecules, nucleic acids, nucleotides, oligonucleotides, proteins, peptides, amino acids, lipids, polysaccharides, quantum dots, nanoparticles, monosaccharides, gold particles, vitamins, and steroids, as well as combinations thereof. The exogenous substance need not have a net charge.

[0096] Preferably, the nucleic acid is selected from the group consisting of: PNA, DNA, RNA, miRNA, and siRNA, and combinations thereof. Preferably, the DNA is an oligonucleotide or a plasmid.

[0097] In a specific embodiment of the invention, the plasmid is an expression vector. The expression vector can be a self-replicating extrachromosomal vector (such as a plasmid) or a vector integrated into the host genome. As used herein, the term "vector" refers to any molecule used as a medium to facilitate the delivery or expression of nucleic acids in a cell. Preferably, the vector expresses DNA, RNA, miRNA, siRNA, or protein. "Vector" refers to a polynucleotide molecule, suitably a DNA molecule derived from, for example, plasmids, bacteriophages, viruses, yeast, or higher eukaryotes (including plants, vertebrates, or invertebrates) into which polynucleotides can be inserted or cloned. The vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell (including target cells or tissues or progenitor cells or tissues thereof), or may be integrated into the genome of a defined host such that the cloned sequence is reproducible. Thus, the vector can be a self-replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a linear or closed circular plasmid, extrachromosomal element, miniature chromosome, or artificial chromosome. The vector may include any elements used to ensure self-replication. Alternatively, the vector can be a vector that integrates into the genome when introduced into a host cell and replicates along with the chromosome into which it has already been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids (which together contain the total DNA of the host cell genome to be introduced), or a transposon. The choice of vector will generally depend on the compatibility of the vector with the host cell to which it will be introduced. In some embodiments, the vector is a virus or viral-derived vector that is operatively functional in vertebrate or invertebrate and suitable mammalian cells. Such a vector may be derived from poxviruses, lentiviruses, retroviruses, adenoviruses, or yeast. The vector may also include selection markers, such as antibiotic resistance genes, that can be used to select suitable transformants. Examples of such resistance genes are known to those skilled in the art and include those conferring resistance to the antibiotics kanamycin and G418. The nptII gene, which confers resistance, and the hph gene, which confers resistance to the antibiotic hygromycin B.

[0098] In other embodiments of the invention, the vector is a viral vector, preferably a lentiviral vector or a retroviral vector. The vector may also be a bacterial artificial chromosome or a yeast artificial chromosome.

[0099] In the context of this invention, the term "dissolved" refers to a cell whose cell wall / cell membrane is sufficiently damaged such that most of the cell contents are no longer contained within the cell wall / cell membrane, and the cell is therefore non-viable. However, even if the cell is not dissolved, it does not necessarily need to be viable for the purposes of this invention. Those skilled in the art will understand that it may be desirable to transfect the cell with an exogenous substance if the cell is not dissolved, without requiring the resulting transfected cell to be viable. For example, in cases where the exogenous substance is a marker or antibody designed to bind to and indicate the location or expression profile of a specific protein in the cell, cell viability may not be a determining factor in the assay results. In another instance, the method of this invention can produce undissolved, but necrotic, and still viable cells of interest.

[0100] In a specific embodiment of the invention, the cells are viable after the transient pressure reduction following exposure to the method of the invention. It should be understood that a "viable" cell is a cell capable of cellular metabolism and / or cell division. A cell capable of cellular metabolism is a cell capable of degrading molecules and releasing energy (commonly referred to as catabolism), producing molecules (such as polysaccharides, lipids, nucleic acids, and proteins), and / or using energy (commonly referred to as anabolism). A living cell can also be a cell capable of cellular metabolism but permanently in the G0 phase of the cell cycle and unable to divide.

[0101] The method of the present invention can be used to transfect cell populations, in which some cells may be dissolved (and therefore not viable), some cells may not be dissolved but may not viable, while other cells may be viable.

[0102] As used herein, the term "pressure reduction," in connection with exposing cells to such a reduced range, refers to exposing the cells to a region having a pressure relatively lower than that of the immediate surrounding region. The pressure in this region may be uniform, or it may be a localized region with varying pressure, as long as these localized regions still have a lower pressure relative to the pressure surrounding the region. The pressure surrounding the region may be uniform, or it may be a localized region with varying pressure, as long as these localized regions have a higher pressure relative to the region.

[0103] "Pressure" refers to the force exerted by a substance on its surrounding environment per unit area, as is known in the art. The SI unit of pressure is the Pascal (Pa). Other commonly used units for measuring pressure include kilopascals (kPa), pounds-force per square inch (PSI), millimeters of mercury (mmHg), millibars (mbar), and atmospheric pressure (atm) of air. Pressure explicitly associated with a vacuum can be measured in Torr. In this application, when the term "kPa" is used, it refers to gauge pressure, not absolute pressure, where a gauge pressure of 0 kPa refers to an absolute pressure of 101.325 kPa.

[0104] Instantaneous pressure drop can be defined in the context of the pressure difference between the region with lower pressure and the surrounding region. Instantaneous pressure drop can also be defined in the context of the minimum pressure in the region with lower pressure and the maximum pressure in the surrounding region. For example, if the minimum pressure in the region with lower pressure is -10 kPa and the maximum pressure in the surrounding region is 100 kPa, the pressure difference will be 110 kPa. In another example, if the minimum pressure in the region with lower pressure is 20 kPa and the maximum pressure in the surrounding region is 500 kPa, the pressure difference will be 480 kPa. In yet another example, the pressure difference between the region with lower pressure and the surrounding region could be 200 kPa, which might be the result of the minimum pressure in the region with lower pressure being in the range of -100 kPa to 1000 kPa and the maximum pressure in the surrounding region being in the range of 100 kPa to 1200 kPa. In another example, the pressure difference between the area with lower pressure and the surrounding area could be 50 kPa. This could be the result of the minimum pressure in the area with lower pressure being in the range of 0 kPa to 150 kPa and the maximum pressure in the surrounding area being in the range of 50 kPa to 200 kPa.

