Method for introducing substances into cells, genetically modified cells and methods for producing the same, and control program for a device for introducing substances into cells

Abstract

The objective is to provide a method for introducing substances into cells that achieves both high substance delivery efficiency and high cell viability. [Solution] A method for introducing a substance into cells, comprising the following steps: a) seeding suspended cells into a gel plate medium of a specific hardness; b) filling the gel plate medium on which the cells were seeded with an electrolyte and positioning a nanopipette filled with the substance at a cell-corresponding position in the electrolyte; c) measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell until the rate of decrease from the steady current reaches a set current decrease rate; d) moving the nanopipette toward the cell at a set puncture distance and puncturing a cell having a cell wall; e) applying a voltage between the inside of the nanopipette and the electrolyte to discharge the substance into the cell; and f) removing the nanopipette.

Description

[Technical Field]

[0001] This disclosure relates to a method for introducing substances into cells, genetically modified cells and methods for producing the same, and a control program for a device for introducing substances into cells. [Background technology]

[0002] Until now, establishing cell lineages with properties suitable for use as host cells for substance production, particularly those with cell walls, has had to rely on inefficient and inflexible methods such as the spontaneous generation of lineages with specific traits or induction through mutagen treatment. Against this backdrop, the introduction of substances such as genome editing tools and nucleic acids into cells is being considered as a more efficient method for modifying targeted genes. As methods that enable the introduction of substances into cells, for example, the electroporation method described in Patent Documents 1 and 2, and the nanopipette method described in Patent Document 3 are being investigated.

[0003] Electroporation typically requires precise pulse conditions to improve the efficiency of substance delivery to cells with cell walls, as the presence of the cell wall usually inhibits substance delivery. However, these precise pulse conditions can easily damage cells, making it difficult to achieve both low cell damage and high delivery efficiency with electroporation.

[0004] For example, in the electroporation method described in Patent Document 1, increasing the number of electroporation pulses to improve introduction efficiency, especially in the case of cells with cell walls such as microalgae, reduces cell viability. Furthermore, increasing the applied voltage to improve introduction efficiency can lead to a decrease in cell viability and aggregation of cells, depending on the applied voltage. Moreover, not only the cells but also the substances to be introduced into the cells (e.g., nucleic acids, proteins, and other substances) are damaged, making substance introduction itself difficult. In addition, in the case of cells containing many ions, such as marine organisms, sparks are likely to occur when pulses are applied, posing a challenge to minimally invasive substance introduction.

[0005] Furthermore, the electroporation method described in Patent Document 2 reduces the inhibitory effect on substance introduction by isolating microalgae lacking the outer shell, which are very rare in nature, and then performing electroporation treatment on these microalgae lacking the outer shell. However, the risk of cell damage during the electroporation process and the resulting unexpected cell behavior remains high. In addition, isolation of strains lacking the outer shell (cell wall) is necessary, making the process numerous and complex. Moreover, strains lacking the outer shell have low environmental tolerance and are susceptible to cell death due to, for example, UV light, temperature changes, and physical stimuli. Microalgae lacking the outer shell can also be obtained by performing cell wall removal using an enzymatic method with cellulase, but this cannot be applied to cell walls other than cellulose (e.g., diatoms). Furthermore, enzymatic treatment becomes difficult for species that secrete large amounts of extracellular polysaccharides. It is also possible to obtain microalgae lacking the outer shell through genetic engineering, but this requires complicated procedures.

[0006] The nanopipette method described in Patent Document 3 has no known application to puncturing floating cells with cell walls. In particular, cells with cell walls, such as microalgae, have rigid cell walls, and when the pipette tip touches the cell, the cell moves, making puncture impossible. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2004-33070 [Patent Document 2] Japanese Patent Publication No. 2015-181402 [Patent Document 3] International Publication No. 2013 / 012452 [Overview of the project] [Problems that the invention aims to solve]

[0008] There is a need for a method to enhance the efficiency of introducing substances into floating cells, particularly a method to enhance the efficiency of introducing substances into floating cells having a cell wall. Substance production utilizing the metabolism of microorganisms such as microalgae, filamentous fungi, Escherichia coli, and yeast has been widely studied as a technique capable of producing compounds difficult to synthesize chemically while suppressing environmental impact. Among them, microalgae are expected as a substance production host accompanied by photosynthesis. However, even when attempting to breed microalgae through genetic modification or the like, due to the presence of the cell wall of microalgae in particular, it has been difficult to achieve both high introduction efficiency of protein complexes such as nucleic acids and genome editing tools into the cell and high cell viability.

[0009] Therefore, an object of the present disclosure is to provide a method for introducing substances into cells that achieves both high substance introduction efficiency and high cell viability. Furthermore, an object of the present disclosure is to provide an automatically controlled substance introduction system into cells using this substance introduction method.

Means for Solving the Problems

[0010] The present inventors predicted that by puncturing with a nanopipette having a tip diameter of several tens of nm and discharging substances by ion voltage, it would be possible to achieve both high substance introduction efficiency into cells and high cell viability. Then, by seeding cells in a floating state on a gel plate medium of a specific hardness and performing substance introduction using a nanopipette, successful substance introduction was achieved that achieved both high substance introduction efficiency and high cell viability. By this technology, it becomes possible to introduce substances such as nucleic acids and genome editing tools into cells, and it becomes possible to contribute to improving breeding efficiency.

[0011] That is, the present disclosure is as follows.

[0012] 〔1〕 A method for introducing a substance into a cell, comprising the following: a) Floating cells are evaluated for hardness according to JIS K6503, and have a breaking load of 2.0 to 30 [N], or 0.5×10 5 ~7×10 5The step of seeding cells on a gel plate medium having a Young's modulus of [[Pa]] or a hardness such that the pressing stress for 1 mm is 1.0 to 8 [N]; b) The step of filling the gel plate medium seeded with cells with an electrolytic solution and positioning a nanopipette filled with a substance at a position corresponding to the cells in the electrolytic solution; c) The step of measuring the current between the inside of the nanopipette and the electrolytic solution and moving the nanopipette in the cell direction to a position where the reduction rate of the set current from the steady current is 2% or more and 50% or less; d) The step of moving the nanopipette in the cell direction at a set piercing distance of 1 μm or more and 50 μm or less to pierce the cells; e) The step of applying a voltage between the inside of the nanopipette and the electrolytic solution to discharge the substance into the cells; and f) The step of removing the nanopipette. [2] A method for introducing a substance into cells, comprising: a') a' - 1) The step of seeding floating cells on a substrate and laminating a gel on the cells; a' - 2) The step of peeling the gel from the substrate to obtain a gel sheet on which the cells are transferred; a' - 3) Laminating the gel sheet on the gel plate medium with the cell transfer surface facing up, and setting the hardness of the entire laminated gel to a breaking load of 2.0 to 30 [N] or a Young's modulus of 0.5×10 5 ~7×10 5 [Pa] or a pressing stress for 1 mm of 1.0 to 8 [N]; b) The step of filling the gel plate medium seeded with cells with an electrolytic solution and positioning a nanopipette filled with a substance at a position corresponding to the cells in the electrolytic solution; c) The step of measuring the current between the inside of the nanopipette and the electrolytic solution and moving the nanopipette in the cell direction to a position where the reduction rate of the set current from the steady current is 2% or more and 50% or less; d) The step of moving the nanopipette in the cell direction at a set piercing distance of 1 μm or more and 50 μm or less to pierce the cells; e) The step of applying a voltage between the inside of the nanopipette and the electrolytic solution to discharge the substance into the cells; and f) The step of removing the nanopipette. [3] The method according to [1] or [2] above, wherein the substance is a protein, a mixture or complex containing a protein, a mixture or complex containing a protein and a nucleic acid, a nucleic acid, or a dye. [4] The method according to [3] above, wherein the substance is a genome editing substance. [5] The substance is either a positively charged substance or a negatively charged substance, If the substance is a positively charged substance, step e) is performed by applying a voltage such that the inside of the nanopipette becomes positively charged and the electrolyte becomes negatively charged. If the substance is a negatively charged substance, step e) is performed by applying a voltage such that the inside of the nanopipette becomes negatively charged and the electrolyte becomes positively charged. The method described in [3] above. [6] The method according to [5] above, wherein step e) is performed by applying a set applied voltage of -11V or more and +11V or less and a set applied time of 0.1 seconds or more and 5.0 seconds or less. [7] Methods for producing genetically modified cells, including the following: a) The floating cells were subjected to a breaking load of 2.0 to 30 [N] or 0.5 × 10⁻¹⁶ according to the hardness evaluation of JIS K6503. 5 ~7×10 5 A step of sowing seeds in a gel plate culture medium with a Young's modulus of [Pa] or a hardness that results in a 1 mm pressure stress of 1.0 to 8 [N]; b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution; c) A step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the decrease in current from the steady current is between 2% and 50% (a set current decrease rate); d) A step of moving a nanopipette in the direction of the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell; e) A process of applying a voltage between the inside of a nanopipette and the electrolyte to expel a substance into a cell; and f) Step of removing the nanopipette. 〔8〕 Method for producing a genetically modified cell, comprising the following: a’) a’-1) Step of seeding floating cells on a substrate and laminating a gel on the cells; a’-2) Step of peeling the gel from the substrate to obtain a gel sheet on which the cells have been transferred to the gel surface; a’-3) Step of laminating the gel sheet on a gel plate medium with the cell transfer surface facing up, and setting the hardness of the entire laminated gel to a breaking load of 2.0 to 30 [N] by hardness evaluation according to JIS K6503, or a Young's modulus of 0.5×10 5 ~7×10 5 [Pa], or a 1-mm pressing stress of 1.0 to 8 [N]; b) Step of filling the gel plate medium seeded with cells with an electrolytic solution and positioning a nanopipette filled with a substance at a position corresponding to the cells in the electrolytic solution; c) Step of measuring the current between the inside of the nanopipette and the electrolytic solution and moving the nanopipette in the cell direction to a position where the set current reduction rate from the steady current is 2% or more and 50% or less; d) Step of moving the nanopipette in the cell direction at a set piercing distance of 1 μm or more and 50 μm or less and piercing the cells; e) Step of applying a voltage between the inside of the nanopipette and the electrolytic solution to discharge the substance into the cells having cell walls; and f) Step of removing the nanopipette. 〔9〕 Genetically modified cell produced by a method comprising the following: a) Step of seeding floating cells on a gel plate medium having a hardness of a breaking load of 2.0 to 30 [N], a Young's modulus of 0.5×10 5 ~7×10 5 [Pa], or a 1-mm pressing stress of 1.0 to 8 [N] by hardness evaluation according to JIS K6503; b) Step of filling the gel plate medium seeded with cells with an electrolytic solution and positioning a nanopipette filled with a substance at a position corresponding to the cells in the electrolytic solution; c) A step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the decrease in current from the steady current is between 2% and 50% (a set current decrease rate); d) A step of moving a nanopipette in the direction of the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell; e) A process of applying a voltage between the inside of a nanopipette and the electrolyte to expel a substance into a cell; and f) The process of removing the nanopipette.

