WO2021000844A1 - Dispositif et procédé d'administration de substances exogènes dans des cellules eucaryotes et application associée - Google Patents

Dispositif et procédé d'administration de substances exogènes dans des cellules eucaryotes et application associée Download PDF

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WO2021000844A1
WO2021000844A1 PCT/CN2020/099054 CN2020099054W WO2021000844A1 WO 2021000844 A1 WO2021000844 A1 WO 2021000844A1 CN 2020099054 W CN2020099054 W CN 2020099054W WO 2021000844 A1 WO2021000844 A1 WO 2021000844A1
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cells
cell
piston
eukaryotic
unit
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PCT/CN2020/099054
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Chinese (zh)
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陈汉
刘玲蓉
杜博
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广州世赛生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present disclosure relates to the field of biotechnology, in particular to a device and method for delivering exogenous substances into eukaryotic cells and their applications.
  • Cytoplasmic and nuclear delivery are key steps in cell engineering, cell therapy research and development, and many biological functions.
  • Existing conventional intracellular delivery technologies include electroporation, viral vectors, or non-viral gene vectors.
  • electroporation often leads to higher cell mortality; viral vectors are immunogenic, and the construction of viral vectors that deliver specific sequence nucleic acids takes a long time and is low in efficiency; non-viral gene vectors have low delivery efficiency in suspension cells and primary cells. It takes a long time and is not suitable for delivering other biological macromolecules except deoxyribonucleic acid DNA and ribonucleic acid RNA (Stewart M P, Sharei A, Ding X, et al.
  • the prior art also has a porous membrane-based macromolecule delivery system that can deliver microcircle DNA to the nucleus of artificial hematopoietic stem cells (WO2018064387A1, CN201780060638.6) and express the GFP gene.
  • this delivery system has the following drawbacks: the single-channel device has a small processing volume of cell suspension (only 50 ⁇ L), high cell density in the suspension (up to -10 7 cells/ml) and the resulting low cell recovery rate (12% to 57%), and minicircle DNA (the number of base pairs is about 0.7kbp) transfection efficiency is extremely low (1% to 8.4%).
  • this disclosure has designed a device with a temperature control module, and conducted various macromolecule delivery and plasmid DNA transfection experiments.
  • the present disclosure provides a device for delivering exogenous substances into eukaryotic cells.
  • the device alleviates the deficiencies of the existing eukaryotic intracellular delivery technology, such as the inability to rupture and perforate the nuclear membrane of the cell.
  • a combination is required
  • Microfluidics and electric fields, as well as carrier-based intracellular delivery methods are only suitable for technical problems such as the delivery of specific molecules such as nucleic acids.
  • the present disclosure provides a device for delivering exogenous substances into eukaryotic cells.
  • This device can process a higher cell suspension volume and reduce cell density in a single delivery test, thereby obtaining better cell recovery and survival rates .
  • the present disclosure provides a device with a temperature control module, which can achieve higher delivery efficiency and higher cell recovery rate by changing the delivery temperature.
  • the present disclosure also provides a method for delivering exogenous substances into eukaryotic cells, the method comprising extruding a mixture of eukaryotic cells and exogenous substances through the device of the present disclosure.
  • the present disclosure provides an application of a high-throughput eukaryotic intracellular delivery device or a method for delivering exogenous substances into eukaryotic cells in regulating cell functions.
  • the present disclosure provides a device for delivering exogenous substances into eukaryotic cells.
  • the device includes an extrusion module in which a microporous membrane with regular pores is arranged, and the pore diameter of the pores is smaller than that of the true cell.
  • the diameter of the nuclear cell also includes a pressure unit for driving eukaryotic cells and exogenous substances through the pores at the same time; in one or more embodiments, the pressure unit includes a piston unit or a compressed gas drive unit.
  • the device further includes a temperature control module for controlling the environmental temperature of the extrusion module and the cell suspension system.
  • the present disclosure also provides a method for delivering exogenous substances into eukaryotic cells, the method comprising: extruding a suspension system containing eukaryotic cells and exogenous substances through the above-mentioned device, so that the exogenous substances are delivered to the eukaryotic cells.
  • Nuclear cell
  • the present disclosure also provides the application of the above-mentioned high-throughput eukaryotic intracellular delivery device, or the above-mentioned method of delivering exogenous substances into eukaryotic cells, in regulating cell functions.
  • the beneficial effects of the present disclosure include at least:
  • the device for delivering exogenous substances into eukaryotic cells is provided with an extrusion module in which a microporous membrane with regular pores is arranged, and the pore diameter of the pores is smaller than the diameter of the eukaryotic cell;
  • the output module also includes a piston unit for driving eukaryotic cells and exogenous substances through the pores at the same time;
  • the device also includes a temperature control module for controlling the environmental temperature of the extrusion module and the cell suspension system.
  • the cells suspended in the solution can be compressed and deformed in the process of passing through the pores, thereby causing the cell membrane and nuclear membrane to perforate, so that the exogenous materials dispersed or dissolved in the cell suspension enter the cytoplasm and nucleus.
  • the eukaryotic intracellular delivery method provided by the present disclosure overcomes the shortcomings of the existing intracellular delivery technology, such as the intracellular delivery technology based on nuclear pore membrane.
