Compound introduction device and compound introduction method

Abstract

To achieve both of the efficient introduction of a compound into a cell and the efficient production of a cell whose properties have been modified by the introduction.SOLUTION: One embodiment of the present invention is a compound introduction device for introducing a compound into a cell, which has: a supply channel for supplying a cell suspension containing the cell and the compound; introduction means for introducing the compound into the cell; and an ejection port for ejecting the cell suspension containing the cell into which the compound has been introduced, where with respect to the flow direction of the cell suspension flowing through the supply channel, the height of the supply channel is more than 1.0 times and less than 2.0 times of the diameter of the cell, the width of the supply channel is greater than 1.0 times and less than 2.0 times of the diameter of the cell, and the diameter of the ejection port is more than 1.0 times and less than 2.0 times of the diameter of the cell.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present disclosure relates to a compound introduction device and a compound introduction method for introducing a compound into a cell. [Background technology]

[0002] There are known techniques for introducing compounds, including genes, into cells. The introduction methods include chemical introduction methods using cationic substances or viruses into living cells, or biological introduction methods. In addition, various methods have been developed, such as electroporation, which is expected to be less toxic, physical introduction methods such as gene gun methods, and microinjection, which has high selectivity for the type of compound to be introduced and is highly reliable in introduction.

[0003] In recent years, the emergence of cell therapy drugs or induced pluripotent stem cells that modify the properties of cells, obtained by introducing genes or other compounds into cells, has created a demand for more efficient compound introduction methods. However, there is no established means for achieving such high efficiency, which has been an issue.

[0004] As one of the means for solving the above problems, Patent Document 1 describes an introduction method using a microchannel. In this method, cells are dispersed in a liquid containing a compound to be introduced and passed through a microchannel of approximately the same size as the cells. In this way, by applying a shear force to the cells, holes are temporarily generated in the cell membrane, and the compound to be introduced outside the cells can be introduced into the cells, making it possible to efficiently introduce the compound into each cell.

[0005] Patent Document 2 describes that a target compound can be effectively introduced into cells by generating pressure and shear force in a micro-sized space using an inkjet device used as an image recording device. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US Patent Application Publication No. 2018 / 0003696 [Patent Document 2] Patent No. 5645657 [Non-patent literature]

[0007] [Non-Patent Document 1] Campbell, A et al. 2020 Frontiers in Bioengineering and Biotechnology, 8 doi: 10.3389 / fbioe.2020.00082 Summary of the Invention [Problem to be solved by the invention]

[0008] An embodiment of the present invention aims to achieve both efficient introduction of a compound into a cell and efficient production of a cell whose properties have been altered by said introduction. [Means for solving the problem]

[0009] One embodiment of the present invention is a compound introduction device for introducing a compound into a cell, comprising: a supply flow path for supplying a cell suspension containing the cells and the compound; an introduction means for introducing the compound into the cells; and an outlet for discharging a cell suspension containing the cells into which the compound has been introduced, wherein the height of the supply flow path relative to the flow direction of the cell suspension flowing through the supply flow path is greater than 1.0 times and less than 2.0 times the diameter of the cells, the width of the supply flow path is greater than 1.0 times and less than 2.0 times the diameter of the cells, and the diameter of the outlet is greater than 1.0 times and less than 2.0 times the diameter of the cells. Effect of the Invention

[0010] According to one embodiment of the present invention, it is possible to achieve both efficient introduction of a compound into a cell and efficient production of a cell whose properties have been altered by said introduction. [Brief description of the drawings]

[0011] [Figure 1] FIG. 2 is a diagram showing an example of a compound introduction device. [Diagram 2] FIG. 2 is a block diagram showing an example of a control system of the compound introduction device. [Diagram 3] FIG. 2 is a perspective view showing the appearance of the ejection head cartridge. [Figure 4] 1 is a diagram showing an example of the layout relationship of each component formed on an electronic substrate; [Diagram 5] 5A and 5B are diagrams illustrating the state of a cell suspension in a flow channel in a discharge head. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] According to the study by the inventors of the present disclosure, when the method described in Patent Document 1 is adopted, it is necessary to prepare a microchannel slightly narrower than the size of the cell for each cell type. In addition, when feeding a liquid into the microchannel, the pressure loss becomes large and a very large force is required. Furthermore, it was found that the channel frequently becomes clogged with cells, which is an issue in terms of the production efficiency of the introduction process.

[0013] It was also found that the method described in Patent Document 2 could not achieve highly efficient compound introduction.

[0014] Therefore, the inventors of the present disclosure have conducted intensive research in order to provide a compound introduction device that is capable of achieving both efficient introduction of a compound into a cell and efficient production of a cell whose properties have been modified by said introduction, and have arrived at the present invention.

[0015] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. Note that the following embodiment does not limit the present invention, and not all of the combinations of features described in the embodiment are necessarily essential. Note that the same components will be described with the same reference numerals.

[0016] [First embodiment] <Compound introduction device> FIG. 1 is a diagram showing an example of a compound introduction device 100 of this embodiment. The compound introduction device 100 of this embodiment is a device equipped with an inkjet type discharge head 101. The discharge head 101 is filled with a liquid containing a compound and cells to which the compound is to be introduced. In this specification, this liquid is called a cell suspension (also called a cell-containing liquid). The discharge head 101 may be called a cell processing head or a liquid discharge head. The cell suspension discharged from the discharge head 101 contains cells to which the compound has been introduced. Such a compound introduction device 100 may be called a discharge device or a cell processing device. The configuration of the compound introduction device 100 will be described below.

[0017] Fig. 1 is a schematic diagram of a compound introduction device 100 of this embodiment. The compound introduction device 100 shown in Fig. 1 includes a drive motor 102, a drive belt 103 connected to the drive motor 102, and a discharge head 101 transported by the drive belt 103. The discharge head 101 can be moved to any location within a transportable area by the drive of the drive motor 102.

[0018] The liquid filled in the discharge head 101 is filled into the flow paths and discharge ports in the discharge head 101 by bringing the discharge head 101 into contact with the suction mechanism 104 and operating the suction motor 105. Even if no discharge has been performed for a certain period of time, the liquid in the discharge head 101 can be discharged by bringing the discharge head 101 into contact with the suction mechanism 104 and operating the suction motor 105. The liquid generated by the suction and discharge is discharged to the outside of the system through the waste liquid tube 106. The suction mechanism 104 can also be used as a cap to prevent the head from drying out during non-introduction processing.

[0019] The cells into which the compound has been introduced are discharged from the discharge head 101 toward a culture dish 107 fixed on a processing stage 108. In this manner, the cells into which the compound has been introduced can be obtained.

