JP2022145590A - Compound introduction device and compound introduction method - Google Patents

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

The present disclosure relates to a compound introduction device and a compound introduction method for introducing a compound into cells.

Techniques for introducing compounds such as genes into cells are known. Methods of introduction include chemical introduction methods using cationic substances or viruses, and biological introduction methods into living cells. In addition, various methods have been developed, including physical introduction methods such as the electroporation method and the gene gun method, which are expected to have low toxicity, and microinjection methods, which have high selectivity for the type of compound to be introduced and high certainty of introduction. .

In recent years, with the emergence of cell therapeutic drugs or induced pluripotent stem cells that modify the properties of cells, which are obtained by introducing compounds such as genes into cells, there is a demand for more efficient methods of compound introduction. It is However, a means to achieve such high efficiency has not been established and has been a problem.

As one means for solving the above problems, Patent Literature 1 describes an introduction method using a microchannel. In this method, cells dispersed in a liquid containing a compound to be introduced pass through a microchannel having approximately the same size as the cells. As a result, by applying a shearing force to the cells, pores are temporarily generated in the cell membrane, and the extracellular to-be-introduced compound can be introduced into the cells. is possible.

Patent Document 2 discloses that an inkjet device used as an image recording apparatus can be used to effectively introduce a compound of interest into cells by generating pressure and shear force in a micro-sized space. is described.

U.S. Patent Application Publication No. 2018/0003696 Patent No. 5645657

Campbell, A et al. 2020 Frontiers in Bioengineering and Biotechnology, 8 doi: 10.3389/fbioe. 2020.00082

One embodiment of the present invention aims to achieve both efficient introduction of a compound into cells and efficient generation of cells whose properties are modified by said introduction.

One embodiment of the present invention is a compound introduction device for introducing a compound into cells, comprising: a supply channel for supplying a cell suspension containing the cells and the compound; and an introduction for introducing the compound into the cells. means, and an ejection port for ejecting a cell suspension containing the cells into which the compound has been introduced, and the direction of the supply channel with respect to the flow direction of the cell suspension flowing in the supply channel. the height is greater than 1.0 times and less than 2.0 times the diameter of the cell, and the width of the feed channel is greater than 1.0 times and less than 2.0 times the diameter of the cell; The compound introduction device is characterized in that the diameter of the ejection port is more than 1.0 times and less than 2.0 times the diameter of the cells.

According to one embodiment of the present invention, it is possible to achieve both efficient introduction of compounds into cells and efficient generation of cells whose properties are modified by said introduction.

It is a figure which shows an example of a compound introduction apparatus. 4 is a block diagram showing an example of a control system of the compound introduction device; FIG. 3 is a perspective view showing the appearance of an ejection head cartridge; FIG. It is a figure which shows the example of the arrangement|positioning relationship of each structure formed on the electronic board. FIG. 4 is a diagram schematically showing the state of cell suspension in a flow path within the ejection head;

According to studies by the inventors of the present disclosure, when adopting the method described in Patent Document 1, it is necessary to prepare a microchannel having a width slightly narrower than the cell size for each cell type. In addition, when the liquid is sent into the microchannel, the pressure loss increases and a very large force is required. Furthermore, it was found that clogging of the flow path by cells frequently occurred, which was a problem in considering the production efficiency of the introduction treatment.

In addition, it was found that the method described in Patent Document 2 also cannot achieve highly efficient compound introduction.

Therefore, the inventors of the present disclosure have developed a compound introduction device capable of achieving both efficient introduction of a compound into cells and efficient generation of cells whose properties have been modified by the introduction. In order to provide the above, the present invention has been achieved.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments according to the present invention will be described with reference to the drawings. It should be noted that the following embodiments do not limit the present invention, and not all combinations of features described in the embodiments are essential. In addition, the same configuration will be described by attaching the same reference numerals.

[First embodiment]
<Compound introduction device>
FIG. 1 is a diagram showing an example of a compound introduction device 100 of this embodiment. A compound introduction device 100 of the present embodiment is a device provided with an ink jet type ejection head 101 . The ejection head 101 is filled with a liquid containing a compound and cells into which the compound is introduced. In this specification, this liquid is referred to as cell suspension (also referred to as cell-containing liquid). The ejection head 101 may also be called a cell processing head or a liquid ejection head. The cell suspension ejected from the ejection head 101 contains cells into which the compound has been introduced. Such a compound introduction device 100 may be called an ejection device or a cell processing device. The configuration of the compound introduction device 100 will be described below.

FIG. 1 is a diagram schematically showing a compound introduction device 100 of this embodiment. A compound introduction device 100 shown in FIG. The ejection head 101 is driven by the drive motor 102 to move to any location within the transportable area.

By bringing the ejection head 101 into contact with the suction mechanism 104 and operating the suction motor 105 , the liquid filled in the ejection head 101 is filled in the flow paths and the ejection ports in the ejection head 101 . Also, even if no liquid is ejected for a certain period of time, the liquid in the ejection head 101 can be discharged by bringing the ejection head 101 into contact with the suction mechanism 104 and operating the suction motor 105 . The liquid generated by suction and discharge passes through the waste liquid tube 106 and is discharged out of the system. The suction mechanism 104 can also be used as a cap to prevent head drying during non-introduction processing.

The compound-introduced cells are ejected from the ejection head 101 toward the culture dish 107 fixed on the processing stage 108 . In this way, cells into which the compound has been introduced can be obtained.

Intake 109 and exhaust 110 with fans and filters can reduce mist and dust in the device. This allows introduction of the compound while reducing contamination.