  • the cell processing volume is too small (50 ⁇ L) and the cell density is too high (up to -10 7 cells).
  • minicircle DNA transfection efficiency is extremely low (1% to 8.4%), and the cell recovery rate (12% to 57%) of minicircle DNA delivery is low, such as intracellular delivery based on microfluidics Technology cannot rupture and perforate the nuclear membrane of the cell; or in order to deliver plasmid DNA into the nucleus, a combination of microfluidics and electric fields are required, and intracellular delivery methods based on non-viral gene vectors are only suitable for the delivery of specific molecules such as nucleic acids.
  • the device for delivering exogenous substances into eukaryotic cells can not only deliver exogenous substances of various molecular weights into the cytoplasm, but also deliver a hydration radius larger than the upper limit of the diffusion of the nuclear pore complex (NPC) Exogenous substances, such as 2MDa FITC-dextran, are delivered into the nucleus with high efficiency. Therefore, it is shown that the present disclosure achieves cytoplasmic and nuclear delivery by simultaneously inducing rupture and perforation of cell membrane and nuclear membrane. Secondly, the device provided by the present disclosure can efficiently deliver plasmid DNA with a larger hydration radius into the cell nucleus and express the encoded protein.
  • NPC nuclear pore complex
  • the present disclosure can deliver various types of exogenous materials into the cytoplasm and nucleus without being restricted by the physical and chemical properties of the materials themselves, such as dextran and plasmid DNA.
  • the present disclosure does not require electric field assistance when delivering plasmid DNA into the nucleus and expressing the corresponding protein.
  • the device provided by the present disclosure is suitable for high-throughput cell-containing system through the microporous membrane, a single treatment of cell-containing suspension can reach at least 0.5 mL, and the total number of cells extruded at one time can reach at least 0.6 ⁇ 10 6 pcs.
  • the present disclosure also provides a method for delivering exogenous substances into eukaryotic cells, the method comprising extruding a mixture of eukaryotic cells and exogenous substances through the aforementioned device. Therefore, it has all the beneficial effects of the above device, which will not be repeated here.
  • Figure 1 is a schematic diagram of the principle of a device for delivering foreign substances into eukaryotic cells provided by the present disclosure
  • FIG. 2A is a polyethylene terephthalate (PET) nuclear porous membrane used in Examples 1-3 of the present disclosure
  • Figure 2B shows the pores on the PET nuclear porous membrane used in Examples 1-3 of the present disclosure
  • 2C is a device for delivering exogenous substances into eukaryotic cells provided in Example 1 of the present disclosure
  • Fig. 3 is a device for delivering exogenous substances into eukaryotic cells provided in Example 2 of the disclosure
  • Example 4 is a device for delivering exogenous substances into eukaryotic cells provided in Example 3 of the disclosure
  • 5A is a confocal picture of 70kDa glucan delivered to CT26 cytoplasm and nucleus in Example 4 of the disclosure;
  • Figure 5B is a confocal picture of 2MDa glucan delivered to CT26 cytoplasm and nucleus in Example 4 of the disclosure;
  • 6A is the flow cytometry data of the delivery efficiency of 70kDa glucan to CT26 cells in Example 4 of the disclosure
  • 6B is the flow cytometry data of the delivery efficiency of 2MDa glucan to CT26 cells in Example 4 of the disclosure
  • Figure 7A is the flow cytometric data of the delivery efficiency of 70kDa glucan to K562 cells in Example 5 of the disclosure
  • Example 7B is the flow cytometry data of the delivery efficiency of 2MDa glucan to K562 cells in Example 5 of the disclosure.
  • Figure 8 is a confocal picture of pCMV-GFP plasmid delivered to K562 cell nucleus and expressing GFP protein in Example 6 of the disclosure;
  • Figure 9 is the flow cytometry data of pCMV-GFP plasmid delivered to K562 cell nucleus and expressing GFP protein in Example 6 of the disclosure;
  • Figure 10 is the flow cytometry data of pCMV-GFP plasmid delivered to CT26 cell nucleus and expressing GFP protein in Example 7 of the disclosure;
  • Figure 11 is a confocal picture of the delivery of 2MDa glucan to K562 cytoplasm and nucleus provided in Example 8 of the disclosure;
  • Figure 12 is the flow cytometric data of 2MDa glucan delivered to CT26 cells provided in Example 9 of the disclosure.
  • FIG. 13 is the cell recovery rate after extrusion treatment of the CT26 suspension with different density provided in Example 10 of the disclosure.
  • Figure 14 shows the intracellular delivery efficiency of K562 cells with different molecular weight exogenous substances provided in Example 11 of the disclosure under the same pore size nuclear pore membrane condition;
  • 15A shows the delivery efficiency and cell recovery rate of 2MDa FITC-dextram in CT26 cells at different temperatures provided in Example 12 of the disclosure
  • 15B shows the transfection efficiency of pDNA in CT26 cells at different temperatures provided in Example 12 of the disclosure
  • the present disclosure provides a device for delivering exogenous substances into eukaryotic cells.
  • the device includes an extrusion module in which a microporous membrane with regular pores is arranged, and the pore diameter of the pores is smaller than that of the true cell.