[0105] The maximum and minimum pressures applicable to any cell type will be readily apparent to a skilled technician. At too low a pressure, the efficiency of the method may be compromised, and at too high a pressure, the cells may rupture. The optimal pressure gradient for a particular cell can be identified by referring to the embodiments of this application and through routine experiments.

[0106] Preferably, the instantaneous pressure reduction of cells exposed to the presence of exogenous substances is a reduction of at least 10 kPa, at least 100 kPa, at least 500 kPa, or at least 1000 kPa. In some implementations, the instantaneous pressure reduction is a pressure (kPa) reduction of at least 15 kPa, at least 20 kPa, at least 25 kPa, at least 30 kPa, at least 35 kPa, at least 40 kPa, at least 45 kPa, at least 50 kPa, at least 60 kPa, at least 70 kPa, at least 80 kPa, at least 90 kPa, at least 100 kPa, at least 150 kPa, at least 200 kPa, at least 250 kPa, at least 300 kPa, at least 350 kPa, at least 400 kPa, at least 450 kPa, at least 500 kPa, at least 550 kPa, at least 600 kPa, at least 650 kPa, at least 700 kPa, at least 750 kPa, at least 800 kPa, at least 850 kPa, at least 900 kPa, at least 950 kPa, or at least 1000 kPa.

[0107] In the context of pressure reduction, the term "transient" refers to a pressure reduction that occurs temporarily, meaning that after a cell is exposed to the reduced pressure, it will subsequently be exposed to a higher pressure. In some embodiments of the invention, transient pressure reduction means that the cell is exposed to a minimum pressure reached during a specific exposure lasting at least 10 nanoseconds but no more than 1 millisecond. It should be understood that this time does not include the time between when the cell is exposed to the maximum pressure in the surrounding area and when the cell is exposed to the minimum pressure in a region with lower pressure relative to the surrounding area. Nor does this time include the time between when the cell is exposed to the minimum pressure in a region with lower pressure and when the cell is exposed to the maximum pressure in the surrounding area.

[0108] The duration of exposure to a transient pressure drop for any cell type will be determined by a skilled technician. Too long an exposure can lead to inefficiency, while too short an exposure may not allow for the introduction of exogenous substances into the cells. The optimal exposure time for a particular cell type can be determined by referring to the embodiments of this application and through routine experiments.

[0109] Preferably, the cells are exposed to a transient pressure decrease in the presence of an exogenous substance for at least 10 nanoseconds, at least 100 nanoseconds, at least 1 microsecond, or at least 1 millisecond. In some embodiments of the invention, the cells are exposed to a transient pressure decrease for at least 15 nanoseconds, at least 20 nanoseconds, at least 25 nanoseconds, at least 30 nanoseconds, at least 35 nanoseconds, at least 40 nanoseconds, at least 45 nanoseconds, at least 50 nanoseconds, at least 60 nanoseconds, at least 70 nanoseconds, at least 80 nanoseconds, at least 90 nanoseconds, at least 100 nanoseconds, at least 150 nanoseconds, at least 200 nanoseconds, at least 250 nanoseconds, at least 300 nanoseconds, at least 350 nanoseconds, or at least 40 nanoseconds. 0 nanoseconds, at least 450 nanoseconds, at least 500 nanoseconds, at least 550 nanoseconds, at least 600 nanoseconds, at least 650 nanoseconds, at least 700 nanoseconds, at least 750 nanoseconds, at least 800 nanoseconds, at least 850 nanoseconds, at least 900 nanoseconds, at least 950 nanoseconds, at least 100 microseconds, at least 200 microseconds, at least 300 microseconds, at least 400 microseconds, at least 500 microseconds, at least 600 microseconds, at least 700 microseconds, at least 800 microseconds, or at least 900 microseconds.

[0110] "Instantaneous" also means that the pressure reduction occurs relatively quickly, meaning the time between when the cell is exposed to the maximum pressure in the surrounding area and when the cell is exposed to the minimum pressure in the relatively low-pressure area is less than one second. In a specific embodiment of the invention, the time between when the cell is exposed to the maximum pressure in the surrounding area and when the cell is exposed to the minimum pressure in the relatively low-pressure area is less than one millisecond. Similarly, once the cell is exposed to the minimum pressure in the relatively low-pressure area, the time between the time the cell is exposed to this minimum pressure and the time the cell is exposed to the maximum pressure in the surrounding area is less than 10 seconds, 100 seconds, or 1 minute. In this invention, the time between the time the cell is exposed to this minimum pressure and the time the cell is exposed to the maximum pressure in the surrounding area is sufficient to permeate the cell membrane without dissolving the cell.

[0111] When performing the method of the present invention, cells may be exposed to more than one transient pressure drop in the presence of an exogenous substance. In some embodiments, cells may be exposed to more than one transient pressure drop, wherein said transient pressure drop is the same or different with respect to the pressure difference between the region having a relatively low pressure and the surrounding region. In other embodiments of the invention, the pressure difference defining the transient pressure drop may be due to the same or different minimum pressure in the region having a relatively low pressure. The pressure difference defining the transient pressure drop may also be due to the same or different maximum pressure in the surrounding region.

[0112] For example, a cell may be exposed to a transient pressure drop of 10 kPa; subsequently exposed to a second transient pressure drop, also of 10 kPa. The first transient pressure drop of 10 kPa may result from a minimum pressure of 50 kPa in a region with relatively low pressure and a maximum pressure of 40 kPa in the surrounding region, while the second transient pressure drop of 10 kPa may result from a minimum pressure of 20 kPa in a region with relatively low pressure and a maximum pressure of 30 kPa in the surrounding region.

[0113] In another example, cells may be exposed to a transient pressure drop of 300 kPa, followed by a second transient pressure drop of 80 kPa. The first transient pressure drop of 300 kPa could be the result of a minimum pressure of 100 kPa in a region with relatively low pressure and a maximum pressure of 400 kPa in the surrounding region, while the second transient pressure drop of 300 kPa could be the result of a minimum pressure of -50 kPa in a region with relatively low pressure and a maximum pressure of 250 kPa in the surrounding region.