[10] Genetically modified cells produced by methods including the following: a') a'-1) A process of seeding suspended cells onto a substrate and layering a gel on top of the cells; a'-2) A step of detaching the gel from the substrate and obtaining a gel sheet on which cells have been transferred to the gel surface; a'-3) Laminate the gel sheets on the gel plate medium with the cell transcription side facing upwards, and evaluate the hardness of the entire laminated gel using the hardness evaluation method of JIS K6503, with a breaking load of 2.0 to 30 [N] or 0.5 × 10 5 ~7×10 5 A process to achieve a Young's modulus of [Pa] or a 1mm pressure stress of 1.0 to 8 [N]; b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution; c) A step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the decrease in current from the steady current is between 2% and 50% (a set current decrease rate); d) A step of moving a nanopipette in the direction of the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell; e) A process of applying a voltage between the inside of a nanopipette and the electrolyte to expel a substance into a cell; and f) The process of removing the nanopipette.

[11] A control program for a cell substance delivery device, including instructions to perform the following: a) A step of positioning a nanopipette filled with a substance at a cell-corresponding position in the electrolyte of a gel plate medium of a specific hardness, in which suspended cells have been seeded and the gel plate is filled with an electrolyte; b) A step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the decrease in current from the steady current is between 2% and 50%; c) The process of moving the nanopipette in the direction of the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell; d) A process of applying a voltage between the inside of a nanopipette and the electrolyte to expel a substance into a cell; and e) The process of removing the nanopipette. [Effects of the Invention]

[0013] This disclosure provides a method for introducing substances into cells that achieves both high substance introduction efficiency and high cell viability. Furthermore, this disclosure enables the provision of an automated system for introducing substances into cells using this substance introduction method. This technology makes it possible to introduce substances such as nucleic acids and genome editing tools into cells, contributing to improved breeding efficiency. [Brief explanation of the drawing]

[0014] [Figure 1] This diagram shows a system for use in the method disclosed herein and a conceptual diagram of the pipette approach step in the method disclosed herein. [Figure 2] This is a conceptual diagram of the pipette insertion process in the method disclosed herein. [Figure 3] This is a conceptual diagram of the pipette substance discharge step in the method disclosed herein. [Figure 4] This figure shows an example of a system control mechanism for use in the method disclosed herein. [Figure 5] This is a conceptual diagram of the overall scheme of the method of this disclosure, which involves seeding cells onto a gel plate medium. [Figure 6] This is a conceptual diagram of the overall scheme of the method of this disclosure, which involves transferring cells onto a gel sheet. [Modes for carrying out the invention]

[0015] The present invention will be described in detail below with reference to the drawings as necessary, but the drawings are illustrative for illustrating the present invention, and the technical scope of the present invention is not limited by the illustrations in the drawings.

[0016] [System for introducing substances into cells] This document describes a system for use in the methods described herein (methods for introducing substances into cells and methods for producing genetically modified cells) (a system for introducing substances into cells, hereinafter referred to as "the System of the Disclosure"). The System of the Disclosure includes, for example, the following components: • Nanopipette; A three-dimensional (xyz) movable pipette holder for moving nanopipettes in three dimensions (sometimes referred to as "xyz" for convenience); • An electrode that can come into contact with the liquid filled inside the nanopipette (hereinafter referred to as the "pipette electrode"). • An electrode that can come into contact with the electrolyte solution in the cell holder (hereinafter referred to as the "reference electrode"); • A current measuring circuit for measuring the current between the pipette electrode and the reference electrode; A voltage application circuit for applying voltage between the pipette electrode and the reference electrode.

[0017] The system of this disclosure may further include a position input device for manually moving a three-dimensional moving module. The system of this disclosure is used mounted on an optical microscope. The system of this disclosure is also used by placing a cell holder that holds cells, a gel plate culture medium, and an electrolyte for immersing the cells on the optical microscope, and holding a solution containing a substance to be introduced into the cells inside a nanopipette.

[0018] The following describes each component.

[0019] (Nanopipette) A "nanopipette" refers to a tubular structure having a nanoscale tip opening. A nanoscale tip opening is, for example, a conical tip opening (i.e., a nanopore) with a diameter of approximately 10 nm to 500 nm, preferably approximately 50 nm (±20%). The material used for the nanopipette is an inert, non-biological material, such as glass or quartz. The inner wall of the nanopipette may also be surface-treated to suppress the adsorption of substances (e.g., nucleic acids, proteins) filled inside the nanopipette. Furthermore, it is preferable that the nanopipette has a shape or scale that allows for the insertion of an electrode that contacts the solution inside the nanopipette.

[0020] A nanopipette has a single channel (sometimes called a "barrel" or "bore") or multiple parallel channels within its tube. A nanopipette with a single channel is sometimes called a "single-barreled nanopipette." A nanopipette with multiple parallel channels is sometimes called a "multi-barreled nanopipette." A nanopipette with two parallel channels is sometimes called a "double-barreled nanopipette." In the method of this disclosure, a single-barreled nanopipette is preferred from the viewpoint of ease and reliability of filling the nanopipette with a substance, and accuracy of current measurement and voltage application.

[0021] Nanopipettes are available commercially (e.g., Yokogawa, part number SU10ACC-NP01, etc.). Alternatively, nanopipettes can be fabricated, for example, by pulling glass or quartz capillaries with a laser.

[0022] Details of the nanopipette are described, for example, in International Publication No. 2014 / 160036 and International Publication No. 2013 / 012452 (Patent Document 3).