  • Nuclear cell diameter; the extrusion module also includes a piston unit for driving eukaryotic cells and foreign substances through the pores at the same time.
  • the extrusion module is driven by the piston unit to deform the cells during the process of passing through the pores, thereby causing the cell membrane and the nuclear membrane to perforate and increase the permeability, so that the foreign substances scattered around the cell enter the cell and the nucleus.
  • Regular pores refer to the uniform shape of the pores on the microporous membrane, which are round or nearly round. Since the device for delivering exogenous substances into cells provided in the present disclosure is suitable for the passage of higher flux cells, since the number of cells passing through the extruded membrane each time is large, if the pores on the microporous membrane are irregular, the cross-section There is a greater difference in shape, the cell death rate will be higher.
  • the "cell” described in the present disclosure also refers to "eukaryotic cells”. It can be understood that the device also includes a storage space before the cells and foreign substances pass through the microporous membrane, and a receiving space for receiving the extrudate after extrusion.
  • the present disclosure does not limit the shape, volume and material of the storage space and the receiving space, as well as the connection mode of the storage space, the microporous membrane and the receiving space, as long as the cells and foreign substances can pass from the storage space through the microporous membrane, and from The flow between the microporous membrane and the receiving space is sufficient. It is understandable that the device can be used independently or in conjunction with other devices to form a system.
  • the temperature of the extrusion module is 28-31°C, such as 28-30°C, such as 30°C. In one or more embodiments, the temperature of the extrusion module is, for example, 28.2°C, 28.4°C, 28.6°C, 28.8°C, 29°C, 29.2°C, 29.4°C, 29.6°C, 29.8°C, or 30°C.
  • the eukaryotic cell concentration in the suspension system containing eukaryotic cells and exogenous substances is 1 ⁇ 10 6 cells/ml to 4 ⁇ 10 6 cells/ml, for example, 1 ⁇ 10 6 cells/ml. 10 6 cells/ml to 2 ⁇ 10 6 cells/ml.
  • the eukaryotic cell concentration in the suspension system containing eukaryotic cells and exogenous substances is 1.2 ⁇ 10 6 cells/ml, 1.4 ⁇ 10 6 cells/ml, 1.6 ⁇ 10 6 cells/ml, 1.8 ⁇ 10 6 cells/ml, 2.0 ⁇ 10 6 cells/ml, 2.2 ⁇ 10 6 cells/ml, 2.4 ⁇ 10 6 cells/ml, 2.6 ⁇ 10 6 cells/ml , 2.8 ⁇ 10 6 cells/ml, 3.0 ⁇ 10 6 cells/ml, 3.2 ⁇ 10 6 cells/ml, 3.4 ⁇ 10 6 cells/ml, 3.6 ⁇ 10 6 cells/ml, 3.8 ⁇ 10 6 Cells/ml or 4.0 ⁇ 10 6 cells/ml.
  • the material of the microporous membrane includes metal or non-metal. In one or more embodiments, the material of the microporous membrane includes non-metal, and the non-metal includes, but is not limited to, polymer, ceramic, or silicon.
  • the material of the microporous membrane is polymer.
  • the microporous membrane prepared by using the polymer as the substrate has uniform pore distribution, and the microporous membrane is lighter and thinner.
  • the polymer includes but is not limited to polyethylene terephthalate, polytetrafluoroethylene, polycarbonate, polyimide, polyamide, cellulose acetate, nitrocellulose, polyethylene, polyethersulfone or Polytetrafluoroethylene; for example, polyethylene terephthalate or polyimide or polycarbonate.
  • the polymer is selected from polyethylene terephthalate, polytetrafluoroethylene, polycarbonate, polyimide, polyamide, cellulose acetate, nitrocellulose, polyethylene, polyethersulfone, and polyethersulfone. At least one of the group consisting of tetrafluoroethylene.
  • the method for preparing the pores on the microporous film includes etching or perforating.
  • the etching is preferably track etching or photoetching, and the photoetching is preferably laser etching; and punching is preferably used for perforating.
  • the microporous film is prepared by track etching, which has better effect.
  • nuclear pore membrane is also called “nuclear track membrane” or “nuclear track-etch membrane”, which generally refers to radiation track-chemical engraving Non-metallic film obtained by etching technology.
  • Nuclear porous membranes have unique microporous characteristics and uniform pore size, and are widely used in the field of membrane separation.
  • the microporous membrane is a nuclear porous membrane.
  • the piston unit applies positive pressure to the cells and exogenous substances, and the piston unit may be manually driven by hand or driven by a driving unit. In one or more embodiments, it can also be driven by compressed gas.
  • the piston unit includes a piston tube and a piston push rod.
  • the piston unit includes a driving unit for driving the piston push rod, and the driving unit can select a conventional unit capable of providing power, for example, using a motor to drive the piston to move, so as to apply cells and foreign substances. Positive pressure towards the microporous membrane.
  • the material of the piston tube body includes metal materials, alloy materials, or non-metallic materials other than glass; in one or more embodiments, the metal material includes aluminum or copper; In various embodiments, the alloy material includes stainless steel or titanium-magnesium alloy.
  • the piston push rod is mainly made of natural polymer materials or synthetic polymer materials.
  • the natural polymer material is rubber.