[0020] The inlet 109 and outlet 110 with fans and filters can reduce mist and dust inside the device, allowing compounds to be introduced with reduced contamination.

[0021] <Control block diagram> 2 is a block diagram showing an example of a control system provided in the compound introduction device 100 of this embodiment. The compound introduction device 100 has a CPU 205, an interface controller 201, a program ROM 202, an image memory 203, a work RAM 204, a discharge head control circuit 206, an output port 207, and a motor drive circuit 208. In this embodiment, the compound introduction device 100 is described as controlling the introduction of various compounds in response to instructions from a host device such as an external device, but is not limited to this. The compound introduction device 100 may be provided with an operation unit or the like, and the introduction of various compounds may be controlled in response to instructions according to the operation of the operation unit.

[0022] Output data and commands are sent to the compound introduction device 100 from a host information terminal (for example, a personal computer, a smartphone, or a tablet device). The control unit, CPU 205, receives the output data and commands via the interface controller 201. The CPU 205 is an arithmetic processing device that controls the entire compound introduction device 100, such as receiving output data, introduction operation, and transportation of the discharge head. After analyzing the received command, the CPU 205 develops control signal data for operating the discharge head 101 and stores it in the image memory 203.

[0023] As an operation process before compound introduction, the CPU 205 reads out control signal data used for introduction control from the image memory 203 in synchronization with the transportation of the discharge head 101. Then, this read out data is transferred to the discharge head 101 via the discharge head control circuit 206. Also, if necessary, the cells are processed in the discharge head 101 and the compound is introduced. Then, directly above the culture dish 107, the cell suspension is discharged from the discharge port 3 of the discharge head 101 (see FIG. 4(a) etc.) toward the culture dish 107. Note that the control signal data may be stored in the work RAM 204, which is a working memory, instead of the image memory 203.

[0024] The operation of the CPU 205 is executed based on a processing program stored in the program ROM 202. The program ROM 202 stores processing programs and tables corresponding to control processing. The work RAM 204 stores the elapsed time from the end of the previous introduction processing, and the CPU 205 executes commands such as capping processing and outlet suction processing according to the elapsed time. The CPU 205 also uses the work RAM 204 as a working memory.

[0025] The motor drive circuit 208 drives various motors such as a head up / down motor 209 , the drive motor 102 , a ventilation fan motor 210 , a capping motor 211 , the suction motor 105 , and a stage transport motor 212 .

[0026] Each component of the compound introduction device 100 of this embodiment will be described in detail below.

[0027] <Discharge head> In the compound introduction device 100 of the present embodiment, at least mechanical energy is applied to the cell suspension by using an energy generating element, which is an introduction means for instantaneously introducing a compound into a cell, thereby enabling the introduction of the compound into the cell. As a method capable of instantaneously applying energy in this manner, a piezoelectric inkjet method can be used, in which a piezoelectric element is placed in a microchannel and a mechanical action is brought about by using a displacement caused by a voltage application. Alternatively, a heater (heating element) with high electrical resistance is placed in the channel, and the heater is heated by applying a voltage to cause the liquid near the heater to bubble in the order of microseconds. This allows the use of a thermal inkjet method that can bring about mechanical and thermal actions. The use of the thermal inkjet method is preferable because it allows mechanical energy to be applied in a very small space and the actuator section to be dense. Therefore, the energy generating element is preferably a heating element. In addition, when heat is generated by the heating element, it is preferable that the stress caused by the foaming caused by the heat is applied to the cell suspension inside the processing chamber. The heating time of the heater is preferably 0.05 μs or more and 10 μs or less since a bubbling phenomenon can be generated, and more preferably 0.1 μs or more and 5 μs or less since cell death can be suppressed.

[0028] Furthermore, when the heater's heating time is 1 μs or more and 10 μs or less, stress acts on the cell suspension over a span of 1 μs or more and 10 μs or less, resulting in droplets being ejected from the outlet at a speed of 0.5 m / s or more and 30 m / s or less, which is preferable.

[0029] The thermal action of the heater has the effect of promoting the expression of heat shock protein genes or genes that promote cell division in cells, as shown by Campbell et al. in Non-Patent Document 1. From the viewpoint of efficiently introducing a compound into dividing cells, the thermal inkjet method is preferable.

[0030] In addition, it is preferable that the energy generating element generates energy in the height direction of the supply flow channel, which makes it possible to efficiently open holes in the membrane on the cell surface.

[0031] The configuration of the ejection head 101 in this embodiment will be described below with reference to FIG. 3 and FIG. 4. FIG. 3 is a perspective view showing the configuration of the cartridge (called the ejection head cartridge) of the ejection head 101. The ejection head cartridge 1 is composed of a liquid tank portion (also called a cell suspension storage portion) that serves as a liquid supply source, and an ejection head in the form of a chip that is integrally provided therewith. The ejection head is attached to the liquid supply holding portion 10 (see FIG. 4) by a second sealing material 4. In addition, an electrical wiring member 5 for electrically connecting the ejection head to the main body side is disposed from the liquid supply holding member 10 to the liquid tank portion. The electrical wiring member 5 has a connection portion (not shown) with the electrode of the ejection head sealed by a first sealing material 2. Coordinate axes are set as shown in FIG. 3. These coordinate axes will also be used in the following drawings.

[0032] Fig. 4 is a diagram showing an example of the arrangement relationship of each component formed on an electronic substrate included in a discharge head. Fig. 4(a) is a vertical cross-sectional view taken along line IVa in Fig. 3. Fig. 4(b) is an enlarged view of a part of Fig. 4(a), specifically, a part indicated by line IVb in Fig. 4(a). Fig. 4(c) is a horizontal cross-sectional view taken along line IVc in Fig. 4(b).

[0033] As shown in Figs. 4(a) to (c), a silicon substrate 9 attached to a liquid supply holding portion 10 by a second sealing material 4 is provided with a liquid supply port 11 for receiving liquid supplied from a liquid tank and supplying the received liquid to the discharge ports 3 arranged in a row. As shown in Figs. 4(b) and 4(c), the liquid supply port 11 is configured to extend over the entire length of the discharge port rows arranged in a row on both sides of the liquid supply port 11. The liquid supply port 11 receives liquid supplied from a liquid supply source, and the liquid is supplied to each of the multiple discharge ports in each of the multiple discharge port rows constituting the discharge portion. As shown in Fig. 4(c), the substrate 9 is provided with a heating element 12 corresponding to each discharge port 3. In addition, a discharge port plate 7 is joined to the substrate 9, and includes a flow path wall 22 for dividing a liquid supply flow path (also simply referred to as a supply flow path) 8 that communicates between the liquid supply port 11 and the discharge port 3 and guides the liquid. The flow path wall 22 is configured of a flow path forming member. The ejection ports 3 are arranged at predetermined intervals along the longitudinal direction of the liquid supply port 11 (Y direction in the figure).