[0114] In embodiments of the invention, when both are in a liquid state, the cells are exposed to a transient pressure reduction in the presence of the exogenous substance. The liquid can be any liquid that generally does not cause cell lysis and is capable of maintaining cell viability for the duration of the method in some embodiments of the invention. Preferably, the exogenous substance will be soluble in, able to suspend in, or dispersible in the liquid. For example, the liquid can be a cell growth culture medium or a buffered saline solution, such as phosphate-buffered saline or Tris-buffered saline. The liquid can be blood, plasma, or serum, or another bodily fluid, such as whole blood, umbilical cord blood, bone marrow, or a fat-derived fluid. Blood or bodily fluids may be fractionated, separated, and / or diluted to improve processing. While the fluid may contain reagents or chemicals that promote the introduction of the exogenous substance into the cells, the liquid does not necessarily contain any additional reagents or chemicals used to promote the introduction of the exogenous substance into the cells. For example, in some embodiments of the invention, the liquid does not contain any additional cationic lipids, cationic polymers, calcium ions (e.g., in the form of calcium chloride or calcium phosphate), magnesium ions (e.g., in the form of magnesium chloride), or dendritic polymers. It should be understood that many of these chemicals and reagents are toxic to cells, and that the absence or substantial absence of added amounts of these chemicals or reagents in the liquids used in the methods of the present invention can prevent undesirable cell lysis or cell death when performing the methods of the present invention.

[0115] "Additional" means any additional amount of chemicals or reagents other than those normally and / or naturally present in liquids. For example, many bodily fluids (such as blood) may naturally contain calcium ions, but in a specific embodiment of the invention, calcium phosphate will not be added to the blood prior to its use as a liquid in the method of the invention. In another example, cell growth media may typically contain magnesium ions, but in a specific embodiment of the invention, magnesium chloride will not be added to the growth media prior to its use as a liquid in the method of the invention.

[0116] In a preferred embodiment of the invention, the instantaneous pressure reduction of cells exposed to fluid within a channel (preferably a closed channel) is achieved, the channel being sized to allow flow of fluid containing exogenous substances and cells. In the context of this invention, "channel" refers to any component having a length and two or more ends, having a hollow space extending the length of the component, allowing fluid flow through the hollow space and through openings at the two or more ends. The channel only needs to be sized to allow flow of the relevant cell type in the fluid. The cross-section of the channel can have any shape. The channel should include at least some closed sections, but is not necessarily sealed along its entire length, as long as areas within the channel where the desired pressure changes may occur.

[0117] It should be understood that the flow of the liquid will be essentially from one end of the channel to the other, and the direction of the flow will determine the direction of "upstream" and "downstream".

[0118] Flow through the channel can be caused by various means, including but not limited to hydrostatic pressure, hydrodynamic pressure, and / or electroosmotic flow. Liquid flow can be driven by a pressure source, including but not limited to pressure pumps, cylinders, compressor pumps, vacuum pumps, syringes, syringe pumps, peristaltic pumps, pistons, capillary pumps, the heart, muscles, or gravity.

[0119] When liquid enters a channel, the pressure source used to generate the liquid flow through the channel will preferably provide a steady flow, such as creeping or laminar flow. Those skilled in the art will understand that creeping flow refers to a liquid flow in which the inertial forces of the liquid are significantly lower than the viscous forces of the liquid. Laminar flow refers to a liquid flow in which the inertial forces within the liquid are greater than or equal to the viscous forces of the liquid, but not large enough to induce transitional or turbulent flow.

[0120] The flow of liquid through the channel will have a velocity, and this velocity can be affected by factors including, but not limited to, the channel configuration, the strength and nature of the pressure source, the viscosity of the liquid, the cell type and cell density in the liquid, and / or the nature and amount of exogenous substances.

[0121] In a preferred embodiment of the invention, the velocity of the liquid fluctuates as it flows through the channel, and the fluctuation velocity can be defined according to the maximum and minimum velocities of the liquid as it flows through the channel. The velocity of the liquid can fluctuate between a specific maximum and minimum velocity as it flows through the channel. Preferably, the fluctuation velocity of the liquid flowing through the channel has a minimum peak velocity of 1 m / s or more preferably 5 m / s. In other preferred embodiments of the invention, the fluctuation velocity of the liquid flowing through the channel has a maximum velocity of 10 m / s, a maximum velocity of 20 m / s, a maximum velocity of 30 m / s, a maximum velocity of 40 m / s, a maximum velocity of 50 m / s, a maximum velocity of 60 m / s, a maximum velocity of 70 m / s, a maximum velocity of 80 m / s, a maximum velocity of 90 m / s, and a maximum velocity of 100 m / s. Therefore, it should be understood that the peak velocity of the liquid flowing through the channel can fluctuate between 1 m / s and 100 m / s.

[0122] As a liquid flows through a channel, and as the flow exhibits fluctuating velocities, the type of flow can change. For example, the flow can alternate between laminar, creeping, and unsteady flow, where unsteady flow refers to laminar vortex streets, transitional vortex streets, turbulent vortex streets, transitional flow, or turbulent flow. Those skilled in the art will understand the differences between creeping, laminar, and unsteady flow. In a specific embodiment of the invention, the channel is configured to influence the flow of the liquid such that there are one or more regions within the channel where the flow of the liquid is laminar, and / or one or more regions within the channel where the flow of the liquid is creeping, and one or more regions within the channel where the flow of the liquid is unsteady.

[0123] The type of flow can be estimated by calculating two different Reynolds numbers: one for flow through a closed channel (Re...). c ) and / or a specific flow in the area between the diverter and the object (Re) o The flow around it. For example, for crepitus, Re c Significantly less than 1 (Re) c <<1), and for laminar flow, Re c Between 1 and approximately 2000 (1 <Re c <2000). For example, for unstable flows around an object, Re o Greater than approximately 40 (Re o >40) or sufficient to induce unstable flow. c It can be defined as the average liquid velocity (ū) and the hydraulic diameter (D) of the liquid. h The ratio of the width to the kinematic viscosity (ν) is defined as follows. For wide channels where the width is significantly greater than the height (or vice versa), D hIt can be replaced by twice the length of the shorter distance. When calculating the Reynolds number (Re) of the flow path between columns... c When using this equation and the hydraulic diameter of the channel (D) h ) refers to the hydraulic diameter of the channel between the columns ( Figure 1 ), and the average liquid velocity (ū) refers to the average velocity between the columns.