  • the synthetic polymer material is polyester.
  • the synthetic polymer material is polytetrafluoroethylene and/or polyethylene.
  • a polyethylene terephthalate material is used to prepare the piston push rod.
  • a piston unit composed of polyethylene or polytetrafluoroethylene is used; in one or more embodiments, a piston unit made of polyethylene is selected. Synthetic polymer materials have good mechanical strength, corrosion resistance and smooth surface, especially polyester, polyethylene and polytetrafluoroethylene.
  • the device further includes a temperature control module, which is used to provide a constant temperature to the extrusion module, so that cells and foreign substances can be extruded under a specified temperature condition.
  • the temperature control module is in contact with the extrusion module, so that the temperature is more directly transferred to the extrusion module to control the temperature of the extrusion module.
  • heating metal wires, ceramic heaters, semiconductor refrigeration fins or fluid circulation temperature control units are arranged on the outer wall of the space for storing cells and exogenous substances in the extrusion module.
  • the fluid circulation temperature control unit is provided with a cavity in contact with the extrusion module, and the cavity is used for fluid circulation in the fluid circulation temperature control unit.
  • the device further includes a temperature control module configured to control the eukaryotic cells and exogenous substances in the form of the extrusion module and the suspension by contacting the extrusion module temperature.
  • the temperature control module includes a fluid circulation temperature control unit or a semiconductor refrigeration unit.
  • the fluid circulation temperature control unit is provided with a cavity in contact with the extrusion module, and the cavity is configured for fluid circulation in the fluid circulation temperature control unit.
  • the pressure unit is a piston unit, and the piston unit is configured to drive eukaryotic cells and foreign substances in a suspension form through the pores at the same time.
  • the piston unit is configured to apply a positive pressure to the cells and the foreign substance, so that the cells and the foreign substance pass through the pores.
  • the piston unit includes a piston tube and a piston push rod.
  • the piston unit includes a driving unit for driving the piston push rod.
  • the microporous membrane is detachably connected to the piston tube body.
  • the material of the piston tube body includes metal materials, alloy materials or non-metal materials other than glass.
  • the non-metallic material other than glass includes plastic.
  • the non-metallic materials other than glass include polymer materials.
  • the material of the piston push rod includes natural polymer materials or synthetic polymer materials.
  • the present disclosure also provides a method for delivering exogenous substances into eukaryotic cells, the method comprising extruding a suspension system of eukaryotic cells and exogenous substances through the above-mentioned device.
  • the suspension system described in the present disclosure refers to a system in which eukaryotic cells and exogenous substances are dispersed.
  • the dispersion medium of the system can be a conventional medium that can maintain the activity of eukaryotic cells, including but not limited to medium, physiological Saline and PBS buffer, etc.
  • the delivery described in the present disclosure refers to the introduction of foreign substances into cells, that is, foreign substances into the cytoplasm, or foreign substances into the cytoplasm and nucleus.
  • the method for delivering exogenous substances into eukaryotic cells provided in the present disclosure can be performed at room temperature, and the room temperature described in the present disclosure refers to an ambient temperature without human intervention.
  • the temperature of the extrusion module is similar to the ambient temperature.
  • the exogenous substance is delivered into eukaryotic cells at a preset temperature.
  • Delivering exogenous substances into eukaryotic cells at a preset temperature has higher delivery efficiency, cell recovery rate and survival rate. Therefore, the method of delivering the exogenous substance into eukaryotic cells is performed at a preset temperature.
  • the preset temperature is regulated by the temperature control module of the device for delivering the exogenous substance into the eukaryotic cells.
  • the suspension system containing cells and exogenous substances is extruded through the device at one time; wherein extruding through the device at one time refers to applying continuous pressure to the mixture to make the mixture Uninterrupted through the microporous membrane.
  • the volume of the suspension system extruded through the device at one time is at least 0.5 mL.
  • the number of cells in the mixture passing through the microporous membrane in a single pass is at least 0.6 ⁇ 10 6 cells.
  • the method can process cells with a larger flux, and has the advantages of a single cell suspension treatment volume (>0.5 ml) and a single treatment cell throughput (>0.6 ⁇ 10 6 cells).
  • the incubation is continued for a period of time, the cell membrane pores and nuclear membrane pores are repaired after the incubation, and the exogenous materials can be retained in the cytoplasm and nucleus.
  • the method further includes separating the exogenous substances in the suspension system before continuing to culture the extruded cells, optionally using a centrifugal method to remove the exogenous substances, and then removing the exogenous substances Continue to cultivate.
  • the present disclosure does not limit the types of foreign substances, and those skilled in the art can use the methods of the present disclosure to deliver any foreign substances into any cell according to actual production and research needs. And based on the beneficial effects of the device for delivering foreign substances into cells provided by the present disclosure, the method of the present disclosure is particularly suitable for delivering larger molecules into the cytoplasm and nucleus.
  • exogenous substances described in the present disclosure may be natural substances, including but not limited to nucleic acids, proteins or glycoproteins from natural sources; they may also be artificially synthesized or modified substances, including but not limited to macromolecular polymers , Artificial synthesis of nucleic acids or nano-components, etc.