[0034] As shown in Figures 4(a) to (c), the ejection head has a substrate 9, a liquid supply port 11, a heating element 12, an ejection port 3, an ejection port plate 7, a liquid supply flow path 8 that connects the liquid supply port 11 and the ejection port 3, and a flow path wall 22 that separates adjacent liquid supply flow paths 8.

[0035] On the substrate 9, in addition to the heating elements 12 (for example, electrothermal conversion elements), a wiring pattern is formed. Then, the ejection port plate 7, which is a structure made of a resin material in which a row of ejection ports 3 and a liquid supply flow path 8 are formed, is formed by photolithography. On the ejection port plate 7, a water-repellent layer 13 is formed except for the ejection ports 3 portion. As a result, this water-repellent layer 13 forms the outermost surface of the ejection head. Furthermore, on the substrate 9, electrical wiring, electrode parts 6, etc. are formed.

[0036] The electrical wiring member 5 is a member for forming an electrical signal path for supplying an electrical signal to the electrode wiring on the substrate 9, and an opening is formed in the electrical wiring member 5 for incorporating the substrate 9. An electrode terminal (not shown) connected to the electrode part 6 of the chip is formed near the edge of this opening. The chip and the electrical wiring member 5 are bonded to a liquid supply holding member 10 formed by resin molding. The electrical connection part between the substrate 9 and the electrical wiring member 5 is sealed by a first sealing material 2 which seals the lower part of the electrical connection part and the part of the chip where the electrodes are not formed, and a second sealing material 4 which seals the upper part of the electrical connection part. The sealing between the first sealing material 2 and the second sealing material 4 protects the electrical connection part from corrosion by the cell suspension and external impact.

[0037] <Flow path height and width of liquid supply section> The height of the liquid supply flow channel 8, which is the liquid supply section, in the Z direction (gravity direction) and the width in the Y direction are greater than 1.0 times the diameter of the cell and less than 2.0 times the diameter of the cell, respectively. Since the height and width of the liquid supply flow channel 8 are greater than 1.0 times the diameter of the cell, the liquid supply flow channel 8 is less likely to be clogged with cells. Therefore, a cell suspension containing a compound and cells can be supplied to the foaming section (also called a processing chamber) in which the heat generating element 12 is located. In addition, since the height of the liquid supply flow channel 8 is less than 2.0 times the diameter of the cell, when the cells are supplied to the foaming section in which the heat generating element 12 is located, the supplied cells are located in the vicinity of the heat generating element 12. Therefore, by adopting such a configuration, it is possible to efficiently perform processing to open holes in the membrane on the cell surface in order to introduce a compound into the cell.

[0038] The height and width of the liquid supply flow channel described above are the shortest in the liquid supply flow channel. For example, the width of the liquid supply flow channel in FIG. 4(c) means the length indicated by the arrow of the supply flow channel immediately before the foaming section at the top of which the heating element 12 is located. The shape of the liquid supply flow channel may be linear or serpentine, but is not limited thereto, and any shape of liquid supply flow channel may be used. The height and width of the liquid supply flow channel may be narrowed from the liquid supply section toward the foaming section, or may be the same length from the liquid supply section toward the foaming section. The height and width of the supply flow channel are relative to the flow direction of the cell suspension flowing through the supply flow channel.

[0039] The height and width of the liquid supply channel may be appropriately selected depending on the diameter of the cells to be processed. Specifically, the height and width of the liquid supply channel are preferably greater than 1 μm and less than 200 μm, respectively.

[0040] <Discharge port> In this embodiment, as shown in Fig. 4(c), a circular outlet 3 is used, but any shape of outlet may be appropriately selected and used. Examples of the shape of the outlet include a circle, an ellipse, a triangle, a rectangle, a square, a polygon, a star, and a shape with a protrusion. However, regarding the shape of the outlet, the longest length is preferably 1.0 times or more and 5.0 times or less than the shortest length, and more preferably 1.0 times or more and 2.0 times or less.

[0041] In addition, the diameter of the discharge port in this embodiment is the length of the shortest part of the discharge port. The diameter of the discharge port is greater than 1.0 times and less than 2.0 times the diameter of the cells. Since the diameter of the discharge port is greater than 1.0 times the diameter of the cells, the cell suspension to which stress is applied due to foaming caused by the heating element can be discharged without clogging of the flow path by the cells. Furthermore, since the diameter of the discharge port is less than 2.0 times the diameter of the cells, the introduction of the compound occurs efficiently due to the stress caused by the discharge.

[0042] The ratio between the diameter of the discharge port and the height of the liquid supply flow path can be any ratio, but it is preferable that the diameter of the discharge port is larger than the height of the liquid supply flow path. The reason for this is that by making the diameter of the discharge port larger than the height of the liquid supply flow path, it becomes relatively easier for the liquid to be discharged toward the discharge port. This reduces the variation in the position of the cells in the flow path, making it possible to perform processing stably.

[0043] <Mechanism of compound introduction> Hereinafter, a presumed mechanism of compound introduction by the compound introduction device 100 in this embodiment will be described with reference to Figures 4(b) and 5. Figure 5 is an enlarged view of the vicinity of the heating element 12 shown in Figure 4(b).

[0044] The cell suspension supplied from the liquid supply port 11 passes through the liquid supply flow path 8 and is filled up to the discharge port 3. When the cell suspension is filled, the water of the cell suspension is rapidly heated and foamed by applying electricity to the heating element 12, forming bubbles. At this time, the cell suspension present in the part of the foamed bubbles 14 by heating is rapidly pushed out mainly toward the discharge port 3 direction (+Z direction in FIG. 5) where the flow resistance is small. At this time, the unprocessed cells 16 contained in the cell suspension supplied through the liquid supply flow path 8 become cells 17 with holes in their cell membranes due to the strong shear flow caused by foaming. In cells in this state, even compounds that normally cannot pass through the cell membrane can temporarily pass through the holes in the cell membrane. Therefore, a part of the compound contained in the cell suspension is taken up into the cells. In addition, the cell suspension pushed out by foaming is discharged through the discharge port 3, which is directly below the heating element 12 and the foaming part (the processing chamber in which the heating element is arranged). In order to easily discharge the cell suspension containing the cells to which the compound has been introduced, it is preferable that the discharge port is arranged directly below the heating element and the processing chamber. When the liquid is pushed out by foaming and has sufficient kinetic energy, the liquid is ejected in the form of flying droplets. In addition, when the cells to which the compound contained in the ejected cell suspension has been introduced are cultured under appropriate conditions, the temporarily opened holes in the cell membrane close, and depending on the case, the cells can survive and be continuously cultured.