[0124] Re c =ūD h / ν

[0125] In some embodiments of the invention, the flow of liquid within the channel is laminar in at least one region where the channel Reynolds number (Re) of the liquid flow is... c The number of the liquid in the channel is at least 100, but not more than 2000. In some embodiments, the flow of liquid within the channel is laminar in at least one region, and the channel Reynolds number (Re) is the number of the liquid flow in that region. c The minimum is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or approximately 2000.

[0126] In a preferred embodiment of the invention, the flow of the liquid is influenced by one or more diverters within the channel. As used herein, a “diverter” is any element or component that causes the flow of liquid through the channel to be diverted in a localized region, thereby creating a localized region of reduced pressure, optionally coupled to an unstable flow.

[0127] In a specific embodiment, a diverter is an obstruction placed within the channel. The term "obstruction" refers to any object placed within the channel that causes the flow of liquid to be diverted around the object, thereby creating a localized area of ​​pressure reduction or a pressure reduction coupled to an unstable flow substantially immediately downstream of the obstruction. The obstruction must allow cells to pass through the channel beyond it. In a preferred embodiment, the obstruction may extend outward from the inner surface of the channel in a direction generally perpendicular to the length of the channel. The obstruction may extend from one side of the channel to the other. Alternatively, the obstruction may extend only partially from one side of the channel.

[0128] In some embodiments of the invention, the obstacle has a width between 10 nanometers and 1 millimeter, and all integer widths therebetween. In preferred embodiments, the obstacle has a width greater than 50 nanometers, greater than 100 nanometers, greater than 500 nanometers, greater than 800 nanometers, greater than 1 micrometer, greater than 10 micrometers, greater than 50 micrometers, greater than 100 micrometers, greater than 200 micrometers, greater than 500 micrometers, greater than 800 micrometers, or about 1 millimeter. In preferred embodiments, the obstacle has a width less than 1 millimeter, less than 800 micrometers, less than 500 micrometers, less than 200 micrometers, less than 100 micrometers, less than 50 micrometers, less than 10 micrometers, less than 1 micrometer, less than 800 nanometers, less than 500 nanometers, less than 100 micrometers, or less than 50 nanometers. In a particularly preferred embodiment, the obstacle width is about 20 μm.

[0129] In a specific implementation, the obstacle is a pillar. In the context of this invention, an obstacle called a "pillar" can be a prism with a height greater than or equal to its maximum width. The pillar can be cylindrical, triangular, square, polygonal, wing-shaped, or any other shape, and a specific shape can be selected to adjust for a given Reynolds number (Re). c The instantaneous pressure reduction and / or the Reynolds number for a given object o Unstable flow. In a particularly preferred embodiment, the column is cylindrical.

[0130] The average velocity of the flow through the channel and directly upstream of the splitter can induce a transient pressure drop just downstream of the splitter, or induce a transient pressure drop and a localized region of unstable flow just downstream of the splitter. In an embodiment of the invention where the splitter is a column, the Reynolds number (Re) of the flow of the liquid surrounding the column can be used. o To calculate the appropriate induced average upstream velocity, a Rei of at least 40 is required for the flow of liquid around a cylindrical column. o (Re o ≥40) to induce unstable flow downstream of the column. For other column geometries, the Re required to generate unstable flow is... o This will depend on the specific shape of the column and will require adjustment of the average upstream liquid velocity to produce (1) a sufficiently large instantaneous pressure drop; or (2) unsteady flow and a sufficiently large instantaneous pressure drop. o It is defined as the ratio of the average upstream velocity (ū) and characteristic length of the column (l) as shown below to the kinematic viscosity (ν) of the fluid.

[0131] Re o =ūl / ν

[0132] In certain embodiments of the invention, the body Reynolds number (Re) of the liquid flow in at least one region where the liquid flow within the channel is unstable. o The number of the object's Reynolds number is at least 40, but not more than 2000. In some embodiments, the flow of the liquid within the channel is unstable in at least one region where the flow of the liquid is of unstable quality. o The number of the liquid flow is at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or about 2000. In a preferred embodiment, the Reynolds number of the liquid flow in at least one region where the liquid flow within the channel is unstable is the object Reynolds number (Re). o The values ​​are less than 50, less than 60, less than 70, less than 80, less than 90, less than 100, less than 200, less than 300, less than 400, less than 500, less than 600, less than 700, less than 800, less than 900, less than 1000, less than 1100, less than 1200, less than 1300, less than 1400, less than 1500, less than 1600, less than 1700, less than 1800, less than 1900, or less than 2000.

[0133] While not wishing to be bound by any particular theory, in embodiments of the invention where the unstable flow is localized substantially immediately downstream of the shunt, the cell may be exposed to a bidirectional increase in pressure: (1) a localized increase in pressure caused by the unstable flow; and (2) a pressure increase following a momentary decrease in pressure. This can generate a pressure drop across the permeable cell membrane, where the extracellular pressure is greater than the intracellular pressure and can facilitate the active delivery of exogenous substances near the cell membrane and / or the introduction of exogenous substances into the cell, for example, by diffusion or flow from the local extracellular environment into the cytosol.

[0134] The placement of any obstructions within the channel will generally create regions of the channel with pathways or gaps through which cells must pass, said regions being smaller in height, width, or diameter than other regions of the channel. However, it should be understood that these smaller regions must still be constructed such that fluid containing exogenous substances and cells can still flow through them. To facilitate this, any gaps created within the channel by the obstructions that allow fluid containing exogenous substances and cells to flow through will preferably be at least 1.01 times the minimum diameter of said cells. It should be understood that cells are generally not perfectly spherical, and therefore, the minimum diameter of the cell will be the minimum width of the cell when the shortest cross-section is made to pass through it.

[0135] In a specific embodiment of the invention, the gap has a width and height or diameter that is at least 1.01 times the minimum diameter of the cell. In other embodiments, the gap has a width and height or diameter that is at least 2, 5, 10, or 100 times the minimum diameter of the cell.