  • the exogenous substance can also be modified by a modifier, including but not limited to the use of fluorescent labels, isotope labels or drug modifiers; in one or more embodiments, the exogenous substance includes nucleic acids modified by fluorescent labels, Isotope-labeled protein or drug modifier modified synthetic macromolecules with therapeutic effects, etc.
  • the foreign substance can be a single kind of substance or a combination of different kinds of substances, which preferably includes one of dextran, DNA, RNA, protein, ribonucleoprotein complex and nanodevice. Species, or a combination of several.
  • foreign substances include a mixture of DNA and ribonucleoprotein complexes.
  • foreign substances include a mixture of DNA and protein.
  • the foreign substance includes a combination of dextran, DNA, and nanodevices.
  • DNA includes biologically derived DNA or synthetic DNA.
  • the biologically derived DNA includes DNA molecules amplified by biosynthesis, including but not limited to plasmids extracted from microorganisms or DNA molecules directly extracted from microorganisms or animals.
  • synthetic DNA refers to a DNA molecule synthesized without biological action, for example, a DNA molecule synthesized directly chemically.
  • the biologically derived DNA includes a plasmid to transform the plasmid with the target gene into the cell, up-regulate or inhibit the expression of the target protein, or allow the cell to express an exogenous protein.
  • RNA includes mRNA, siRNA, miRNA, or lncRNA to study the regulatory effect of RNA-based molecules on eukaryotic cells.
  • the device for delivering exogenous substances into eukaryotic cells provided in the present disclosure can deliver exogenous substances with a hydration radius larger than the upper limit of the diffusion of the nuclear pore complex (NPC) into the nucleus. Therefore, in one or more embodiments, at least one of the exogenous substances cannot cross the nuclear membrane nuclear pore complex of the cell nuclear membrane by free diffusion.
  • NPC nuclear pore complex
  • the substance that cannot cross the nuclear membrane nuclear pore complex by free diffusion includes dextran, and the molecular weight of the dextran is at least 41kDa, for example 70kDa-2MDa, such as 2MDa dextran. Glycans.
  • the substance that cannot cross the nuclear membrane nuclear pore complex by free diffusion includes DNA, and the molecular weight of the DNA is at least 1 kbp.
  • the present disclosure does not limit the types of eukaryotic cells to which exogenous substances are delivered, and exogenous substances can be delivered to eukaryotic cells according to actual development and production needs.
  • the eukaryotic cells to which the exogenous substance is delivered are, for example, derived from mammals, including but not limited to humans, pigs, cows, monkeys, mice, or sheep, such as humans or mice.
  • the eukaryotic cells include primary cells or cell lines.
  • Primary cells refer to cells isolated directly from organisms, including but not limited to cells isolated from various tissues and organs of animals such as blood, skin, bones, heart or tendons, and may also include these primary cells Passage cells that have not yet constituted a stable cell line.
  • Cell lines refer to passage cells with a stable shape. These passage cells can be obtained from primary cells after stable passage, or they can be derived from commercial cell lines, including but not limited to CHO cells, PK15 cells, Hela cells, K562 Cells or CT26 cells.
  • the types of primary cells include, but are not limited to, stem cells, immune cells, tumor cells, fibroblasts, skin cells, or neurons, for example, immune cells, tumor cells, or stem cells.
  • immune cells include T cells, B cells, DC cells, NK cells, monocytes, mast cells, eosinophils, basophils, neutrophils, or giant cells. Phages.
  • the stem cells include hematopoietic stem cells, mesenchymal stem cells, or skin stem cells.
  • the immune cells, tumor cells or stem cells are derived from mammals, such as humans or mice.
  • the immune cells are human immune cells, which are more suitable for routine production and scientific research needs.
  • the present disclosure also provides the application of the above-mentioned device or the above-mentioned method in regulating cell function.
  • the present disclosure up-regulates or down-regulates the expression of certain proteins by eukaryotic cells by delivering exogenous substances to eukaryotic cells, or allows eukaryotic cells to express foreign proteins.
  • the regulation described in the present disclosure can be transient regulation, even if the cells are relatively Changing physiological and biochemical characteristics in a short period of time can also be continuous regulation, even if cells continue to change physiological and biochemical characteristics.
  • the regulation of cellular functions includes transient regulation or continuous regulation.
  • the regulating cell function includes down-regulating or up-regulating the expression of a specific protein in the cell.
  • down-regulating the expression of a specific protein in a cell includes down-regulating the expression of programmed death receptor-1, T cell receptor or major histocompatibility complex.
  • the regulation of cell functions includes allowing cells to express foreign proteins.
  • the foreign protein includes a chimeric antigen receptor, a T cell receptor that recognizes a specific antigen, and beta globulin. Up-regulation, down-regulation, or expression of foreign proteins by eukaryotic cells can be used to produce protein drugs, such as monoclonal antibodies, fusion proteins, or antigens for vaccines.
  • This embodiment provides a device for delivering exogenous substances into eukaryotic cells.
  • the device uses PET nuclear porous membrane (purchased from the Institute of Modern Physics, Chinese Academy of Sciences (Jiang Bitou)) as the microporous membrane.