[0045] Thus, in this embodiment, the height 20 and width of the liquid supply flow path are each greater than 1.0 times the diameter of the cell. This allows the cell suspension to be supplied to the foaming section in which the heat generating element is located without clogging the flow path with cells. In addition, since the height 20 of the liquid supply flow path is less than 2.0 times the diameter of the cell, when the cells are supplied to the foaming section in which the heat generating element 12 is located, the supplied cells are located in the vicinity of the heat generating element 12. Therefore, this configuration allows for efficient processing.

[0046] In addition, the diameter 21 of the discharge port is larger than 1.0 times the diameter of the cells. This allows the cell suspension to be discharged without clogging the flow path due to the cells being stressed by the foaming of the heating element 12. Furthermore, the diameter 21 of the discharge port is less than 2.0 times the diameter of the cells, which is not too large. Therefore, the compound is efficiently introduced due to the stress applied during discharge.

[0047] <Compound introduction method> The compound introduction method in this embodiment has at least the following three steps.

[0048] The process includes a step (first step) of supplying a cell suspension containing cells and a compound through a supply flow path, a step (second step) of introducing the compound into the cells, and a step (third step) of discharging the cell suspension containing the cells to which the compound has been introduced from a discharge port. Each of these steps will be described below, taking as an example a case in which the introduction means for introducing the compound is a heating element.

[0049] In the first step, the cell suspension containing the compound and the cells to which the compound is introduced is passed through a liquid supply flow path included in the liquid supply unit to reach the foaming unit (processing chamber) where the heat generating element is present. The height of this liquid supply flow path is greater than 1.0 times and less than 2.0 times the diameter of the cells, and the width of the liquid supply flow path is greater than 1.0 times and less than 2.0 times the diameter of the cells. The cell suspension may be introduced into the discharge head 101 from a cell suspension storage unit of the discharge head cartridge 1, or may be introduced directly into the discharge head 101 using a micropipette or the like. When the cell suspension is smoothly filled up to the discharge port of the discharge head 101 due to the wetting and spreading of the cell suspension caused by surface tension, the introduction operation is performed immediately after filling. When the cell suspension does not fill up to the discharge port of the discharge head 101, it can be filled by sucking from the discharge port using the suction mechanism 104 or an external suction pump. Alternatively, it can be filled by pressurizing the storage unit that stores the cell suspension using an external pressure pump.

[0050] In the second step, the heating element is heated in the foaming section where the heating element is located. As a result of the second step, the cell suspension foams, and stress is applied to the cell suspension due to the stress generated by the foaming, thereby allowing the compound to be introduced into the cells.

[0051] In the third step, the cell suspension containing the compound-introduced cells is discharged from a discharge port having a diameter greater than 1.0 times and less than 2.0 times the diameter of the cells.

[0052] The discharged cell suspension is discharged into the substrate or culture solution. In selecting the substrate, the overall load on the cells must be considered, and the substrate is selected according to the purpose. Specifically, when discharging onto the substrate for introduction processing, in the case of an adhesive substrate such as glass or polystyrene, which has a high affinity for the substrate surface, it is preferable to immerse the substrate in a medium containing serum or extracellular matrix beforehand, and discharge the substrate after processing. In addition, in the case of using a fluororesin such as PTFE, which has a low affinity for cells, as the substrate, it is preferable to transfer the discharged liquid to the medium after discharging. In the case of performing the introduction operation on a culture dish, it is preferable to contact the culture dish with a medium containing serum or extracellular matrix beforehand. In addition, it is also possible to discharge the cells directly from the discharge head 101 onto the medium filled in the culture dish, and the method is selected according to the purpose.

[0053] The discharged cell suspension can be immediately transferred to a dish containing a medium optimal for the cells, or it can be kept in a saturated water vapor environment. If the amount of introduction is to be increased, it is preferable to leave it as it is.

[0054] When cells are discharged onto the substrate, it is possible to either remove the cell culture solution on the substrate with a pipette or the like and seed the cells onto a dish, or to place the substrate together with the substrate in a dish, add a medium, and culture the cells. Furthermore, the compound introduction operation can be repeated by injecting the cell suspension once discharged into the discharge head 101 again and discharging it. In this case, it is preferable to use a substrate that has a low affinity between the cells and the substrate surface.

[0055] In the following explanation, a compound to be introduced into a cell is also referred to as a "compound to be introduced."

[0056] <Cell diameter> The diameter of the cells in the cell suspension is obtained by placing the cell suspension immediately before processing on a hemocytometer or the like and measuring it with an optical microscope or the like equipped with an image sensor. The diameter of the cells can be obtained based on the distance information corresponding to the image stored in advance using the image recorded by the image sensor. It is preferable to focus on the cells so that the diameter is maximized, and then record the image and measure the length.

[0057] <Target compound for introduction> The compound to be introduced can be appropriately selected according to the purpose. Examples of compounds that can be introduced include nucleic acids, proteins, and labeling substances. However, the compounds are not limited to these examples as long as they are large enough to be encapsulated in the cells to be introduced. However, from the viewpoint of minimizing damage to cells, the size of the compound is preferably 1 / 5 or less, more preferably 1 / 10 or less, of the average diameter of the cells.

[0058] The size of the cells can be measured by treating the cells with an enzyme or the like, and then using a laser diffraction particle size distribution meter or the like to form a cell suspension. Furthermore, the cells can be placed on a hemocytometer or the like and measured using an optical microscope. Similarly, the size of the introduced compound can also be measured. In particular, for compounds with a size of 1 μm or less, a dynamic light scattering particle size distribution meter can be used. A representative compound that can be applied in this embodiment is nucleic acid.

[0059] <Nucleic acid> For the purpose of transient and stable expression of nucleic acids or interference with genes, exogenous RNA (ribonucleic acid) or DNA (deoxyribonucleic acid) different from that derived from the cells to be introduced can be used as the compound to be introduced. As the higher-order structure of the nucleic acid, a single-stranded primary structure, a hairpin-shaped stem-loop structure, a secondary structure such as a helix structure, etc. can be used. In addition, a tertiary structure such as an A-form, B-form, or Z-form can be used. In addition, a quaternary structure such as a supercoiled structure can be used. These higher-order structures can be suitably used depending on the purpose. In addition, these nucleic acids may be labeled with a fluorescent compound or a radioisotope and can be used depending on the purpose.