[0136] The present invention also relates to an apparatus for introducing exogenous substances into cells in a liquid, the apparatus comprising a channel having dimensions configured to allow the cells and exogenous substances suspended in the liquid to flow through therethrough; and one or more diverters within the channel; wherein the diverters result in at least one region of pressure reduction immediately downstream of the diverters.

[0137] In a specific embodiment of the present invention, the device is a microfluidic device.

[0138] Suitably, the pharmaceutical compositions of the present invention comprise suitable pharmaceutically acceptable carriers, diluents, cryoprotectants, or excipients. Preferably, the pharmaceutically acceptable carriers, diluents, or excipients are suitable for administration to mammals, and more preferably to humans. “Pharmaceutically acceptable carrier” means a pharmaceutical mediator comprising a substance that is not biologically or otherwise undesirable, i.e., said substance can be administered to a subject together with a selected active agent without causing any or substantially adverse reactions. The carrier may include excipients and other additives such as diluents, detergents, colorants, wetting agents or emulsifiers, pH buffers, preservatives, etc. Helpful references describing pharmaceutically acceptable carriers, diluents, and excipients are Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991) and Remington: The Science and Practice of Pharmacy (Pharmaceutical Press, London, 22nd edition, 2012), which are incorporated herein by reference.

[0139] In embodiments considering cell suspensions, it should be understood that the liquid in the suspension can be the liquid used in the method of performing the invention thereon, with or without additional components. Cell suspensions can also refer to dried or alternatively freeze-dried formulations as understood in the art.

[0140] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods and materials are described. For the purposes of this invention, the following terms are defined below.

[0141] The article “a / an” is used in this text to refer to one or more of the grammatical objects of the article (i.e., “at least one”). By way of example, “an element” means one element or more elements. Unless otherwise explicitly stated, as used herein, the singular includes the plural (and vice versa).

[0142] “Approximately” means that the quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length varies from a reference quantity, level, value, number, frequency, percentage, size, quantity, weight, or length by as much as 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0143] In the context of this invention, the terms “comprise” and “comprising” should be interpreted in their inclusive meaning rather than their exclusive meaning, that is, in the sense of “including but not limited to”. Brief description of the attached diagram

[0145] Figure 1 An overview of the unit geometry of the device according to an embodiment of the present invention is shown.

[0146] Figure 2 An overview of the pressure changes that occur during simulation tests of an embodiment of the method of the present invention is shown.

[0147] Figure 3 This is an overview of experimental transfection data obtained at 20x magnification using fluorescence microscopy (left) and optical microscopy (right), in which HEK293 cells were transfected with pcDNA 3.1 according to the parameters shown in Table 1. The upper image is a color illustration, while the lower image is a black-and-white illustration of the color image.

[0148] Figure 4 According to one embodiment of the present invention, it includes three columns (n c =3) and four-row pillars (n r A schematic diagram of a microfluidic device (=4).

[0149] Figure 5 According to another embodiment of the invention, it includes three columns (n) c=3) and four-row pillars (n r A schematic diagram of a microfluidic device (=4).

[0150] Figure 6 According to another embodiment of the invention, it includes three columns (n) c =3) and four-row pillars (n r A schematic diagram of a microfluidic device (=4).

[0151] Figure 7 According to another embodiment of the invention, it includes three columns (n c =3) and four-row pillars (n r A schematic diagram of a microfluidic device (=4).

[0152] Figure 8 Figure A is a cross-sectional view of a device design according to a preferred embodiment of the present invention. Figure B is an exploded view (3x magnification) of the array design, while Figure B is an exploded view (9x magnification) of the column design present on the array.

[0153] Figure 9 Figure A is a cross-sectional view of the device design according to another preferred embodiment of the present invention. Figure A is an exploded view of the array design (3x magnification), while Figure B is an exploded view of the column design present on the array (9x magnification).

[0154] Figure 10 Figure A is a cross-sectional view of another preferred embodiment of the invention. Figure A is an exploded view of the array design (3x magnification), while Figure B is an exploded view of the column design present on the array (9x magnification). Figure 10 The illustrations in the image are not drawn to scale.

[0155] Some drawings include color representations or solids. Color illustrations are available upon request from the applicant or the appropriate patent office. If obtained from the patent office, a fee may be charged.

[0156] Detailed Description of Preferred Embodiments of the Invention

[0157] While the invention has been described with reference to certain embodiments detailed herein, other embodiments may achieve the same or similar results. Variations and modifications of the invention will be apparent to those skilled in the art, and the invention is intended to cover all such modifications and equivalents.

[0158] The invention is further described through the following non-limiting embodiments.

[0159] Example 1

[0160] By using pcDNA 3.1 (Invitrogen) expressing green fluorescent protein (GFP) TMThe method and apparatus of the present invention were evaluated using transfected cell models. The apparatus used was a microfluidic device configured with an array of columns, wherein the gaps between the columns were larger than the cell diameter.

[0161] method

[0162] Simulation experiments and analysis

[0163] Simulations were conducted using computational fluid dynamics (CFD) with the finite volume method to target the parameters shown in Table 1 and Figure 1 The geometry of the device shown examines the microenvironment around the gaps between the columns. Figure 1 An overview of the unit geometry of the device according to the invention is shown, wherein a liquid having a velocity (Q) and a suspension in the liquid having a diameter (d) c The cells together enter the closed channel at the entrance, where dc is less than the gap width (g). Other variables represent the column diameter (d). p The channel width (w) and channel height (h) were determined. For the purpose of solving the problem, a 3D geometry was constructed in SolidWorks with an inlet length of 100 μm and an outlet length of 1000 μm. A structured mesh was built in ICEM CFD 14.5, and element quality was checked using determinant, angle, and aspect ratio. The solution was obtained using ANSYS FLUENT 14.5 on a Windows 7 Enterprise 64-bit computer with a 3.20 GHz Intel Core i5-3470 CPU and 16.0 GB RAM. A coupled pressure-velocity solver was used to solve for the velocity, pressure, and shear stress profiles. The channel Reynolds number (Re) was calculated using the contracted internal dimensions and the following equation based on the parameters in Table 1. C ):

[0164] Re C =2ρQ / μ(g+h)

[0165] The boundary conditions for the top, bottom, and column-defined walls of the channel are set to no slip. The boundary conditions for the fluid sidewalls are set to zero shear. The inlet velocity is defined by the average velocity, and the outlet is set to zero pressure boundary conditions.