[0023] (Three-dimensional (xyz) movable pipette holder) A "three-dimensional (xyz) moving pipette holder" is a pipette holder on which a nanopipette is mounted and which moves the mounted nanopipette in three dimensions by driving a rough actuator and a fine actuator. Of the three dimensions, two directions (sometimes referred to as the "x-axis direction" and the "y-axis direction" for convenience) are perpendicular or nearly perpendicular to the long axis of the nanopipette, and the remaining direction (sometimes referred to as the "z-axis direction" for convenience) is parallel or nearly parallel to the long axis of the nanopipette when mounted on the three-dimensional moving pipette holder. The three-dimensional moving pipette holder may consist, for example, a holder stage driven by a rough actuator and a holder head mounted on the holder stage and on which the nanopipette is mounted, driven by a fine actuator.

[0024] A "rough actuator" is a three-dimensional actuator capable of rough positioning. Examples include three-dimensional actuators with a stroke of approximately 10 to 100 mm and a setting resolution of approximately 0.1 to 1 μm. Examples of rough actuators include electromagnetically driven actuators such as motor (rotary motor, linear motor) based actuators.

[0025] A "fine actuator" is a three-dimensional actuator capable of precise positioning. Examples include a three-dimensional actuator with a stroke of approximately 100-500 μm and a setting resolution of approximately 1 nm. Examples of fine actuators include piezoelectric actuators and other piezoelectrically driven actuators. If the XY axis setting resolution of a rough actuator is sufficient for the target cell size, the fine actuator may be limited to one dimension, specifically the Z axis, for precise approach and puncture of cells.

[0026] (Position input device) A position input device is a device for manually instructing the position of a nanopipette. Examples of position input devices include pointing devices such as joysticks, key input devices such as keyboards, and combinations of these.

[0027] (Pipette electrode, reference electrode) Examples of pipette electrodes and reference electrodes include gold electrodes, silver electrodes (e.g., silver tetrakis(4-chlorophenyl) borate (AgTBACI) electrodes, Ag / AgCl electrodes, etc.), and platinum electrodes. The reference electrode may be used in contact with the electrolyte in the cell holder. Furthermore, if the nanopipette is a multi-barrel type (e.g., a double-barrel type), the pipette electrode may be placed in the channel that holds the substance to be introduced into the cells, and the reference electrode may be placed in a separate channel.

[0028] (Current measurement circuit, voltage application circuit) The current measurement circuit is a circuit for measuring the ionic current between the pipette electrode and the reference electrode (i.e., the current between the inside of the nanopipette and the electrolyte). The current measurement circuit should have a current measurement range of approximately 100 pA to 100 nA, and for cell surface detection, a circuit capable of measuring current changes (reductions) of about 2% to 50% is preferable. For example, in a case where the current at a sufficient distance from the cell surface (steady-state current) is 10 nA, if cell surface detection is set to a 20% decrease in current, the point at which the current becomes 8 nA is measured. In addition, a low-noise amplification circuit can be used to accurately detect a very small steady-state reference current and its changes. In some cases, low noise can be achieved by using software-based digital filtering technology.

[0029] The current measured by the current measurement circuit is used as an indicator of the distance between the cell and the tip of the nanopipette, based on the principle of scanning ion conductance microscopy (SICM) technology. When the tip of the nanopipette comes into contact with the electrolyte in the cell holder, an ionic current begins to flow between it and the reference electrode. Subsequently, even if the tip of the nanopipette is moved closer to the cell, there is no significant change in the current value as long as there is a sufficient distance from the cell surface. This current value is called the "steady-state current." Then, when the tip of the nanopipette approaches a very close distance to the cell, the current drops sharply in proportion to the distance between the cell and the tip of the nanopipette. This is because the cell membrane is a highly insulating film. Therefore, by automatically controlling the nanopipette to pause when the rate of decrease of the current from the steady-state current reaches a preset value (hereinafter referred to as the "set current decrease rate"), the tip of the nanopipette can be automatically paused in close proximity to the cell.

[0030] (Voltage Injection Circuit) The voltage application circuit is a circuit for applying a voltage between the pipette electrode and the reference electrode (between the inside of the nanopipette and the electrolyte). Preferably, the voltage application circuit is capable of applying a voltage of approximately -11 to +11V with a time control of approximately 0.01 seconds.

[0031] (cell) The method disclosed herein is particularly applicable to cells having a cell wall, and especially to cells having a cell wall that can become a suspension cell (hereinafter sometimes referred to as "suspension cells having a cell wall"). Notwithstanding the foregoing, the present invention is also applicable to cells that lack a cell wall or have a partially missing cell wall. Microalgae are given as examples of suspension cells having a cell wall or lacking a cell wall to which the present invention is applicable, but any suspension cell can be applied. Examples of applicable cell types include microorganisms such as microalgae cells, yeast cells, Escherichia coli, and filamentous fungal cells. Examples of microalgae include, for example, genera such as Tetraselmis, Nannochloropsis, Dunaliella, Phaeodactylum, Isochrysis, Chlorella, Haematococcus, Spirulina, Scenedesmus, and Chlamydomonas. Examples of yeasts include, for example, the genera Saccharomyces, Candida, Pichia, Kluyveromyces, Zygosaccharomyces, Schizosaccharomyces, Debaryomyces, Hansenula, Torulopsis, and Yarrowia. Examples of Escherichia include, for example, the genus Escherichia. Examples of filamentous fungi include, for example, the genera Aspergillus, Penicillium, Rhizopus, Fusarium, Trichoderma, Mucor, Neurospora, Alternaria, Beauveria, and Cladosporium. Cells with no cell wall or partially missing cell wall that are applicable to the method disclosed herein include cells that originally lack a cell wall, such as those of the genus Euglena. Furthermore, cells having the cell wall described above can be subjected to genetic modification or protoplast treatment (degradation and removal of the cell wall using enzymes or drugs) to create cells without a cell wall or with a partially missing cell wall, which can then be applied to the method of this disclosure.

[0032] (cell holder) While there are no particular limitations on the cell holder used to hold cells, gel plate culture medium, and electrolyte, a transparent holder with an open top is usually used. Examples of such holders include cell culture dishes, cell culture vessels covered with openable lids such as multiwell plates, and flat plates such as microscope slides.

[0033] (cell immersion electrolyte) Examples of electrolytes used to immerse cells in the cell holder include general culture media for the cell type being used, and buffered salines (e.g., phosphate-buffered saline (PBS), HEPES-buffered saline (HBS), Hanks equilibrium salt solution (HBSS), etc.).

[0034] The appropriate culture medium for microalgae can be used alone or in combination, depending on the type of strain used and whether the target microalgae inhabit freshwater or saltwater. Examples of culture media for freshwater microalgae include BG11 medium, Z8 medium, ASM-11 medium, TAP medium, CHU-10 medium, WC medium, BBM medium, and AAP medium. Examples of culture media for saltwater microalgae include f / 2 medium, ESM medium, and MNK medium.

[0035] Suitable culture media for E. coli include LB medium, TSA medium, NA medium, MacConkey medium, EMB medium, M9 minimal medium, SOB medium, and TB medium, depending on the type of strain used. These can be used individually or in combination.

[0036] Suitable culture media for yeast include YPD medium, YPG medium, SD medium, SGal medium, PDA medium, SC medium, YMM medium, and YM medium, depending on the type of yeast strain used. These can be used individually or in combination.

[0037] Suitable culture media for filamentous fungi include PDA medium, SDA medium, Czapek-Dox medium, MEA medium, CMA medium, V8 Juice Agar medium, YES medium, OA medium, Czapek yeast extract medium, Emerson YpSs medium, etc., which can be used individually or in combination.

[0038] (Gel plate culture medium) Gel plate media are culture media that have been gelled to a specific hardness, and can be obtained, for example, from a mixture of culture medium and a gelling agent. Gelated media are generally used for sorting cell colonies, but there are no reports of their use for optimizing injection into suspension cells. In the present invention, by using gel plate media for injection into suspension cells, it is possible to achieve the unexpectedly excellent effect of achieving both high substance introduction efficiency and high cell viability. Examples of culture media include those mentioned above. Examples of gelling agents include agarose, agar, λ-carrageenan, κ-carrageenan, ι-carrageenan, gellan gum, xanthan gum, guar gum, gum arabic, gellite, pectin, methylcellulose, glucomannan, gelatin, collagen, corn starch, sodium alginate, calcium alginate, etc., and can be added to the culture medium as a gelling agent, either alone or in combination.