  • the PET nuclear pore membrane is a PET film that is irradiated with high-energy heavy ions to form a cylindrical pipe with a diameter of about 10 nm, and then chemically etched to produce a larger specific diameter pore; provided by liposome extruder LF1 (Canada AVESTIN Company)
  • the positive pressure pressure unit puts the PET nuclear pore membrane in the liposome extruder to assemble the device for delivering exogenous substances into eukaryotic cells according to the embodiment.
  • the PET nuclear porous membrane is shown in Figure 2A and Figure 2B, and the liposome extruder LF1 is shown in Figure 2C.
  • This embodiment provides a device for delivering exogenous substances into eukaryotic cells, as shown in FIG. 3, where icon 110 is a microporous membrane, 120 is a piston tube body, and 130 is a piston push rod.
  • the device uses PET nuclear porous membrane (purchased from the Institute of Modern Physics, Chinese Academy of Sciences (Jiang Bitou)) as the microporous membrane 110.
  • the extrusion module of the device uses a piston unit as a pressure unit to apply positive pressure to eukaryotic cells and exogenous substances.
  • the specific structure is as follows:
  • the piston unit includes a piston tube body 120 and a piston push rod 130, the piston tube body 120 and the piston push rod 130 are detachably connected by sliding and sealing; a detachable piston tube body 120 is provided at the bottom of the piston tube body 120 that is far from the piston push rod 130 and enters one end.
  • the PET nuclear porous film serves as the microporous film 110.
  • This embodiment provides a device for delivering exogenous substances into eukaryotic cells, as shown in Figure 4, where icon 110 is a microporous membrane, 120 is a piston tube, 130 is a piston push rod, and 210 is a cavity. 221 is the inflow passage, and 222 is the outflow passage.
  • the device uses PET nuclear porous membrane (purchased from the Institute of Modern Physics, Chinese Academy of Sciences (Jiang Bitou)) as the microporous membrane 110.
  • the extrusion module of the device uses a piston unit as a pressure unit to apply positive pressure to eukaryotic cells and exogenous substances.
  • the device uses a temperature control module including a fluid circulation temperature control unit to control the temperature, and the specific structure is as follows:
  • the piston unit includes a piston tube body 120 and a piston push rod 130, the piston tube body 120 and the piston push rod 130 are detachably connected by sliding and sealing; a detachable piston tube body 120 is provided at the bottom of the piston tube body 120 that is far from the piston push rod 130 and enters one end.
  • the PET nuclear porous film serves as the microporous film 110.
  • the outer wall of the piston tube 120 is provided with a cavity 210, which is used for fluid circulation in the fluid circulation temperature control unit, and the outer wall of the cavity 210 is provided with passages for fluid to enter and flow out. In this way, the passage close to one end of the microporous membrane 110 is the inflow passage 221, and the passage close to the entry end of the piston push rod 130 is the outflow passage 222.
  • the two passages on the cavity 210 are connected with the outer circulation of the constant temperature water bath, so that the liquid with a constant temperature can flow in and out of the cavity 210 continuously and stably.
  • 70kDa and 2MDa glucan were delivered to the cytoplasm and nucleus of CT26 (mouse colon cancer cell line).
  • This embodiment uses the device provided in Embodiment 1.
  • 70kDa or 2MDa FITC-labeled dextran FITC-dextran, hereinafter referred to as dextran
  • dextran FITC-dextran
  • the cell suspension was passed through PET nuclear pore membranes with different pore diameter channels, and the results of intranuclear delivery and intracellular delivery efficiency were evaluated by confocal microscope and flow cytometry.
  • the test materials are shown in Table 1:
  • Collect the processed cell suspension incubate for a certain period of time, then centrifuge to discard the supernatant, resuspend the cells in the cell culture medium and continue culturing for 24 hours. After incubation, add Hoechst33342 solution, incubate for 15 minutes, discard the supernatant, wash twice with PBS, add 20 ⁇ L PBS, observe and take pictures with the oil lens (630 ⁇ ) of a confocal microscope. And collect the processed cell suspension, incubate for a certain period of time, centrifuge to discard the supernatant, resuspend the cells in PBS and wash twice, and analyze the delivery efficiency of the cells resuspended in PBS with a flow cytometer.
  • FIG. 6A and Figure 6B Flow cytometry analysis of the intracellular delivery efficiency of dextran in CT26 is shown in Figure 6A and Figure 6B, showing the intracellular delivery efficiency of 70kDa and 2MDa dextran, and the relationship between delivery efficiency and PET nuclear pore membrane pore diameter Under the condition of 8 micron pore diameter, both 70kDa and 2MDa glucan achieved the highest intracellular delivery efficiency.
  • Centrifuge the K562 single cell suspension add serum-free medium to resuspend the cells (cell concentration 1.2 ⁇ 10 6 cells/ml), and then add 70kDa or 2MDa dextran; nuclear pore membranes of different pore sizes are installed in the liposome for extrusion
  • the instrument LF1 transfer 1 ml of the K562 cell and dextran suspension into the device provided in Example 1 at room temperature (25 degrees Celsius), and then use the liposome extruder to push the cell suspension through the PET nuclear pore membrane.
  • the cell recovery rate is between 70-80% under the conditions of 7, 8 and 9 micron pore size; while under the condition of 10 micron pore size, the cell recovery rate reaches about 90%.