[0166] Table 1: Overview of Experimental Parameters

[0167]

[0168]

[0169] transfection

[0170] The master mold of the microfluidic device was fabricated using standard photolithography, while the device was replicated using soft photolithography and bonded to glass using oxygen plasma. An overview of the device design and transfection parameters is provided in [the respective sections]. Figure 1 As shown in Table 1.

[0171] HEK293 (human embryonic kidney 293) cells were loaded at a rate of 1 x 10⁻⁶. 5 cells / ml -1 The density was suspended in cell culture medium and at 890 ng / ml -1 The pcDNA 3.1GFP plasmid was inoculated at a density of [insert density here]. This suspension was loaded into a syringe and dispensed at 5 ml / min. -1 The flow rate is pumped into the microfluidic device, which corresponds to a Re of 375 at the gap between the columns. c This is because the flow cell contains an array of 8 units separated by pillars with a diameter of 20 μm, and the gap between the pillars is 30 μm. This also corresponds to Re 131. o Subsequently, the cells were incubated for 6 days, and then examined for green fluorescent protein gene expression using fluorescence and optical microscopy imaging.

[0172] result

[0173] Simulation experiments and analysis

[0174] Simulation tests show that the high-voltage region is exactly in Figure 1 The decompression zone occurs upstream of the column in the apparatus, and precisely downstream of the column. This means that as cells flow through the column, they are exposed to sudden and transient pressure drops. Furthermore, these simulations are run as transients to determine if unstable flow occurs. Figure 2 An overview of the pressure changes occurring during simulation tests of an embodiment of the method of the present invention is shown, illustrating (a) pressure profiles, (b) velocity magnitudes, (c) the x-direction velocity of the liquid, which can be used to approximate cellular velocities, and (d) the y-direction velocity of the liquid with alternating jets due to unsteady flow. Figure 2 As shown, there is a significant flow velocity in the x direction of the overall flow perpendicular to the y direction within the closed channel, which means that an unstable flow has occurred.

[0175] According to simulation experiments, when cells pass through the gaps between columns positioned within the closed channels of the device, they migrate from the surrounding area under a localized pressure of 43.5 kPa, and are exposed to a transient pressure drop of 94.3 kPa as they enter a relatively low-pressure region (with a minimum pressure of -50.8 kPa). The magnitude of the transient pressure drop can vary depending on the phase of the oscillation. Furthermore, during this transient pressure drop, the cell velocity in the liquid is estimated to be 15 ms. -1 For -35.4x106 kPa s -1 Instantaneous pressure reduction (dP / d t It occurs within a distance of approximately 40 μm, where dP / d t It is relative to the change over time (d) t The pressure change (dP) is the pressure change between a local maximum and a local minimum. t It is the time variation between the local maximum pressure and the local minimum pressure.

[0176] Subsequently, as Figure 2 As shown in d, the unsteady flow condition causes the cell to rapidly change its flow velocity in the y-direction orthogonal to the direction of cell movement (x-direction), with the peak y-direction velocity occurring at -8.5 ms. -1 Up to 8.3ms -1 Within a certain range, and these localized unstable flows are approximately 20 μm wide. The amplitude of the localized unstable flows decays as the cell moves away from the column, and within approximately 500 μm (in this space, the cell is subjected to approximately 5 unstable flow pulses with velocity amplitudes between 3.4 ms), the range is wide. -1 With 8.5ms -1 After a period of time (between 10 ms), the cell rate completely decays. During this period, the cell rate is estimated to be between 10 ms. -1 The pulse duration is between 15 m s⁻¹ and approximately 20 μm, indicating that the pulse duration is in the range of 2.0 μs to 1.3 μs. After the cell is pulsed with an instantaneous pressure decrease and an unstable flow, the pressure increases to the same level as the outlet pressure when the cell leaves the microfluidic device or moves away from the gap.

[0177] Simulation experiments show that exposure to unsteady flow generates a pressure drop across the cell membrane, where the local extracellular pressure is greater than the local intracellular pressure, thereby promoting active (mechanical) delivery. Furthermore, the increase in pressure as the cell moves toward the device outlet is conducive to promoting active delivery due to the pressure drop across the permeable cell membrane.

[0178] transfection

[0179] like Figure 3 As shown, transient pressure reduction and unstable flow conditions through the column array can be used to transfect HEK293 cells with the pcDNA3.1 GFP plasmid. The images in the top row and bottom row are taken from the same field of view. The bright spots in the images on the left side of the figure represent HEK293 cells successfully transfected with pcDNA 3.1, which are viable and continue to express green fluorescent protein 6 days after transfection.

[0180] The simulation experiments allow for unsteady flow, and preliminary simulation experiments are used to determine the parameters for calculating Re based on the transition from laminar to unsteady flow conditions. o The most appropriate speed. Used to calculate Re. o The velocity of the liquid varies in the literature; however, previous simulations have confirmed that the average upstream velocity is appropriate. For example, for liquid flow around a cylindrical column, the Reynolds number of the object (Re) is... o The following equation can be used to calculate:

[0181] Re o =ρv∞d / μ

[0182] Where v∞ refers to the velocity of the liquid relative to the bulk of the cylindrical column, and in this case, it refers to the average upstream velocity of the liquid ahead of the cylindrical column. For the parameters shown in Table 1, this would be 8.68 ms. -1 This results in a Re of 131.2. o .

[0183] To estimate the frequency of the oscillation, the correlation shown below is used because it applies to liquid flow around a cylindrical column, where Re o Between 40 and 190. The Stockhal number (Sr) (a dimensionless number used to describe unstable flow) can be obtained from Re. o The calculations, where the flow around the cylindrical column has the following correlations:

[0184] For (40) <Re o <190), Sr=0.2665–1.018 / √Re o

[0185] This calculation yields a Sr of 0.17, and can be expressed using the following equation with the liquid velocity (v) and characteristic length (L) (which is equal to the diameter of the column (d)). p )) Calculate the oscillation frequency (f):

[0186] f = Sr v / d p

[0187] For the parameters described above, the unstable flow is estimated to oscillate at a frequency of 44.4 kHz. These unstable oscillations are also known to induce structural vibrations within the column itself. Therefore, it is believed that the cell can be exposed to transient pressure drops, an unstable flow of 44.4 kHz, and induced structural vibrations.