[0039] The specific hardness of a gel plate medium is the hardness optimized for injection of suspension cells using a nanopipette, and can be indicated by, for example, the breaking load, Young's modulus, or pressure stress. The breaking load, Young's modulus, and pressure stress can be measured by hardness evaluation according to JIS K6503. When using the breaking load as an indicator, the specific hardness of the gel plate medium is a breaking load of 2.0 N to 30 N, preferably 2.2 N or more, more preferably 5 N or more, preferably 27 N or less, and more preferably 20 N or less. When using Young's modulus as an indicator, the specific hardness of the gel plate medium is 0.5 × 10⁻⁶ 5 Pa or more 7×10 5 The Young's modulus is less than or equal to Pa, preferably 0.75 × 10⁻⁶.5 Pa or higher, more comfortably 2.5 × 10 5 Pa or higher, preferably 5.6 × 10 5 Pa or less, more comfortable 5 x 10 5 The Young's modulus is Pa or less. When using pressure as an indicator, the specific hardness of the gel plate medium is a 1mm pressure of 1.0N to 8N, preferably 1.3N or higher, more preferably 3N or higher, preferably 7.2N or lower, and more preferably 7N or lower. The hardness of the gel plate medium can be appropriately adjusted by the type and concentration of the gelling agent. The gel plate medium may be a single layer or a multilayer with the same or different gels stacked on top of each other, as long as the hardness of the entire medium is within the above range. If stable hardness evaluation is difficult due to reasons such as the thinness of the gel plate medium, the hardness measurement value obtained by separately preparing a gel plate medium sample for hardness measurement with the same medium composition but a film thickness of 2mm to 10mm can be applied.

[0040] (Substances introduced into cells) The substances introduced into cells are not particularly limited, but examples include proteins, mixtures or complexes containing proteins, mixtures or complexes containing proteins and nucleic acids, nucleic acids, and dyes. The substances introduced into cells are not particularly limited, but substances that dissolve or suspend in an electrolyte solution are preferred. Furthermore, it is preferable that they be electrostatically charged substances. Electrostatically charged substances may be a single charged substance, a mixture or complex of multiple substances that are charged as a whole, or a substance that charges the entire solution when dissolved or suspended in an electrolyte solution (a single substance or a mixture or complex of multiple substances). Examples of electrostatically charged substances include positively charged substances and negatively charged substances. It is preferable that the substances introduced into cells (e.g., proteins, mixtures or complexes containing proteins, mixtures or complexes containing proteins and nucleic acids, nucleic acids, and dyes) are either positively charged or negatively charged substances. For example, proteins, mixtures or complexes of proteins, mixtures or complexes containing nucleic acids and proteins, etc., may be either positively charged or negatively charged substances, for example, they may be positively charged substances. Examples of mixtures or complexes containing nucleic acids and proteins include genome editing substances. Examples of genome editing substances include CRISPR-Cas9, CRISPR-Cas3, ZFN, TALEN, and PPR systems. Furthermore, nucleic acids (e.g., DNA, RNA, etc.) may be either positively charged or negatively charged, for example, they may be negatively charged. In addition, the substance introduced into the cell may be a marker substance such as a dye, or it may contain a marker substance.

[0041] Substances introduced into cells are usually in liquid form (e.g., solution). When the substance introduced into cells is a solution, the solvent can be an aqueous electrolyte or a non-aqueous electrolyte, but an aqueous electrolyte is preferred. Examples of aqueous electrolytes include those exemplified above as the electrolyte in which cells are immersed.

[0042] [Methods for introducing substances into cells] In one embodiment, the method for introducing a substance into cells according to the present disclosure includes the following steps. a) A process of seeding suspended cells onto a gel plate medium of a specific hardness (suspension cell seeding process); b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution (pipette positioning step); c) A step (pipette approach step) in which the current between the inside of the nanopipette and the electrolyte is measured and the nanopipette is moved toward the cell to a position where the decrease in current from the steady current is between 2% and 50% (set current decrease rate); d) The process of moving a nanopipette towards the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell (pipette puncture process); e) A process of applying a voltage between the inside of the nanopipette and the electrolyte to expel substances into the cells (substance expulsion process); and f) The process of removing the nanopipette (pipette removal process). The overall scheme of the method disclosed in this embodiment is shown in Figure 5. Cells (211) are cultured in suspension (first scheme in Figure 5), the suspension-cultured cells (211) are seeded onto a gel plate medium (213) in a cell holder (201) (second scheme in Figure 5), a substance is introduced into the cells (211) using a nanopipette (101) (third scheme in Figure 5), and colonies (211') of cells into which the substance has been introduced are obtained (fourth scheme in Figure 5). In the method disclosed, by fixing suspension cells on a gel plate of a specific hardness and combining it with specific injection conditions using a nanopipette, it is possible to achieve both high efficiency of substance introduction into suspension cells and high cell viability. By fixing suspension cells on a gel plate of a specific hardness, it is possible to inject the cells without them moving when the nanopipette comes into contact with them.

[0043] In another embodiment, the method for introducing a substance into cells according to the present disclosure includes the following steps: a') (Transfer process of suspension cell gel sheet) a'-1) A process of seeding suspended cells onto a substrate and layering a gel on top of the cells; a'-2) A step of detaching the gel from the substrate and obtaining a gel sheet on which cells have been transferred to the gel surface; a'-3) Laminate the gel sheets on the gel plate medium with the cell transcription side facing upwards, and evaluate the hardness of the entire laminated gel using the hardness evaluation method of JIS K6503, with a breaking load of 2.0 to 30 [N] or 0.5 × 10 5 ~7×10 5 A process to achieve a Young's modulus of [Pa] or a 1mm pressure stress of 1.0 to 8 [N]; b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution (pipette positioning step); c) A step (pipette approach step) in which the current between the inside of the nanopipette and the electrolyte is measured and the nanopipette is moved toward the cell to a position where the decrease in current from the steady current is between 2% and 50% (set current decrease rate); d) The process of moving a nanopipette towards the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell (pipette puncture process); e) A process of applying a voltage between the inside of the nanopipette and the electrolyte to expel substances into the cells (substance expulsion process); and f) The process of removing the nanopipette (pipette removal process). The overall scheme of the method disclosed in this embodiment is shown in Figure 6. Cells (211) are seeded on a substrate (221) (first scheme in Figure 6), a gel is layered onto the cells (211) on the substrate (221) to form a gel sheet (213') (second scheme in Figure 6), the gel sheet (213') is peeled off from the substrate (221) to transfer the cells (211) to the gel sheet (213') (third scheme in Figure 6), and the gel sheet (213') onto which the cells (211) have been transferred is layered onto a gel plate medium (213), and a substance (111) is introduced into the cells (211) using a nanopipette (101) (fourth scheme in Figure 6). In this method of layering a gel sheet onto a gel plate, cell migration is more easily suppressed, and the success rate of substance introduction can be increased.

[0044] (Step a): Suspension cell seeding step) Step a) is the step of seeding cells that are in a suspension state onto a gel plate medium. Cells can be made into a suspension state by, for example, culturing them in a liquid medium. Seeding of suspension cells may be performed, for example, by seeding a culture medium containing suspension cells onto a gel plate medium.

[0045] (Step a'): Transfer of suspended cell gel sheet) Step a') is a step of transferring cells in a suspension state to a gel sheet and stacking the gel sheet with the transferred cells on a gel plate culture medium. The substrate is not particularly limited, but examples include glass slides, plastic plates, cell culture dishes, etc., but it is preferable to use a material with low cell adhesion. The gel used for the gel sheet may be one of those exemplified as gel plate culture medium above, or it may be the same type of gel as the gel plate culture medium, or a different gel from the gel plate culture medium.

[0046] (Step b): Pipette positioning step) Step b) is a step in which a nanopipette filled with a substance is positioned at a cell-corresponding location in the electrolyte. In this step, by arbitrarily selecting the puncture site of a nanopipette with a tip diameter of several tens of nanometers, it becomes possible to introduce the substance into any single cell. Furthermore, it is also possible to arbitrarily select whether the puncture site in a single cell is the cell nucleus or the cytoplasm. This is a minimally invasive method for cells, and the ability to introduce substances at the single-cell level and in a positionally selective manner within the cell is a unique feature of this invention that cannot be achieved by existing methods. Methods for filling the inside of the nanopipette with a substance include, for example, centrifugation and aspiration. Centrifugation may be performed by attaching the nanopipette to a centrifuge holder, filling it with a substance from the base opening of the nanopipette, and centrifuging the centrifuge holder with the nanopipette attached in a centrifuge. Aspiration may be performed by aspirating the substance from the tip opening. Nanopipettes filled with material may be mounted, for example, on a three-dimensional (xyz) moving pipette holder as described above, for positioning. The "cell-corresponding position" refers to the position on the "z-axis" relative to the location of the target cell. Positioning may be performed using a rough actuator, a fine actuator, or a combination of both. The rough actuator and fine actuator may be driven by manual control via a position input device or by programmed automatic control. Furthermore, positioning can also be performed using a microscope stage for the XY axes.