[0060] <rna> The RNA used in this embodiment includes messenger RNA, which copies and carries sequences from DNA to ribosomes, the site of protein synthesis. In addition, there are ribosomal RNA, which is a component of ribosomes, and transfer RNA, which carries amino acids corresponding to the sequence to ribosomes. In addition, there are small nuclear RNAs, small nucleolar RNAs, microRNAs, and siRNAs with interference effects, but the RNAs are not limited to these and can be used as appropriate depending on the purpose.

[0061] <dna> The DNA handled in this embodiment can be selected from single-stranded DNA, double-stranded DNA, triple-stranded DNA, and quadruple-stranded DNA. Linear or circular shapes are generally used as shapes, but in recent years, as typified by DNA origami, any shape may be used, and the shape is not important. Double-stranded DNA is preferred in terms of the stability of the substance, and circular plasmid DNA is more preferred in terms of ease of culturing in E. coli or yeast. Furthermore, in order to be introduced into a cell, it is necessary to introduce it into the cell from the cell membrane, so it is preferable that the surface area is as small as possible. For example, even if the DNA has the same sequence, circular DNA is more preferable than linear DNA, and supercoiled DNA guided by the twisting of DNA is more preferable.

[0062] <Proteins> The proteins handled in this embodiment include proteins that are dissolved, dispersed, or supported and dispersed on a substrate for the purpose of introduction into a cell suspension. The structure may be a primary structure including a polypeptide, a secondary structure such as an α-helix or β-sheet, a tertiary structure including these secondary structures, or a quaternary structure such as hemoglobin, and any higher-order structure may be used according to the purpose. Specific examples include enzyme proteins such as amylase, structural proteins such as collagen and keratin, transport proteins such as albumin, storage proteins such as fetilitin, and contractile proteins such as actin and myosin. Other examples include defense proteins such as globulin, regulatory proteins such as calmodulin, various other membrane proteins, zinc finger nucleases for genome editing, and Cas9 proteins used in CRISPR / Cas9.

[0063] <Labeling substance> The labeling substance used in this embodiment may be any substance that can be confirmed to be labeled from outside the cell when introduced into the cell, and may be introduced by chemically or physically modifying the aforementioned nucleic acids or proteins. The labeling substance may have a recognizable absorption or emission wavelength different from that of the cell into which it is introduced. In addition, the labeling substance may be present in a dissolved, dispersed, or supported and dispersed state on a substrate for the purpose of introduction into the cell suspension. Specific examples include stable isotope substances such as deuterium, C13, and N15, radioactive substances, dyes, fluorescent dyes, pigments, fluorescent pigments, quantum dots, nanodiamonds, fullerenes, carbon nanosheets, and carbon nanotubes.

[0064] <Cell type> The cells used in this embodiment include adhesive cells, floating cells, spheroids (aggregate cells), etc. Specific examples of cells include human cervical cancer cell lineage cells, nerve cells, hepatocytes, fibroblasts, myoblasts, smooth muscle cells, cardiac muscle cells, skeletal muscle cells, stem cells, mesodermal stem cells, embryonic stem cells, glial cells, fetal stem cells, and hematopoietic stem cells. Other examples include mast cells, adipocytes, neural stem cells, and blood cells.

[0065] Other examples include microbial cells and plant cells. More specifically, examples include prokaryotic cells such as Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus, and Staphylococcus, eukaryotic cells such as yeast and Aspergillus, and insect cells such as Drosophila S2 and Spodoptera Sf9. Among these, it is preferable that the cells are eukaryotic cells. The diameter of the cells (average value of all cells) is a size that can be discharged from the discharge port, and is preferably, for example, 1 μm or more and 100 μm or less.

[0066] <Cell suspension> The cell suspension has at least a compound and cells to which the compound is introduced. In the present invention, the cell suspension is a suspension in which cells are dispersed in a liquid. The cells in the cell suspension may be in a state in which they can be dispersed in a liquid by stirring, and may be in a state in which the cells precipitate in the liquid when left to stand. In addition, it is preferable to appropriately contain other components in order to enable the survival of the cells during and after the introduction process. Examples of other components include salts, sugars, ribonucleotides, growth factors or hormones, pH buffers, surfactants, chelating agents, water-soluble organic solvents, proteins and amino acids, antibacterial agents, moisturizing agents, and thickeners.

[0067] <Salts> Examples of salts used in this embodiment may be inorganic or organic salts used in cell culture, specifically, sodium chloride, potassium chloride, sodium citrate, etc.

[0068] <Sugars> Examples of sugars used in this embodiment include sugars that can be used as nutrients for cells or for the purpose of adjusting osmotic pressure, etc. Specific examples include glucose, sucrose, and fructose.

[0069] <Ribonucleotides> Examples of ribonucleotides used in this embodiment include ribonucleotides that can be used to assist the metabolism of cells, specifically, adenosine triphosphate and guanosine triphosphate.

[0070] <Growth factors or hormones> Examples of growth factors or hormones handled in this embodiment include, for example, human growth hormone. Also included are other animal growth hormones (such as bovine, porcine or chicken growth factors), insulin, oxytocin, angiotensin, methionine enkephalin, substance P. Also included are ET-1, FGF, KGF, EGF, IGF, PDGF, LHRH, GHRH, FSH, DDAVP, PTH, vasopressin, glucagon, somatostatin, etc.

[0071] <pH buffer> Examples of pH buffers handled in this embodiment include, for example, citrate buffer, phosphate buffer, Tris buffer, or HEPES buffer, etc.

[0072] <Surfactant> Examples of surfactants handled in this embodiment include water-soluble anionic, cationic, or amphoteric or nonionic surfactants, and one or more types can be added.

[0073] <Chelating agent> Examples of chelating agents handled in this embodiment specifically include ethylenediaminetetraacetic acid (EDTA) or glycol ether diamine tetraacetic acid (EGTA), etc.

[0074] <Water and water-soluble organic solvents> For the cell suspension handled in this embodiment, an aqueous liquid medium containing water or a mixture of water and a water-soluble organic solvent can be used. A cell suspension can be obtained by adding cells and a compound to be introduced to the aqueous liquid medium.