  • the cell recovery rate data shows that as the pore size of the nuclear pore membrane decreases, the cell recovery rate also decreases.
  • Comprehensive pore size, delivery efficiency and cell recovery rate it can be found that reducing the pore size can enhance the delivery efficiency, but will also lead to a decline in cell recovery rate. Therefore, the delivery test needs to maximize the delivery efficiency by optimizing the pore size while maintaining a certain cell recovery rate.
  • the pCMV-GFP plasmid was delivered to the nucleus of K562 cells and expressed the GFP protein.
  • the pCMV-GFP plasmid (Plasmid#11153, 4.4kbp) was combined with K562 cells using the device provided in Example 3.
  • the mixed suspension is passed through the microporous membrane 110, and then the green fluorescent protein GFP expression of K562 cells is detected by a confocal microscope, or the GFP expression level of K562 is analyzed by a flow cytometer.
  • the test materials are shown in Table 3:
  • Centrifuge the K562 single cell suspension resuspend the cells in serum-free medium (cell concentration 1.2 ⁇ 10 6 cells/ml), and then add pCMV-GFP plasmid; use the device provided in Example 3 with a PET nuclear pore membrane of 7 ⁇ m, At 30°C, transfer 2 mL of K562 cell and plasmid mixture into the device, use piston push rod 130 to push the cell suspension through the regular channels of PET nuclear membrane, collect the processed cell suspension, incubate for a certain time, and centrifuge Discard the supernatant, resuspend the cells in serum-containing medium and continue to culture for 2 hours, observe the expression of green fluorescent protein GFP with a confocal microscope; or continue to culture for 48 hours, analyze the expression of green fluorescent protein GFP with a flow cytometer.
  • CLSM observed the expression of pCMV-GFP plasmid in K562.
  • Figure 8 2 hours after the K562 and pCMV-GFP plasmid mixed suspension was extruded by the device provided in Example 3, GFP began to express, indicating that the pCMV-GFP plasmid was delivered into the K562 cell nucleus and transcribed into GFP-encoding mRNA, and finally the GFP protein is expressed in the nuclear ribosomes.
  • the expression of pCMV-GFP plasmid in K562 was analyzed by flow cytometry. As shown in Figure 9, 24 hours after the K562 and pCMV-GFP plasmid mixed suspension was extruded by the device provided in Example 3, the positive rate of GFP expression reached 49%, indicating that the delivery of pCMV-GFP plasmid into a larger proportion of K562 Cell nucleus, and express GFP protein.
  • the pCMV-GFP plasmid (Plasmid#11153, 4.4kbp) was delivered to the nucleus of CT26 cells and expressed the GFP protein.
  • 2 mL of the pCMV-GFP plasmid and CT26 single cell suspension were extruded using the device provided in Example 3.
  • the GFP expression level of CT26 at 48 hours was analyzed by flow cytometry.
  • the test materials are shown in Table 4:
  • 2MDa glucan is delivered into K562 cytoplasm and nucleus.
  • a suspension of 2MDa FITC-labeled dextran mixed with K562 cells was extruded using the device provided in Example 3.
  • the test materials are shown in Table 5:
  • Centrifuge the K562 single cell suspension add serum-free medium to resuspend the cells (cell concentration 1.2 ⁇ 10 6 cells/ml), then add 2MDa dextran; install the nuclear pore membrane in a device with temperature control accessories At 30°C, transfer 2 mL of K562 cell and dextran suspension into the device, and then use the plunger 130 to push the cell suspension through the regular channels of the PET nuclear membrane, collect the processed cell suspension, and add Hoechst 33342 Cell nuclear dye, incubate for a certain time, centrifuge to discard the supernatant, resuspend the cells in serum-free medium and wash twice, then resuspend in serum-free medium and add dropwise to a confocal dish, observe 2MDa dextran with a confocal microscope The distribution of sugar in the cytoplasm and nucleus.
  • the 2MDa dextran was delivered to CT26 (mouse colon cancer cell line) cells.
  • CT26 mae colon cancer cell line
  • This embodiment uses the device provided in Embodiment 3.
  • a cell suspension mixed with 2MDa dextran and CT26 was passed through a PET nuclear pore membrane with 9 micron pores at 4 degrees Celsius.
  • the test materials are shown in Table 6:
  • CT26 affects the cell recovery rate after extrusion.
  • CT26 cell suspensions with different cell densities were extruded using the device provided in Example 3. The cells were collected and the Invitrogen Countess II cell counter was used for cell counting and cell viability Detection.
  • Cell recovery rate (the number of living cells in the collected cell sample/the number of living cells in the cell suspension before treatment) ⁇ 100%
  • the cell recovery rate data is shown in Figure 13, showing that the higher the cell concentration, the lower the cell recovery rate.
  • the cell recovery rate under the conditions of 1 ⁇ 10 6 cells/ml and 2 ⁇ 10 6 cells/ml is greater than 80%.
  • a suitable cell concentration range needs to be selected to ensure a high cell recovery rate.