[0047] (Process c): Pipette approach process) Step c) is a process in which the current between the inside of the nanopipette and the electrolyte is measured and the nanopipette is moved toward the cell to a position where the rate of decrease from the steady current is the set current decrease rate (the "pipette pause position (P2)" in Figure 1). The "direction of approaching the cell" refers to the direction toward the cell on the "z axis". The current (ionic current) between the inside of the nanopipette and the electrolyte can be measured by a current measurement circuit as the current between the pipette electrode and the reference electrode. In step b), in order to measure the current, it is preferable to apply a low voltage (set approach voltage) between the inside of the nanopipette and the electrolyte (i.e., between the pipette electrode and the reference electrode) set so as not to cause the filling solution to flow out due to electroosmotic flow. The change in current between the inside of the nanopipette and the electrolyte will be explained with reference to Figure 1. When the tip of the nanopipette is outside the electrolyte, the inside of the nanopipette and the electrolyte are disconnected, and the current (I) is zero (I0). When the tip of the nanopipette reaches the electrolyte surface (P0) and enters the electrolyte, a connection is established between the inside of the nanopipette and the electrolyte, and the current (I) increases. For a while thereafter, even if the nanopipette is advanced, the current (I) does not change significantly and remains in a steady state. This current (I) is called the steady-state current (I1). When the tip of the nanopipette reaches a position very close to the cell surface (P3) ("current drop start point position (P1)"), the current begins to drop sharply in proportion to the distance between the cell and the tip of the nanopipette. Subsequently, the nanopipette is advanced to a position where the current (I) is reduced by a set current drop rate (R) from the steady-state current (I1) to a current (I2) ("pipette pause position (P2)"). The nanopipette may pause at the pipette pause position (P2). The movement of the nanopipette in step b) may be performed using a rough actuator, a fine actuator, or a combination of both. At least the movement after the current drop start point (P1) is preferably performed by a fine actuator. The movement of the nanopipette in step b) is preferably performed by automated control by a program. By pre-setting the current drop rate according to the cell type, it is possible to suppress damage to cells and achieve highly efficient introduction of substances into cells.

[0048] <Current Drop Rate> The set current reduction rate is a value optimized for the cell type. The set current reduction rate is preferably 2-50%, more preferably 10-40%, and most preferably 15-20%.

[0049] <Set Approach Voltage> The set approach voltage is a value optimized for the cell type. The set approach voltage is preferably -2 to +2V, more preferably -1.0 to +1.0V.

[0050] (Step d): Pipette insertion process) Step d) is the step of moving the nanopipette from the position (i.e., the pipette pause position (P2)) toward the cell at a set puncture distance and puncturing the cell. "Along the cell" means the direction toward the cell side (the inside of the cell) on the "z axis". The movement of the nanopipette in the pipette puncture step is usually performed by a fine actuator under automatic control by a program. Furthermore, it is preferable that the movement of the nanopipette in the pipette puncture step is performed at a faster speed than in the pipette approach step. By pre-setting the puncture distance according to the cell type, it is possible to suppress damage to the cell and achieve highly efficient introduction of substances into the cell.

[0051] <Setting Penetration Distance> The set puncture distance is a value optimized for the cell type. The set puncture distance is preferably 1 to 50 μm, more preferably 3 to 40 μm.

[0052] (Process e): Substance discharge process) Step e) is a step in which a voltage is applied between the inside of the nanopipette and the electrolyte to expel the substance into the cell. Depending on the type of substance to be introduced into the cell (e.g., protein, a mixture or complex containing protein, a mixture or complex containing protein and nucleic acid, nucleic acid, and dye, etc.), the voltage is applied so that the inside of the nanopipette becomes positively charged and the electrolyte becomes negatively charged, or so the inside of the nanopipette becomes negatively charged and the electrolyte becomes positively charged. If the substance to be introduced into the cell is a positively charged substance, the voltage is applied so that the inside of the nanopipette becomes positively charged and the electrolyte becomes negatively charged. If the substance to be introduced into the cell is a negatively charged substance, the voltage is applied so that the inside of the nanopipette becomes negatively charged and the electrolyte becomes positively charged. The voltage is applied using a set injection voltage (set applied voltage: V I It is preferable to perform the procedure with the set injection time (set application time: T1).

[0053] <Setting Injection Voltage> The set injection voltage is a value optimized for the cell type. For positively charged materials, the set injection voltage is preferably -11 to +11V, more preferably -10 to +10V, and most preferably 4V to 10V. Furthermore, the set injection voltage can be a value optimized for the cell type, as described in "Examples of Setting Parameters" below.

[0054] <Set Injection Time> The set injection time is a value optimized for the cell type. The set injection time is preferably 0.1 to 10 seconds, more preferably 0.5 to 5.0 seconds.

[0055] (Process f): Pipette removal process) Step f) is the step of withdrawing the nanopipette. "Away from the cell" refers to the direction away from the cell (inside the cell) on the "z-axis". The movement of the nanopipette may be performed at a set withdrawal distance. The movement of the nanopipette in the pipette withdrawal step is usually performed by a fine actuator under automatic control by a program.

[0056] <Setting Retraction Distance> The set extraction distance is a value optimized for the cell type. Preferably, the set extraction distance is within the range of 40 μm or more.

[0057] [Method for producing genetically modified cells] In one embodiment, the method disclosed herein can also be used as a method for producing genetically modified cells. The method for producing genetically modified cells according to this disclosure includes the following steps. a) A process of seeding suspended cells onto a gel plate medium of a specific hardness (suspension cell seeding process); b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution (pipette positioning step); c) A step (pipette approach step) in which the current between the inside of the nanopipette and the electrolyte is measured and the nanopipette is moved toward the cell to a position where the decrease in current from the steady current is between 2% and 50% (set current decrease rate); d) The process of moving a nanopipette towards the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell (pipette puncture process); e) A process of applying a voltage between the inside of the nanopipette and the electrolyte to expel substances into the cells (substance expulsion process); and f) The process of removing the nanopipette (pipette removal process).

[0058] In another embodiment, the method for introducing a substance into cells according to the present disclosure includes the following steps: a') (Transfer process of suspension cell gel sheet) a'-1) A process of seeding suspended cells onto a substrate and layering a gel on top of the cells; a'-2) A step of detaching the gel from the substrate and obtaining a gel sheet on which cells have been transferred to the gel surface; a'-3) Laminate the gel sheets on the gel plate medium with the cell transcription side facing upwards, and evaluate the hardness of the entire laminated gel using the hardness evaluation method of JIS K6503, with a breaking load of 2.0 to 30 [N] or 0.5 × 10 5 ~7×10 5 A process to achieve a Young's modulus of [Pa] or a 1mm pressure stress of 1.0 to 8 [N]; b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution (pipette positioning step); c) A step (pipette approach step) in which the current between the inside of the nanopipette and the electrolyte is measured and the nanopipette is moved toward the cell to a position where the decrease in current from the steady current is between 2% and 50% (set current decrease rate); d) The process of moving a nanopipette towards the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell (pipette puncture process); e) A process of applying a voltage between the inside of the nanopipette and the electrolyte to expel substances into the cells (substance expulsion process); and f) The process of removing the nanopipette (pipette removal process).

[0059] Genetic modification materials include positively charged and negatively charged materials. Positively charged materials include, for example, gene modification materials based on mixtures or complexes containing proteins and nucleic acids, such as genome editing materials (e.g., the Cas9-sgRNA-RNP complex used in the CRISPR-Cas9 system, as well as materials used in genome editing systems such as CRISPR-Cas3, ZFN, TALEN, and PPR). Negatively charged materials include, for example, nucleic acids (DNA, RNA). Examples of nucleic acids include DNA such as plasmid vectors, antisense RNA, RNA such as siRNA, etc.

[0060] The conditions for the method of producing genetically modified cells can be those described in the method for introducing substances into cells in this disclosure.

[0061] [Control program for a device that delivers substances to cells] This disclosure also provides a control program for a device for introducing substances into cells. The program of this disclosure includes instructions for performing the following steps: a) A process of positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte of a gel plate medium of a specific hardness, in which suspended cells have been seeded and the gel plate is filled with an electrolyte (pipette positioning process); b) A step (pipette approach step) in which the current between the inside of the nanopipette and the electrolyte is measured and the nanopipette is moved toward the cell to a position where the decrease in current from the steady state is between 2% and 50% (a set current decrease rate); c) The process of moving the nanopipette towards the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell (pipette puncture process); d) A process of applying a voltage between the inside of the nanopipette and the electrolyte to expel substances into the cells (substance expulsion process); and e) The process of removing the nanopipette (pipette removal process).