[0075] The solvent used in the present embodiment is not particularly limited, and examples thereof include water and physiological saline. Other examples include buffer solutions such as phosphate buffered saline (hereinafter, PBS) or Tris, and Dulbecco's Modified Eagle Medium (hereinafter, D-MEM). Other examples include Iscove's Modified Dulbecco's Medium (hereinafter, IMDM) and Hanks' Balanced Salt Solutions (hereinafter, HBSS). Other examples include Minimun Essential Medium-Eagle, Earle's Salts Base, with Non-Essential Amino Acid (hereinafter, MEM-NEAA). Other examples include cell culture media such as RPMI (Roswell Park Memorial Institute Medium) 1640. Other examples include infusions such as commercially available electroporation buffers, commercially available FACS analysis buffers, and lactated Ringer's solution. These solvents preferably contain 50% or more water. Two or more of these solvents can also be used in combination. The water is preferably water that has been deionized by ion exchange or the like and sterilized by heating in an autoclave or the like. The water content in the cell suspension medium is preferably 30% by mass or more and 99% by mass or less, based on the mass of the cell suspension.

[0076] The type of water-soluble organic solvent is not particularly limited, and any known organic solvent can be used depending on the purpose.Specific examples include glycerin, polyethylene glycol, and dimethyl sulfoxide.The content of the water-soluble organic solvent in the cell suspension is preferably 0.001% by mass or more and 50% by mass or less based on the total mass of the cell suspension.

[0077] <Proteins and amino acids> Examples of proteins and amino acids handled in this embodiment include serum such as fetal bovine serum (hereinafter, FBS) or horse serum.

[0078] <Antibacterial agent> Examples of antibacterial agents used in this embodiment include antibiotics such as sodium azide or penicillin / streptomycin, which can be added to the cell suspension. In particular, physiological saline, buffer solutions such as PBS or Tris, cell culture media such as D-MEM, IMDM, HBSS, commercially available FACS analysis buffers, or infusions such as lactated Ringer's solution are preferably used to control the salt concentration or pH suitable for the cells.

[0079] <Moisturizer> Examples of moisturizing agents used in this embodiment include polyhydric alcohols such as glycerin, propylene glycol, butylene glycol, and sorbitol. In addition, mucopolysaccharides such as hyaluronic acid and chondroitin sulfate, protein hydrolysates such as soluble collagen, ullastin, and keratin, etc. can be used alone or in combination.

[0080] <Thickener> Examples of the thickener used in this embodiment include starches such as oxidatively modified starch, enzyme modified starch, thermochemically modified starch, cationic starch, amphoteric starch, or esterified starch. Other examples include cellulose derivatives such as carboxymethyl cellulose, hydroxyethyl cellulose, or ethyl cellulose, and natural or semi-synthetic polymers such as casein, gelatin, or soy protein. Other examples include water-soluble polymer compounds that are completely or partially saponified. Examples of water-soluble polymer compounds include polyvinyl alcohol, acetoacetylated polyvinyl alcohol, and carboxy-modified polyvinyl alcohol. Other examples include polyvinyl alcohols such as olefin-modified polyvinyl alcohol and silyl-modified polyvinyl alcohol. At least one water-soluble polymer compound can be appropriately selected from these and used. In this embodiment, the preferred viscosity of the liquid used is 0.1 Pa·s (Pascal seconds) or more, and the above-mentioned thickener may be added as necessary.

[0081] <How to prepare cell suspension> In the cell suspension of this embodiment, cells cultured by adhesion culture or suspension culture are separated into single cells or small clusters of cells by the action of enzymes, etc., and then only the cells are precipitated by utilizing the difference in specific gravity using a centrifuge, etc. After that, the supernatant medium other than the cells is removed, a medium containing the compound to be introduced is added, and the mixture is stirred using a pipette or a stirrer to prepare a cell suspension.

[0082] The concentration of the cell suspension prepared may be adjusted according to the purpose, but from the standpoint of production efficiency, it is preferably 1,000 cells / mL or more and 100 million cells / mL or less, and more preferably 100,000 cells / mL or more and 10 million cells / mL or less.

[0083] In addition, before introducing the cell suspension into the discharge head, it is preferable to pass the cell suspension through a cell strainer having a diameter approximately equal to the minimum diameter of the liquid supply flow path in the discharge head 101 to be used. This makes it possible to remove large cell clumps from the cell suspension.

[0084] <Culture operation method> The processed cells can be cultured in the desired culture system. Specifically, in the case of animal cells, it is preferable to culture them in an incubator that maintains a culture temperature of 37 degrees Celsius and saturated water vapor with a carbon dioxide concentration of 5%. During the culture, it is preferable to check the growth state of the cells and replace the medium or perform subculture.

[0085] <Method for confirming compound delivery to cells> The method for confirming the introduction of a compound into a cell varies depending on the compound to be introduced, so a confirmation method appropriate for each compound may be used. For example, when a plasmid DNA expressing GFP, a fluorescent protein, is introduced into the cell, the amount of introduction can be confirmed semi-quantitatively by confirming the presence or absence of GFP emission using a fluorescent microscope for a sample after a certain period of time. In addition, the cultured cells can be dispersed into a single cell using an enzyme or the like, and then the number of emitting cells can be counted using flow cytometry to quantitatively confirm the introduction. Furthermore, the cells can be disintegrated and measured using a fluorescence spectrophotometer or a luminometer. In addition, an ELISA method or an immunostaining method using an antigen-antibody reaction can be used. In addition, it is also possible to measure the introduced DNA and amplified DNA in the cell using a real-time PCR device or the like. When the introduced compound is a labeled compound, it can be analyzed using analytical means used in general chemical analysis.

[0086] [Example] Examples of the present embodiment will be described below. Note that the following examples are merely examples and are not intended to be limiting. Note that "%" in the text is based on mass unless otherwise specified.

[0087] First, the discharge head 101 was washed with a large amount of sterilized water. Furthermore, the inside and outside of the discharge head 101 were cleaned and sterilized in a bioclean bench using an aqueous solution with an ethanol concentration of 70%. After removing the excess aqueous ethanol solution, the inside and outside of the discharge head 101 were washed using 15 mL of 1x phosphate buffered saline (1xPBS) (manufactured by Thermo Fisher Scientific, pH=7.4). Furthermore, an additional 5 mL of 1xPBS was injected into the reservoir of the discharge head 101. In this state, the 1xPBS was sucked out from the connected discharge port surface of the discharge head using an external aspirator connected to a sterilized tube. This operation was performed three times to obtain a sterilized discharge head 101.

[0088] The cell suspension and the compound to be introduced used in the compound introduction device 100 of FIG. 1 were prepared as follows.