[0188] One or more flow dividers, such as (but not limited to) laminar flow between columns (Re >> 1), can be used to create a region of instantaneous pressure reduction substantially immediately downstream of the columns. This can be used when cell flow passes through, for example... Figure 1 and Figures 4 to 10The devices shown in the diagram suddenly and temporarily reduce the environmental stress around the cell. Additionally, if Re... o >40, then these flow characteristics are known to induce unstable flow, and in the above examples, this is manifested by (1) instantaneous pressure reduction and (2) unstable flow pulses in the cell. Furthermore, this can be achieved using channel sizes larger than the cell size (g>d). c This is achieved to alleviate clogging problems. This facilitates the transfer of exogenous substances across the cell membrane and into the cytoplasm. According to Pawell et al. (Pawell RS, et al. (2013). Limits of parabolic flow theory in microfluidic particle separation: a computational study. 4th ASME International Conference on Micro / Nanoscale Heat and Mass Transfer, Hong Kong, December 11-14, 2013). For channels between columns with a Reynolds number greater than 100, c >100), which produces a region of negligible shear stress. That is, under these conditions, membrane permeation is not due to shear stress, suggesting that transfection may be the result of transient pressure reduction and unsteady flow conditions along with any conditions induced by unsteady flow, such as structural oscillations in the column, as observed by Renfer et al. (Renfer A., ​​et al. (2013) Vortex shedding from confined micropostarrays. Microfluidics and Nanofluidics. 15(2):231-242).

[0189] Example 2

[0190] Experiments were conducted to investigate how the magnitude and duration of stress reduction affected the degree of transfection.

[0191] method

[0192] Two cultures of HEK293 cells were cultured at a concentration of 100,000 cells per ml. -1 The density of inoculation was such that culture 1 contained approximately 900 ng of 10 -5 HEK293 cells were seeded at a density of 3 cells per cell and green fluorescent protein pcDNA 3.1 was added. Culture 2 contained 100 ng of 10 -5 HEK293 cells and 25-base-pair oligonucleotides were seeded at a density of [number] cells. Both cultures were placed in a vacuum desiccator, and the pressure was reduced to -95 kPa over a 2-minute process. The vacuum was then released and restored to atmospheric pressure over a 10-second process.

[0193] Results and discussion

[0194] This experiment using prolonged pressure reduction resulted in no transfection. No cells expressed GFP, and colocalization of oligonucleotides with cells was negligible. The magnitude of the pressure reduction was significantly larger (95 kPa reduction, compared to the 20 kPa reduction in Example 1). However, the rate of decrease was significantly slower. In Example 1, the estimated rate of transient pressure reduction (dP / dt) was -35.4 x 10⁻⁶. 6 kPa s -1 In this embodiment, dP / dt is approximately -0.8 kPa·s. -1 Therefore, when the cell membrane is gas-permeable, dP / dt plays a role in permeation of the cell membrane, such that if dP / dt is too low, gas transfer will occur naturally across the cell membrane without requiring permeation. Once dP / dt is sufficient, it is assumed that the physical properties of the cell membrane will not be able to accommodate rapid gas transfer from the intracellular to the extracellular environment. Therefore, the cell membrane can be pressurized to the points where pores form, thereby allowing the introduction of exogenous substances into the cell.

[0195] Example 3

[0196] Figure 4 It includes three columns (n) c =3) and four-row pillars (n r A schematic diagram of a microfluidic device (d = 4). The array is configured such that the diameter of the column (d) p ) equals the gap between the columns (g)(d) p =g), and shift the column of each column by a sufficient distance to cause the flow from the previous gap to bifurcate, where the displacement distance (s) is equal to half the row spacing (s = p). r / 2), and column spacing (p c ) equals line spacing (p c =p r ). Channel width, number of columns (n) c ) and the number of rows (n r This will vary depending on the specific device using this or a similar design.

[0197] Figure 5 It includes three columns (n) c =3) and four-row pillars (n r A schematic diagram of a microfluidic device (d = 4). The array is configured such that the diameter of the column (d) p The distance between the columns is greater than the gap (g), and the columns in each column are shifted a sufficient distance to cause the flow from the previous gap to bifurcate, where the displacement distance (s) is equal to half the row spacing (s = p). r / 2), and column spacing (p c ) equals line spacing (p c =pr )。 The width of the channels, the number of columns (n c ) and the number of rows (n r ) will vary for each specific device using this or a similar design.

[0198] Figure 6 is a schematic diagram of a microfluidic device including three columns of pillars (n c = 3) and four rows of pillars (n r = 4). The array is constructed such that the diameter of the pillars (d p ) is less than the gap (g) between the pillars (d < g), and the pillars in each column are slightly shifted from the previous gap, where the displacement distance (s) is less than half of the row pitch (s < p r / 2), and the column pitch (p c ) is greater than the row pitch (p c > p r )。 The width of the channels, the number of columns (n c ) and the number of rows (n r ) will vary for each specific device using this or a similar design.

[0199] Figure 7 is a schematic diagram of a microfluidic device including three columns of pillars (n c = 3) and four rows of pillars (nr = 4). The array is constructed such that the diameter of the pillars (d p ) is less than the gap (g) between the pillars (d p < g), and the pillars in each column are slightly shifted from the previous gap, where the displacement distance (s) is less than half of the row pitch (s < p r / 2), and the displacement direction changes in each row. The column pitch (p c ) is greater than the row pitch (pc > p r )。 The width of the channels, the number of columns (n c ) and the number of rows (n r ) will vary for each specific device using this or a similar design.