[0062] The program of this disclosure may further include instructions for controlling the approach voltage, injection voltage (applied voltage), injection time (applied time), and withdrawal distance.

[0063] The conditions controlled by the program of this disclosure can be those described in the method for introducing substances into cells of this disclosure. [Examples]

[0064] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited thereto.

[0065] <Acquisition of shares> The microalgae strains Tetraselmis sp. and Haematococcus sp. used in this example were obtained as isolated specimens from the Microbial Lineage Preservation Facility of the National Institute for Environmental Studies. In addition, Chlamydomonas reinhardtii Dangeard (ATCC PRA-142), a microalgae strain lacking a cell wall, was obtained from the American Type Culture Collection (ATCC).

[0066] <Preparation of gel plates for freshwater microalgae> Gel plates for Haematococcus sp. strains and Chlamydomonas reinhardtii Dangeard (ATCC PRA-142) strains, which mainly inhabit freshwater, were prepared using the following procedure. Agarose (Agarose LM low melting point, Nakalai) was added to BG11 medium (Thermo Fisher Scientific) at the concentrations shown in Table 1. Subsequently, the mixture was sterilized using an autoclave at 121°C for 20 minutes. Antibiotics (50 μg / mL final concentration of ampicillin and kanamycin) were then added. Next, approximately 5.8 mL was dispensed into a 3.5 cm diameter polystyrene petri dish with a positioning grid, and allowed to stand to prepare a gel plate with a gel thickness of 6 mm.

[0067] <Preparation of gel plates for freshwater microalgae used in gel transfer methods> In the preparation of the freshwater microalgae gel plate described above, a freshwater microalgae plate with a gel thickness of 4.5 mm was prepared by dispensing approximately 4.3 mL of a solution containing BG11 medium, agarose, and antibiotics into a polystyrene petri dish.

[0068] <Preparation of gel plates for marine microalgae> A gel plate for Tetraselmis sp. strain mainly living in seawater was prepared by the following procedure. Agarose (Agarose LM low melting point, manufactured by Nacalai) was added to the IMK medium (manufactured by Fujifilm Wako Pure Chemical Corporation) so as to reach the concentration described in Table 1. Subsequently, sterilization treatment was performed at 121 °C for 20 min using an autoclave. Subsequently, antibiotics (ampicillin and kanamycin with a final concentration of 50 μg / mL) were added. Subsequently, about 5.8 mL was dispensed into a polystyrene petri dish with a grid for position confirmation with a diameter of 3.5 cm, and by allowing it to stand, a gel plate with a gel thickness of 6 mm was prepared.

[0069] <Preparation of Gel Plate for Marine Microalgae Used in Gel Transfer Method> In the preparation of the gel plate for marine microalgae used in the above gel transfer method, by setting the dispensing volume of the solution containing the IMK medium, agarose and antibiotics into a polystyrene petri dish to about 4.3 mL, a gel plate for marine microalgae used in the gel transfer method with a gel thickness of 4.5 mm was prepared.

[0070] <Inoculation of a Single Strain of Haematococcus sp.> 15 μL of the microalgae suspension was dropped onto a gel plate for freshwater microalgae (gel thickness: 6 mm) and spread with a spreader. It was incubated at 25 °C for several days to form colonies. Subsequently, one colony was collected with a sterilized inoculation loop and inoculated onto a fresh gel plate. It was incubated at 25 °C and a photosynthetic photon flux density of 25 - 100 μmol photons / m 2 / s for several days to form colonies consisting of a single strain.

[0071] <Inoculation of a Single Strain of Tetraselmis sp.> 15 μL of the microalgae suspension was dropped onto a gel plate for marine microalgae (gel thickness: 6 mm) and spread with a spreader. It was incubated at 25 °C for several days to form colonies. Subsequently, one colony was collected with a sterilized inoculation loop and inoculated into a fresh gel plate of IMK medium. 25 °C, photosynthetic photon flux density 25 - 100 μmol photons / m 2Incubated for several days at / s to form colonies consisting of single strains.

[0072] <Inoculation of a single strain of Chlamydomonas reinhardtii Dangeard (ATCC PRA-142)> 15 μL of the microalgae suspension was dropped onto a gel plate for freshwater microalgae (gel thickness: 6 mm) and spread with a spreader. Incubated at 25°C for several days to form colonies. Subsequently, one colony was collected with a sterilized inoculation loop and inoculated into a fresh gel plate IMK medium. 25°C, photosynthetic photon flux density 25 - 100 μmol photons / m 2 Incubated for several days at / s to form colonies consisting of single strains.

[0073] <Liquid culture of a single strain of Haematococcus sp.> 50 mL of sterilized BG11 liquid medium (manufactured by Thermo Fisher Scientific) was added to a sterilized 100 mL Erlenmeyer flask, and one colony was collected from the gel plate with an inoculation loop and inoculated into the liquid medium. Sealed with a sterilized silicon stopper, 25°C, photosynthetic photon flux density 25 - 100 μmol photons / m 2 Shaken culture was carried out at / s for 3 days.

[0074] <Liquid culture of a single strain of Tetraselmis sp.> 50 mL of sterilized IMK liquid medium (manufactured by Fujifilm Wako Pure Chemical Corporation) was added to a sterilized 50 mL Erlenmeyer flask, and one colony was collected from the gel plate with an inoculation loop and inoculated into the liquid medium. Sealed with a sterilized silicon stopper, 25°C, photosynthetic photon flux density 25 - 100 μmol photons / m 2 Shaken culture was carried out at / s for 3 days.

[0075] <Liquid culture of a single strain of Chlamydomonas reinhardtii Dangeard (ATCC PRA-142)> 50 mL of sterilized IMK liquid medium (manufactured by Fujifilm Wako Pure Chemical Corporation) was added to a sterilized 50 mL Erlenmeyer flask. One colony was picked up from a gel plate with a platinum loop and inoculated into the liquid medium. It was stoppered with a sterilized silicon stopper and cultured with shaking at 25 °C under a photon flux density of 25 - 100 μmol photons / m 2 / s for 3 days.

[0076] <Preparation of gel plate with Haematococcus sp. cells fixed: One-layer gel structure> 2 mL of the microalgae cell suspension was collected from the Erlenmeyer flask used for the liquid culture with a pipette or the like and placed in a sterilized tube container. The concentration of the cell suspension was estimated using a hemocytometer, and a cell suspension adjusted by adding BG11 liquid medium (manufactured by Thermo Fisher Scientific) was obtained so that the final concentration became 1.0×10 5 cells / mL. Subsequently, 15 μL of the cell suspension was dropped onto a gel plate for freshwater microalgae (gel thickness: 6 mm) on a previously prepared petri dish with a diameter of 3.5 cm, spread with a spreader, and left standing for 1 day to obtain a gel plate with microalgae cells fixed.

[0077] <Preparation of gel plate with Tetraselmis sp. cells fixed: One-layer gel structure> 2 mL of the microalgae cell suspension was collected from the Erlenmeyer flask used for the liquid culture with a pipette or the like and placed in a sterilized tube container. The concentration of the cell suspension was estimated using a hemocytometer, and a cell suspension adjusted by adding IMK liquid medium (manufactured by Fujifilm Wako Pure Chemical Corporation) was obtained so that the final concentration became 1.0×10 5 cells / mL. Subsequently, 15 μL of the cell suspension was dropped onto a gel plate for marine microalgae (gel thickness: 6 mm) on a previously prepared petri dish with a diameter of 3.5 cm, spread with a spreader, and left standing for 1 day to obtain a gel plate with microalgae cells fixed.

[0078] <Preparation of gel plate with Chlamydomonas reinhardtii Dangeard (ATCC PRA-142) cells fixed: One-layer gel structure> 2 mL of microalgae cell suspension was collected from the Erlenmeyer flask used for the liquid culture using a pipette and placed in a sterile tube container. The concentration of the cell suspension was estimated using a hemocytometer, and the final concentration was 1.0 × 10⁶. 5 A cell suspension was obtained by adding IMK liquid medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to a cell / mL concentration. Subsequently, 15 μL of the cell suspension was dropped onto a pre-prepared 3.5 cm diameter petri dish containing a gel plate for marine microalgae (gel thickness: 6 mm), spread with a spreader, and left to stand for 1 day to obtain a gel plate with fixed microalgae cells.