[0089] (Liquid composition 1 containing a compound to be introduced) As shown in Table 1 below, 500 μl of a trypan blue solution containing trypan blue, which is a compound to be introduced, and 500 μl of Gene Pulser electroporation buffer (Lonza) were mixed in a microtube to obtain Liquid Composition 1.

[0090] (Liquid composition 2 containing the compound to be introduced) A DNA solution containing DNA, which is the compound to be introduced, was prepared as follows.

[0091] First, 0.2 mL of 0.5 mol / L EDTA solution (pH 8.0) (manufactured by Nacalai Tesque) and 1 mL of 1 mol / L Tris-HCl buffer solution (pH 8.0) (manufactured by Nacalai Tesque) were mixed to obtain a mixed solution.

[0092] Next, 98.8 mL of sterilized pure water was added to this mixture to prepare TE buffer (10 mM Tris 1 mM EDTA (pH 8)). Lyophilized CMV-Fresno RFP (ATUM, base pair number 5.5 kbp) and the prepared TE buffer were mixed and stirred in a microtube, and DNA was dissolved therein to obtain a DNA solution. A portion of the obtained DNA solution was further diluted with TE buffer and then filled into a quartz cell, and the concentration was identified using a DNA concentration measuring device (Biochrom, GeneQuant 1300) to determine the DNA solution concentration. The DNA concentration was 2.0 μg / μL.

[0093] As shown in Table 1 below, 500 μl of the obtained DNA solution and 500 μl of Gene Pulser electroporation buffer (manufactured by Lonza) were mixed in a microtube to obtain Liquid Composition 2.

[0094] [Table 1]

[0095] The cell suspension was prepared as follows.

[0096] RAW264.7, a cell line established from mouse monocytic leukemia, purchased from the American Type Culture Collection, was subjected to the following procedure to obtain a cell density of 2.0 million cells / mL.

[0097] The cell line RAW264.7 was dispersed in 20 mL of D-MEM medium containing 10% FBS, 1% penicillin-streptomycin, and 1% MEM-NEAA at a concentration of 2.0 million cells / mL, and then seeded at approximately 2.0 million cells per 100 mm polystyrene dish.

[0098] 10% FBS was manufactured by Global Life Science Technologies Japan. 1% penicillin-streptomycin was manufactured by Sigma-Aldrich. 1% MEM-NEAA was manufactured by Thermo Fisher Scientific. D-MEM was manufactured by Thermo Fisher Scientific. 100 mm polystyrene dishes were manufactured by Corning.

[0099] The dishes with D-MEM medium containing the seeded cells were incubated at 5% CO 2 The cells were grown by incubation at 37°C in the presence of 0.1% EDTA. After 2 to 4 days, it was confirmed that the cells covered approximately 70% of the bottom surface of the dish, and the supernatant medium was removed and rinsed with PBS. The cells were detached from the dish using PBS containing 0.25% trypsin and 1 mM EDTA (Thermo Fisher Scientific). Then, the cell suspension containing RAW264.7 cells was collected from the dish.

[0100] In a sterilized centrifuge tube, the above D-MEM medium was added to the cell suspension collected from the dish to a total volume of 50 mL, and then the mixture was treated for 5 minutes in a centrifuge (Hitachi Koki, CF16RX II) set at a temperature of 4 degrees Celsius and a centrifugal force of 90 G to sediment the cells. The supernatant of the sedimented cell pellet was gently removed, and the above D-MEM medium was added to the cell pellet to obtain a cell suspension. This cell suspension was subjected to the above cell culture operation twice again.

[0101] In the third operation, the number of cells required for the desired cell concentration was separated into another centrifuge tube. Then, the mixture was centrifuged, and the above liquid composition 1 was added instead of the above D-MEM medium, and stirred using a micropipette. The mixture was then passed through a cell strainer (manufactured by Corning, mesh 40 μm) to obtain cell suspension 1 (2.0 million cells / μL) to be used for introducing a compound into cells. Cell suspension 2 was obtained in the same manner as cell suspension 1, except that liquid composition 2 was used instead of liquid composition 1.

[0102] The obtained cell suspensions 1 and 2 were filled into a Countless Cell Counting Chamber Slide (manufactured by Thermo Fisher Scientific), and images were recorded using a phase contrast microscope (manufactured by Olympus, model number: CKX41). The recorded images were then used to measure the longest diameter of 1000 cells to determine the cell diameter. The cell diameter was obtained as the diameter of the 50% cell based on the number in the cell diameter distribution obtained by the above measurement.

[0103] <Example 1> In this example, a compound was introduced into cells using the compound introduction device 100 of FIG. 1 as follows.

[0104] First, 200 μL of the cell suspension 1 prepared by the above operation was injected into the discharge head 101 using a micropipette. After that, the discharge head 101 was transported to the suction mechanism 104 and grounded, and then the suction motor 105 was operated to fill the cell suspension 1 into each flow path and the discharge port 3 of the discharge head 101. After filling, the introduction operation program was executed. By executing the introduction operation program, the discharge head 101 was moved away from the suction mechanism 104, and then transported to the top of a glass bottom dish (manufactured by Iwaki Co., Ltd.) whose inner bottom surface was wetted with 100 μL of the above D-MEM medium that had been installed in advance. Then, using all the discharge ports 3, a liquid discharge operation was performed to output an image with a processing duty of 100% (specifically, a solid image of 1.5 cm x 1.5 cm), and the cell suspension 1 was discharged into the glass bottom dish to obtain a sample. This liquid discharge operation was repeated 40 times to confirm the continuous processability (the number of continuous printable times in Table 2). In the compound introduction device used in this embodiment, "processing duty is 100%" means that one droplet of cell suspension of 23.0 n (nano) g is applied to a unit area of ​​1 / 600 inch x 1 / 600 inch with a resolution of 600 dpi x 600 dpi. During the liquid ejection operation, the ventilation fan motor 210 was driven to operate the intake fan of the intake port 109.

[0105] The sample thus obtained was covered with the upper plate of a glass bottom dish, removed from the apparatus, and 2.5 mL of the above D-MEM medium warmed to 37°C was gently poured into the sample using a micropipette. The cells were then observed using a phase contrast microscope (Olympus, model number: CKX41) to determine the processing rate.