  • the molecular weight of foreign substances affects the efficiency of intracellular delivery. Delivery of 70kDa and 2MDa dextran to K562 cells. In order to evaluate the influence of the molecular weight of exogenous materials on the intracellular delivery efficiency, a suspension of 70kDa or 2MDa FITC-labeled dextran mixed with K562 cells was passed through the device provided in Example 1, and the intracellular delivery was analyzed by flow cytometry. effectiveness. The test materials are shown in Table 8:
  • Centrifuge the K562 single cell suspension add serum-free medium to resuspend the cells (cell concentration 1.2 ⁇ 10 6 cells/ml), and then add 70kDa or 2MDa dextran; nuclear pore membranes of different pore sizes are installed in the liposome for extrusion
  • the instrument LF1 transfer 1 ml of the K562 cell and dextran suspension into the device provided in Example 1 at room temperature (25 degrees Celsius), and then use the liposome extruder to push the cell suspension through the PET nuclear pore membrane.
  • Test results Flow cytometry analysis of the intracellular delivery efficiency of dextran in K562 is shown in Figure 14, showing the intracellular delivery efficiency of 70kDa and 2MDa dextran, as well as the relationship between dextran molecular weight and delivery efficiency.
  • the intracellular delivery efficiency of 2MDa glucan (FITC positive rate, MFI average fluorescence intensity) is less than 70kDa glucan, indicating exogenous The higher the molecular weight of the substance, the lower the efficiency of intracellular delivery.
  • 2MDa dextran was delivered to CT26 (mouse colon cancer cell line) cells.
  • CT26 mae colon cancer cell line
  • This embodiment uses the device provided in Embodiment 3.
  • 2MDa FITC-labeled dextran (FITC-dextran, hereinafter referred to as dextran) or pCMV-GFP plasmid DNA mixed with CT26 cell suspension was passed through Pass the PET nuclear pore membrane with 8 ⁇ m pores, use a cell counter to detect the cell recovery rate, and use a flow cytometer to analyze the intracellular delivery efficiency.
  • the dextran delivery test was performed under 5 temperature conditions (15°C, 20°C, 25°C, 30°C, 35°C), and the plasmid transfection test was performed under two temperature conditions (25°C, 30°C).
  • the test materials are shown in Table 9:
  • Collect the processed cell suspension incubate for a certain period of time, take samples for cell count detection, then centrifuge to discard the supernatant, resuspend the cells in PBS and wash twice, and analyze the delivery efficiency of the cells resuspended in PBS by flow cytometry.
  • For plasmid DNA transfection experiments collect the processed cell suspension, incubate for a certain period of time, centrifuge to discard the supernatant, resuspend in RPMI1640 medium containing FBS, culture for 48 hours, trypsinize the cells to prepare a single cell suspension, Flow cytometry was used to detect GFP expression.
  • the intracellular delivery efficiency and cell recovery rate of dextran in CT26 are shown in Figure 15, showing that temperature affects the intracellular delivery efficiency and cell recovery rate of 2MDa dextran. Under the condition of 8 micron pore size, as the temperature increases, The cell recovery rate increased, and 2MDa glucan achieved the highest intracellular delivery efficiency at 30°C, indicating that 30°C is the best delivery condition under the set 5 temperature conditions, which can obtain the highest cells.
  • the internal delivery efficiency can also obtain an optimized cell recovery rate.
  • CT26 has a higher GFP expression level (GFP positive rate and the average fluorescence intensity MFI of GFP expression) at 30°C, indicating the comparison It has better transfection effect at 25°C and 30°C.
  • the piston tube body in Example 2 is prepared from stainless steel by CNC machine tool processing; a PTFE rod is used to make a piston push rod that can be sealed with the tube body; the microporous membrane is detachably connected to the piston tube body Assembled into a piston unit in a way, the microporous membrane is located at the bottom of the piston tube.
  • the assembled piston unit and piston push rod are sterilized under high temperature, humidity and heat conditions (121.3°C, 20 minutes). After the sterilization is completed, they can be used for intracellular delivery test after drying and cooling.
  • the device provided by the present disclosure can deliver various foreign substances into eukaryotic cells.
  • the delivery process does not require the use of microfluidics and electric fields.
  • the present disclosure can deliver large foreign substances including plasmid DNA into the nucleus and express encoded proteins.
  • the present disclosure can realize high-throughput transfer of foreign substances to eukaryotic cells.

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Abstract

Dispositif et procédé d'administration de substances exogènes dans des cellules eucaryotes, et application associée. Le dispositif d'administration de substances exogènes dans des cellules eucaryotes contient un module d'extrusion, le module d'extrusion étant pourvu d'une membrane microporeuse ayant des pores formés régulièrement, le diamètre de chaque pore étant inférieur à celui d'une cellule eucaryote, et le module d'extrusion comprenant également une unité de pression pour entraîner des cellules eucaryotes et des substances étrangères à passer à travers les pores en même temps. Les cellules en suspension dans une solution peuvent être comprimées et déformées lors du passage à travers les pores, de sorte que la perforation de la membrane cellulaire et de la membrane nucléaire soit provoquée de manière à permettre aux substances exogènes dispersées ou dissoutes dans la suspension cellulaire d'entrer dans le cytoplasme et le noyau.
PCT/CN2020/099054 2019-07-01 2020-06-29 Dispositif et procédé d'administration de substances exogènes dans des cellules eucaryotes et application associée WO2021000844A1 (fr)

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