[0200] Preferred embodiments of the device design of the present invention are shown in Figure 8 , 9 and 10. Both embodiments include a single inlet and a single outlet with different internal pillar shapes, which are specifically shown in FIGS. A and B of each figure. In these embodiments, all substrates are fused silica with a substrate thickness (t s ) of 700 μm. The unit includes a cover having 2 through-holes, each hole having a diameter (D h ) of 700 μm. The bonding strength or burst pressure of the cover and the substrate should be greater than (>>) 10 atmospheres, and once bonded, the total thickness of the device (t dThe 4.80mm x 9.80mm device footprint represents a 200μm cut width. Considering a 7x6 arrangement of devices across a 70mm x 30mm fixture, a total of 42 devices are possible. The bottom component of the device is deep reactive ion etched fused silica, which is bonded to the laser-mechanized fused silica wafer using bulk material bonding. For Figure 8 and Figure 9 In the embodiment shown, the etched substrate will form a channel with a width of 1.5 mm, a length of 7.5 mm, and a depth of 40.0 μm. For Figure 10 In the embodiment shown, the etched substrate will form a channel with a width of 0.6 mm, a length of 5.5 mm, and a depth of 40.0 μm.

[0201] according to Figure 8 The embodiment shown, the array design (Figure A), includes thirty (30) pillars (n) in the x-direction. x ) and a row of columns (n) in the y direction (1) y ), where the array spacing in the x-direction is 50.0 μm (P x Also known as column spacing p c In this embodiment, the column design shown in Figure B is configured such that the diameter (d) of the column is... p =20μm) is smaller than the 30.0μm gap between columns present in this embodiment (gap = P x -d p ).

[0202] according to Figure 9 The embodiment shown, the array design (Figure A), includes thirty (30) pillars (n) in the x-direction. x ) and three (3) rows of columns (n) in the y direction y ), where the array spacing in the x-direction is 50.0 μm (P x Also known as column spacing p c ), and the array spacing in the y direction is 750 μm (Py; also known as row spacing p). r In this embodiment, the column design shown in Figure B is configured such that the diameter (d) of the column is... p =20μm) is smaller than the 30.0μm gap between columns present in this embodiment (gap = P x -d p ).

[0203] according to Figure 10 The embodiment shown, the array design (Figure A), includes twelve (12) pillars (n) in the x-direction. x ) and three (3) rows of columns (n) in the y direction y), where the array spacing in the x-direction is 50.0 μm (P x Also known as column spacing p c ), and the array spacing in the y direction is 500 μm (Py; also known as row spacing p). r In this embodiment, the column design shown in Figure B is configured such that the diameter (d) of the column is... p =20μm) is smaller than the 30.0μm gap between columns present in this embodiment (gap = P x -d p ).

[0204] Suitable scope for the particularly preferred embodiments of the invention as shown in the figures is provided below:

[0205] Column diameter range (d) p ): 10nm–5mm;

[0206] Number of columns (n) c ): 1–10,000;

[0207] Number of rows (n r ): 3–10,000;

[0208] Gap range (g): 10nm–5mm;

[0209] Displacement (s): 0–5 mm;

[0210] Column spacing (p) c ): 30nm–50mm; and

[0211] line spacing (p) r 30nm–50mm

[0212] Every patent, patent application, and publication cited in this document is hereby incorporated herein in its entirety by reference.

[0213] Any reference cited herein shall not be construed as an admission that the reference is available as "prior art" of this application.

[0214] Throughout this specification, the purpose is to describe preferred embodiments of the invention without limiting it to any single embodiment or particular set of features. Therefore, those skilled in the art will understand that, in view of this disclosure, various modifications and variations can be made to the illustrative specific embodiments without departing from the scope of the invention. All such modifications and variations are intended to be included within the scope of the appended claims.

Claims

1. A method for introducing an exogenous substance into cells, the method comprising: Liquid is introduced into the flow channel of a microfluidic device, the channel including one or more diverters, each diverter having a gap between each diverter, the gap between each diverter having a width greater than the diameter of the cell; and As the cells flow through the diverter, they are exposed downstream of the diverter to a transient pressure drop and unstable flow, thereby introducing the exogenous substance into the cells; wherein, The Reynolds number of the fluid flowing around the distributor is 40-2000; The cells survived after exposure to the transient pressure drop and unstable flow; The instantaneous pressure reduction is a reduction of at least 10 kPa; The cells are exposed to the instantaneous pressure reduction in the presence of the exogenous substance for at least 10 nanoseconds.

2. The method of claim 1, wherein the cell is selected from the group consisting of bacterial cells, mammalian cells, yeast cells, plant cells, and insect cells.

3. The method of claim 1 or 2, wherein the exogenous substance is selected from the group consisting of: small organic molecules, nucleic acids, nucleotides, oligonucleotides, proteins, peptides, amino acids, lipids, polysaccharides, quantum dots, carbon nanotubes, nanoparticles, gold particles, monosaccharides, vitamins, and steroids.

4. The method of claim 3, wherein the exogenous substance is introduced into the cytoplasm of the cell.

5. The method of claim 1, wherein the shunt is a column.

6. The method of claim 5, wherein the column is cylindrical.

7. The method of claim 6, wherein the diameter of the column is greater than the gap between the columns.

8. The method of claim 1, wherein the shunt has a maximum width of 1 mm.

9. A microfluidic device for introducing exogenous substances into cells in a liquid, said cells having a diameter, said device comprising: A channel that is at least partially closed, the channel having a configuration that allows the cells and the exogenous substance suspended in the liquid to flow through it; as well as One or more diverters are located within the channel, each diverter having a gap between it, the width of which is configured to be greater than the diameter of the cell; The shunt causes at least one region of transient pressure drop and unstable flow immediately downstream of the shunt, such that the exogenous substance is introduced into the cell after the transient pressure drop; The Reynolds number of the fluid flowing around the distributor is 40-2000; The instantaneous pressure reduction is a reduction of at least 10 kPa; The cells are exposed to the transient pressure reduction in the presence of the exogenous substance for at least 10 nanoseconds, and the cells survive the exposure to the transient pressure reduction and the unstable flow.

10. The microfluidic device of claim 9, wherein the shunt is a column.

11. The microfluidic device of claim 10, wherein the column is cylindrical.

12. The microfluidic device of claim 10, wherein the diameter of the columns is larger than the gap between the columns.

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