[0106] <<Evaluation of processing rate>> As described above, the processing rate of the cells into which the compound was introduced using the cell suspension 1 was measured. In detail, the cells were observed using a phase contrast microscope with a 10x objective lens in bright field mode, and the number of cells stained purple with trypan blue and the total number of cells were counted. The processing rate was calculated according to the formula: processing rate = number of purple cells / total number of cells x 100, and the calculated processing rate was evaluated according to the following five-level criteria. The results of the evaluation are shown in Table 2. In this example, rank C or lower was determined to be an unacceptable level in terms of production efficiency. AA: Machining rate 50% or more A: Processing rate is 40% or more and less than 50% B: Processing rate is 20% or more and less than 40% C: Processing rate 10% or more and less than 20% D: Processing rate less than 10%

[0107] <Example 3, Comparative Example 1 and Comparative Example 2> As shown in Table 2, samples of Example 3 and Comparative Examples 1 and 2 were produced by carrying out the same operations as in Example 1, except that the diameter of the ejection port, the height and width of the liquid supply flow path, and the nozzle thickness were changed. The processing rate of the obtained samples was then evaluated in the same manner as in Example 1. The results of such evaluation are shown in Table 2. Note that in Comparative Example 1, since the liquid ejection operation could not be repeated 40 times, the number of continuous prints possible in Table 2 is indicated as "C," and the processing rate was not evaluated.

[0108] <Example 2> A sample was prepared in the same manner as in Example 1, except that cell suspension 2 was used instead of cell suspension 1. The obtained sample was then incubated in 5% CO 2 The cells were grown by incubation at 37°C in the presence of the vector, and gene expression was evaluated after 24 hours using a fluorescent microscope to determine whether DNA had been introduced into the cells.

[0109] <<Evaluation of gene expression>> The CMV-Fresno RFP used in this example contains a gene that produces a protein that emits red fluorescence when introduced into cells, and therefore DNA introduction was evaluated using the expression of this fluorescent protein.

[0110] Specifically, the medium was removed from the sample incubated for 24 hours, rinsed with 1×PBS, and then 2 mL of 1×PBS was added. After replacing the medium with 1×PBS, the sample was observed using a fluorescence microscope (Keyence Corporation, model number: BZ-8000) with a 10x objective lens, bright field mode, and fluorescence mode (TRITC: excitation 540±12.5 nm, fluorescence 605±27.5 nm, cutoff 565 nm). Then, the number of cells emitting fluorescence and the total number of cells were counted, and the introduction rate was calculated according to the formula introduction rate=(number of cells emitting fluorescence / total number of cells)×100, and the calculated introduction rate was evaluated according to the following criteria. The results of such evaluation are shown in Table 2. Yes: Introduction rate of 1% or more None: Less than 1% adoption rate

[0111] <Example 4> A sample of Example 4 was produced in the same manner as in Example 2, except that the diameter of the discharge port, the height and width of the liquid supply channel, and the nozzle thickness were changed as shown in Table 2. Cells were grown in the obtained sample in the same manner as in Example 2, and gene expression was evaluated in the same manner as in Example 2. The results of this evaluation are shown in Table 2.

[0112] <Comparative Examples 3 and 4> Below, Comparative Example 3 and Comparative Example 4 will be described as comparative examples of Example 1 and Example 2. Specifically, in Comparative Examples 3 and 4, in order to confirm the foaming effect, foaming was not performed by a heating element, and instead of a discharge operation, the cell suspension was taken out by external suction through a discharge port and added to a glass bottom dish. Other than this, the same operations as in Examples 1 and 2 were performed to prepare samples that were not subjected to foaming treatment. Using the prepared samples, the processing rate was evaluated in Comparative Example 3, and gene expression was evaluated in Comparative Example 4. The results of such evaluations are shown in Table 2.

[0113] [Table 2]

[0114] <Other embodiments> The present invention can also be realized by a process in which a program for realizing one or more functions of the above-mentioned embodiments is supplied to a system or device via a network or a storage medium, and one or more processors in a computer of the system or device read and execute the program. The present invention can also be realized by a circuit (e.g., ASIC) for realizing one or more functions.< / dna> < / rna>

Claims

1. A compound introduction device for introducing a compound into a cell, comprising: a supply flow path for supplying a cell suspension containing the cells and the compound; A means for introducing the compound into the cell; a discharge port for discharging a cell suspension containing the cells into which the compound has been introduced; having A height of the supply flow channel relative to a flow direction of the cell suspension flowing through the supply flow channel is greater than 1.0 times and less than 2.0 times the diameter of the cells, and a width of the supply flow channel is greater than 1.0 times and less than 2.0 times the diameter of the cells; The diameter of the outlet is greater than 1.0 times and less than 2.0 times the diameter of the cell. A compound introduction device characterized by:

2. The introduction means is an energy generating element that generates energy in a height direction of the supply flow path.

2. The compound introduction device according to claim 1 .

3. The introducing means is a heating element.

3. The compound introduction device according to claim 1 or 2.

4. Further comprising a processing chamber in which the heating element is disposed.

4. The compound introduction device according to claim 3.

5. The discharge port is disposed directly below the heat generating element and the processing chamber.

5. The compound introduction device according to claim 4.

6. When the heating element generates heat, stress caused by foaming due to the heat is applied to the cell suspension inside the processing chamber.

6. The compound introduction device according to claim 4 or 5.

7. The stress acts for 1 μs or more and 10 μs or less, so that the droplets of the cell suspension are discharged from the outlet at a speed of 0.5 m / s or more and 30 m / s or less.

7. The compound introduction device according to claim 6.

8. The heating time of the heating element is 0.1 μs or more and 5 μs or less.

8. The compound introduction device according to claim 3, wherein the compound introduction device is a compound injection device.

9. The diameter of the outlet is greater than the height of the supply flow path.

9. The compound introduction device according to claim 1, wherein the compound introduction device is a compound injection device.

10. The diameter of the cells is 1 μm or more and 100 μm or less.

10. The compound introduction device according to claim 1 ,

11. The height and width of the supply channel are each greater than 1 μm and less than 200 μm; 11. The compound introduction device according to claim 1 ,

12. The cell is a eukaryotic cell.

12. The compound introduction device according to claim 1, wherein the compound introduction device is a compound injection device.

13. The height of the supply flow path means the length in the direction of gravity.

13. The compound introduction device according to claim 1, wherein the compound introduction device is a compound injection device.

14. A method for introducing a compound into a cell, comprising the steps of: supplying a cell suspension containing the cells and the compound through a supply channel; introducing said compound into said cells; Discharging a cell suspension containing the cells into which the compound has been introduced from a discharge port; having A height of the supply flow channel relative to a flow direction of the cell suspension flowing through the supply flow channel is greater than 1.0 times and less than 2.0 times the diameter of the cells, and a width of the supply flow channel is greater than 1.0 times and less than 2.0 times the diameter of the cells; The diameter of the outlet is greater than 1.0 times and less than 2.0 times the diameter of the cell. A method for introducing a compound, comprising:

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