[0079] <Preparation of a gel plate with fixed Haematococcus sp. cells using gel transfer method: Two-layer gel structure> Cell fixation of Haematococcus sp. strains, which mainly inhabit freshwater, was performed by gel transfer using the following procedure: 2 mL of microalgae cell suspension was collected from the Erlenmeyer flask used for the liquid culture using a pipette and placed in a sterile tube container. The concentration of the cell suspension was estimated using a hemocytometer, and the final concentration was 1.0 × 10⁶. 5A cell suspension was obtained by adding BG11 liquid medium (Thermo Fisher Scientific) to a concentration of cells / mL. Next, 10 μL of the cell suspension was dropped onto an 18 mm × 18 mm coverslip and spread by gravity. Subsequently, 200 μL of a solution containing agarose (Agarose LM low melting point, Nakalai) at the concentrations listed in Table 1, and 50 μg / mL each of Ampicillin (Fujifilm Wako Pure Chemical Industries, Ltd.) and Kanamycin (Fujifilm Wako Pure Chemical Industries, Ltd.) was dropped onto the microalgae cells spread on the coverslip. A new 18 mm × 18 mm coverslip was placed on top, sandwiching the microalgae cells and the gel-like medium containing agarose, and it was left to stand at room temperature for 30 minutes. Next, the coverslip on the side where the microalgae cell suspension was dropped was removed, and the microalgae cells were transferred to the gel sheet. Next, the cover slip on the opposite side was removed from the gel sheet, and a single 1.5 mm thick gel sheet with microalgae cells transferred to its surface was obtained. The obtained single gel sheet was then layered onto a pre-prepared gel plate for marine microalgae (gel thickness: 4.5 mm) used in the gel transfer method, placed on a 3.5 cm diameter petri dish, and left to stand for 30 minutes to obtain a gel plate with microalgae cells fixed by the gel transfer method.

[0080] <Preparation of a gel plate with Tetraselmis sp. cells fixed by gel transfer method: Two-layer gel structure> Cell fixation of Tetraselmis sp. strains, which mainly inhabit seawater, was performed by gel transfer using the following procedure: 2 mL of microalgae cell suspension was collected from the Erlenmeyer flask used for the liquid culture using a pipette and placed in a sterile tube container. The concentration of the cell suspension was estimated using a hemocytometer, and the final concentration was 1.0 × 10⁶. 5A cell suspension was obtained by adding IMK liquid medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to a concentration of cells / mL. Next, 10 μL of the cell suspension was dropped onto an 18 mm × 18 mm coverslip and spread by gravity. Subsequently, 200 μL of a solution containing agarose (Agarose LM low melting point, manufactured by Nakalai) at the concentrations shown in Table 1, and 50 μg / mL each of Ampicillin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and Kanamycin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dropped onto the microalgae cells spread on the coverslip. A new 18 mm × 18 mm coverslip was placed on top, sandwiching the microalgae cells and the gel-like medium containing agarose, and it was left to stand at room temperature for 30 minutes. Next, the coverslip on the side where the microalgae cell suspension was dropped was removed, and the microalgae cells were transferred to the gel sheet. Next, the cover slip on the opposite side was removed from the gel sheet, and a single 1.5 mm thick gel sheet with microalgae cells transferred to its surface was obtained. The obtained single gel sheet was then layered onto a pre-prepared gel plate for marine microalgae (gel thickness: 4.5 mm) used in the gel transfer method, placed on a 3.5 cm diameter petri dish, and left to stand for 30 minutes to obtain a gel plate with microalgae cells fixed by the gel transfer method.

[0081] <Preparation of Reagent Solution> FITC dextran (Merck) was diluted with PBS to a concentration of 10 mg / mL to obtain a FITC dextran / PBS solution. GFP (Merck) was diluted with PBS to a concentration of 0.4 mg / mL to obtain a GFP / PBS suspension. FAM-Oligo DNA (90 mer, 29.7 kDa, Eurofins Genomics) was diluted with PBS to a concentration of 100 μM to obtain a FAM-Oligo DNA / PBS suspension.

[0082] <Preparation of nanopipettes filled with reagent solution> 5 μL of FITC dextran / PBS solution, GFP / PBS suspension, or FAM-Oligo DNA suspension was placed in a microloader mounted on a centrifuge holder, filled into a nanopipette from the top, and centrifuged in a benchtop centrifuge for 30-60 seconds. The silver wire attached to the nanopipette was inserted through the hole at the top where the reagent solution was filled and secured with a dedicated jig. At this time, it was confirmed that the silver wire was immersed in the reagent solution.

[0083] <Injection of reagent solution into microalgae cells> 3 mL of PBS was added as an electrolyte solution to a gel plate medium (3.5 cm diameter petri dish) on which microalgae cells were immobilized. The gel plate sample with immobilized microalgae cells was placed in a microscope (Ti2E, Nikon). A nanopipette filled with reagent solution was attached to the head of a substance delivery device for microalgae cells (YOKOGAWA Single Cellome™ System UNIT SU10), and the rotary angle was adjusted. The reference electrode attached to the SU10 head was placed in the PBS electrolyte solution of the gel plate sample with immobilized microalgae, and the SU10 software (measurement mode) was started. Using manual or Liquid detect mode, the nanopipette was brought into contact with the PBS electrolyte solution, and it was confirmed in the software that the current value had risen from 0 nA. The SU10 joystick was operated to position the nanopipette at the location corresponding to the target cells in the sample. The SU10 software was switched from measurement mode to delivery mode. The parameters according to the microalgae samples listed in Table 1 were set. The SU10 software was used to start the process, measuring the current between the inside of the nanopipette and the electrolyte. The nanopipette was then moved towards the cells to a position where the current reduction rate from the steady state was between 2.5% and 49.5% (a set current reduction rate). The nanopipette was then moved towards the cells at a set puncture distance of between 1.5 μm and 49.5 μm to introduce the reagent solution into the target cells. Next, the joystick was operated to position the nanopipette to the next target cell, and the substance was introduced into 40 cells on the gel plate sample per condition.

[0084] <Method for evaluating gel plate hardness> The hardness of the gel plate was measured using a rheometer under conditions compliant with the gel strength measurement described in JIS K6503. Specifically, the gel plate was placed in a rheometer (CR-100, manufactured by Sun Science Co., Ltd.), and the Young's modulus [Pa], breaking load [N], and stress at 1 mm penetration [N] were evaluated as indicators of gel plate hardness when a plunger (φ12.7 mm, height 35 mm) was inserted into the gel plate at a speed of 1 mm / sec.

[0085] <Method for evaluating the success rate of substance introduction> The efficiency of substance introduction into microalgae cells was evaluated by counting the number of cells out of 40 treated cells that exhibited fluorescence emission originating from the introduced substance. Specifically, a gel plate sample was placed in a microscope (Ti2E, Nikon), and the number of cells exhibiting fluorescence emission around 518 nm (excitation wavelength 475 nm) from FITC dextran or around 507 nm (excitation wavelength 475 nm) from GFP was counted in fluorescence observation mode. The substance introduction efficiency (success rate of substance introduction) was calculated using the formula "number of cells exhibiting fluorescence emission / 40 cells × 100". The evaluation index for substance introduction efficiency is as follows. 0% or more but less than 2% ··× 2% to less than 10% ··△ 10% or more but less than 30% ··○ 30% or more and 100% or less ◎

[0086] <Method for evaluating the maintenance of cell division function> Cell viability is determined by treating cells with a substance and placing them on a gel plate at 25°C with a photon flux density of 25-100 μmol photons / m². 2 After static culture under / s conditions for 3 days, microscopic observation was performed, and the presence or absence of cleaved cells derived from cell division was evaluated using the following indicators. Out of 40 cells, 4 or fewer cells underwent cell division. Out of 40 cells, 5 or more cells underwent cell division, and 29 or fewer cells underwent division. Out of 40 cells, 30 or more cells underwent cell division.

[0087] As shown in the examples, when suspended cells are fixed to a gel plate of a specific hardness and combined with specific puncture conditions using a nanopipette, an effect is achieved that combines high efficiency of substance introduction into suspended cells with high cell viability. By fixing suspended cells to a gel plate of a specific hardness, it is possible to introduce substances without the cells moving when the nanopipette comes into contact with them, even for cells with particularly hard cell walls. Furthermore, in a method in which gel sheets with cells transferred to the gel are stacked on a gel plate, cell movement is more easily suppressed, and a tendency for the success rate of substance introduction to be higher was observed.

[0088] Furthermore, the comparative examples show that simply inhibiting cell migration during nanopipette puncture by setting the gel plate hardness to a specific range is insufficient, as even if substance introduction into cells with cell walls is successful, a decrease in cell viability after puncture may be observed. As per the present invention, cell viability can be improved by setting a specific gel plate hardness and specific nanopipette puncture conditions. This is thought to be because, by setting a specific gel plate hardness and nanopipette puncture conditions, cells are appropriately fixed to the gel plate medium, the resilience of the gel plate medium and the puncture pressure of the nanopipette device minimize physical impact on the cells, a weak ionic current reduces cell load, and the weak current flowing through the cells during puncture under the medium hardness of the present invention contributes to the repair of cell damage.

[0089] [Table 1] [Industrial applicability]

[0090] This disclosure provides a method for introducing substances into cells that achieves both high substance introduction efficiency and high cell viability. Furthermore, this disclosure enables the provision of an automated system for introducing substances into cells using this substance introduction method. This technology makes it possible to introduce substances such as nucleic acids and genome editing tools into cells that have cell walls, thereby contributing to improved breeding efficiency. [Explanation of symbols]

[0091] 101 Nanopipette 102 Pipette electrodes 103 Three-dimensional (xyz) movable pipette holder 111 Substances introduced into cells 201 Cell holder 202 Reference electrode 211 cells 211' Colony 212 Electrolyte 213 Gel plate culture medium 301 Current measurement circuit 302 Voltage Application Circuit 401 Observation section of optical microscope 402 Optical microscope stage D Setting puncture distance Ionic current I0 Base Current I1 steady current Current reduced by I2 setting current reduction rate P0 Electrolyte surface position P1 Current drop starting point position P2 Pipette pause position P3 cell surface position P4 Material discharge position R Set current reduction rate T time T1 Injection time (application time) V Voltage V A Approach voltage V I Injection voltage (applied voltage)

Claims

1. Methods for introducing substances into cells, including the following: a) The floating cells were subjected to a breaking load of 2.0 to 30 [N] or 0.5 × 10⁻¹⁶ according to the hardness evaluation of JIS K6503. ５ ~7 x 10 ５ A step of sowing seeds in a gel plate medium with a Young's modulus of [Pa] or a hardness that results in a 1 mm pressure stress of 1.0 to 8 [N]; b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution; c) A step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the decrease in current from the steady state is between 2% and 50%; d) A step of moving a nanopipette in the direction of the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell; e) A step of applying a voltage between the inside of the nanopipette and the electrolyte to expel a substance into the cell; and f) The process of removing the nanopipette.

2. Methods for introducing substances into cells, including the following: a') a'-1) A step of seeding suspended cells onto a substrate and layering a gel on top of the cells; a'-2) A step of peeling the gel from the substrate and obtaining a gel sheet on which cells have been transferred to the gel surface; a'-3) Laminate the gel sheets on the gel plate medium with the cell transfer surface facing upwards, and evaluate the hardness of the entire laminated gel according to JIS K6503, determining a breaking load of 2.0 to 30 [N] or 0.5 × 10 ５ ~7 x 10 ５ A process to achieve a Young's modulus of [Pa] or a 1 mm pressure stress of 1.0 to 8 [N]; b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution; c) A step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the decrease in current from the steady state is between 2% and 50%; d) A step of moving a nanopipette in the direction of the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell; e) A step of applying a voltage between the inside of the nanopipette and the electrolyte to expel a substance into the cell; and f) The process of removing the nanopipette.

3. The method according to claim 1 or 2, wherein the substance is a protein, a mixture or complex containing a protein, a mixture or complex containing a protein and a nucleic acid, a nucleic acid, or a dye.

4. The method according to claim 3, wherein the substance is a genome editing substance.

5. The substance is either a positively charged substance or a negatively charged substance, If the substance is a positively charged substance, step e) is performed by applying a voltage such that the inside of the nanopipette becomes positively charged and the electrolyte becomes negatively charged. If the substance is a negatively charged substance, step e) is performed by applying a voltage such that the inside of the nanopipette becomes negatively charged and the electrolyte becomes positively charged. The method according to claim 3.

6. The method according to claim 5, wherein step e) is performed by applying a set applied voltage of -11V or more and +11V or less and a set applied time of 0.1 seconds or more and 5.0 seconds or less.

7. Methods for producing genetically modified cells, including the following: a) The floating cells were subjected to a breaking load of 2.0 to 30 [N] or 0.5 × 10⁻¹⁶ according to the hardness evaluation of JIS K6503. ５ ~7 x 10 ５ A step of sowing seeds in a gel plate medium with a Young's modulus of [Pa] or a hardness that results in a 1 mm pressure stress of 1.0 to 8 [N]; b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution; c) A step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the decrease in current from the steady state is between 2% and 50%; d) A step of moving a nanopipette in the direction of the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell; e) A step of applying a voltage between the inside of the nanopipette and the electrolyte to expel a substance into the cell; and f) The process of removing the nanopipette.

8. Methods for producing genetically modified cells, including the following: a') a'-1) A step of seeding suspended cells onto a substrate and layering a gel on top of the cells; a'-2) A step of peeling the gel from the substrate and obtaining a gel sheet on which cells have been transferred to the gel surface; a'-3) Laminate the gel sheets on the gel plate medium with the cell transfer surface facing upwards, and evaluate the hardness of the entire laminated gel according to JIS K6503, determining a breaking load of 2.0 to 30 [N] or 0.5 × 10 ５ ~7 x 10 ５ A process to achieve a Young's modulus of [Pa] or a 1 mm pressure stress of 1.0 to 8 [N]; b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution; c) A step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the decrease in current from the steady state is between 2% and 50%; d) A step of moving a nanopipette in the direction of a cell at a set puncture distance of 1 μm to 50 μm to puncture a cell having a cell wall; e) A step of applying a voltage between the inside of the nanopipette and the electrolyte to expel a substance into the cell; and f) The process of removing the nanopipette.

9. Genetically modified cells produced by methods including the following: a) A step of seeding floating cells in a gel plate medium having a hardness such that, in the hardness evaluation according to JIS K6503, the breaking load is 2.0 to 30 [N], or the Young's modulus is 0.5 × 10 ５ to 7 × 10 ５ [Pa], or the 1 mm pressing stress is 1.0 to 8 [N]; b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution; c) A step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the decrease in current from the steady state is between 2% and 50%; d) A step of moving a nanopipette in the direction of the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell; e) A step of applying a voltage between the inside of the nanopipette and the electrolyte to expel a substance into the cell; and f) The process of removing the nanopipette.

10. Genetically modified cells produced by methods including the following: a') a'-1) A step of seeding suspended cells onto a substrate and layering a gel on top of the cells; a'-2) A step of peeling the gel from the substrate and obtaining a gel sheet on which cells have been transferred to the gel surface; a'-3) Laminate the gel sheets on the gel plate medium with the cell transfer surface facing upwards, and evaluate the hardness of the entire laminated gel according to JIS K6503, determining a breaking load of 2.0 to 30 [N] or 0.5 × 10 ５ ~7 x 10 ５ A process to achieve a Young's modulus of [Pa] or a 1 mm pressure stress of 1.0 to 8 [N]; b) A step of filling a gel plate medium on which cells have been seeded with an electrolyte solution, and positioning a nanopipette filled with a substance at the cell-corresponding position in the electrolyte solution; c) A step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the decrease in current from the steady state is between 2% and 50%; d) A step of moving a nanopipette in the direction of the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell; e) A step of applying a voltage between the inside of the nanopipette and the electrolyte to expel a substance into the cell; and f) The process of removing the nanopipette.

11. A control program for a cell substance delivery device, including instructions to perform the following: a) A step of positioning a nanopipette filled with a substance at a cell-corresponding position in the electrolyte of a gel plate medium of a specific hardness, in which suspended cells have been seeded and the gel plate is filled with an electrolyte; b) A step of measuring the current between the inside of the nanopipette and the electrolyte and moving the nanopipette toward the cell to a position where the decrease in current from the steady state is between 2% and 50%; c) A step of moving a nanopipette in the direction of the cell at a set puncture distance of 1 μm to 50 μm to puncture the cell; d) A step of applying a voltage between the inside of the nanopipette and the electrolyte to expel a substance into the cell; and e) The process of removing the nanopipette.

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