<Control block diagram>
FIG. 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 , an ejection 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 a device that controls the introduction of various compounds according to instructions from a host device such as an external device, but the present invention is not limited to this. An operation unit or the like may be provided in the compound introduction device 100, and the introduction of various compounds may be controlled according to instructions according to the operation of the operation unit.

Output data and commands are sent to the compound introduction device 100 from an information terminal (for example, a personal computer, a smart phone, a tablet device, etc.) that serves as a host. The CPU 205 as a control unit receives these output data and commands via the interface controller 201 . The CPU 205 is an arithmetic processing unit that controls the entire compound introduction apparatus 100, such as reception of output data, introduction operation, and transport of the ejection head. After analyzing the received command, the CPU 205 develops control signal data for operating the ejection head 101 and stores it in the image memory 203 .

As an operation process before compound introduction, the CPU 205 reads control signal data used for introduction control from the image memory 203 in synchronization with the conveyance of the ejection head 101 . Then, the read data is transferred to the ejection head 101 via the ejection head control circuit 206 . Also, if necessary, the cells are processed within the ejection head 101 to introduce a compound. Then, directly above the culture dish 107 , the cell suspension is ejected toward the culture dish 107 from the ejection port 3 (see FIG. 4A and the like) of the ejection head 101 . The control signal data may be stored not in the image memory 203 but in the work RAM 204 which is a working memory.

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 process, and the CPU 205 executes commands such as capping process and ejection port suction process according to the elapsed time. The CPU 205 also uses the work RAM 204 as a work memory.

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

Hereinafter, each part constituting the compound introduction device 100 of the present embodiment will be described in detail.

<Ejection head>
In the compound introduction device 100 of the present embodiment, at least mechanical energy is applied to the cell suspension by using the energy generating element, which is an introduction means for introducing the compound into the cells, in an instant. Allows introduction of compounds. As a method capable of instantaneously applying energy in this way, a piezo ink jet system can be used, in which a piezo element is arranged in a microchannel and displacement due to voltage application can be used to produce a mechanical action. Alternatively, a heater (heat generating element) with high electric resistance is arranged in the flow path, and voltage is applied to heat the heater, causing the liquid in the vicinity of the heater to bubble on the order of microseconds. From this, a thermal ink jet system can be used which can bring about mechanical action and thermal action. The use of the thermal ink jet method is preferable because it is possible to apply mechanical energy in a very small space and to make the actuator portion dense. Therefore, the energy generating element is preferably a heating element. Further, when heat is generated by the heating element, it is preferable that stress due to bubbling generated by the heat generation 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 because it can generate a bubbling phenomenon, and more preferably 0.1 μs or more and 5 μs or less because it can suppress cell death.

Further, when the heat generation time of the heater is 1 μs or more and 10 μs or less, the stress on the cell suspension acts over a span of 1 μs or more and 10 μs or less, and as a result, 0.5 m/s or more and 30 m/s or less from the ejection port. is preferable because the droplets are ejected at a speed of

As shown by Campbell et al. in Non-Patent Document 1, the thermal action of the heater has the effect of promoting the expression of intracellular heat shock protein genes or genes promoting cell division. From the viewpoint of efficiently introducing compounds into dividing cells, the thermal inkjet method is preferred.

Moreover, it is preferable that the energy generating element generates energy in the height direction of the supply channel. As a result, pores can be efficiently opened in the cell surface membrane.

The configuration of the ejection head 101 according to this embodiment will be described below with reference to FIGS. 3 and 4. FIG. FIG. 3 is a perspective view showing the configuration of a cartridge (called an ejection head cartridge) of the ejection head 101. As shown in FIG. The ejection head cartridge 1 is composed of a liquid tank portion (also referred to as a storage portion for cell suspension) serving as a liquid supply source, and a chip-shaped ejection head integrally provided therewith. The ejection head is attached by the second sealing material 4 to the liquid supply holder 10 (see FIG. 4). An electrical wiring member 5 for electrical connection between the ejection head and the main body is arranged from the liquid supply/holding member 10 to the liquid tank portion. The electric wiring member 5 is sealed with the first sealing material 2 at the connecting portion (not shown) with the electrode of the ejection head. Coordinate axes are set as shown in FIG. These coordinate axes are also used in subsequent drawings.

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

As shown in FIGS. 4(a) to 4(c), the silicon substrate 9 attached to the liquid supply holder 10 by the second sealing material 4 receives the liquid supplied from the liquid tank. A liquid supply port 11 is provided for supplying the received liquid to the ejection 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 ejection port rows arranged in rows on both sides thereof. The liquid supply port 11 receives liquid supplied from a liquid supply source, and the liquid is supplied to each of the plurality of ejection ports in each of the plurality of ejection port arrays forming the ejection portion. As shown in FIG. 4C, the substrate 9 is provided with heat generating elements 12 corresponding to the ejection ports 3 . Further, in the substrate 9, the liquid supply port 11 and the ejection port 3 are communicated with each other, and the ejection port is formed with the channel wall 22 for partitioning the liquid supply channel (simply referred to as the supply channel) 8 for guiding the liquid. A plate 7 is joined. The channel wall 22 is composed of a channel forming member. The discharge ports 3 are arranged at predetermined intervals along the longitudinal direction of the liquid supply port 11 (the Y direction in the drawing).

4A to 4C, the ejection head includes a substrate 9, a liquid supply port 11, a heating element 12, an ejection port 3, an ejection port plate 7, and a liquid supply port 11. It has a liquid supply channel 8 communicating with the ejection port 3 and a channel wall 22 partitioning the adjacent liquid supply channels 8 .

A wiring pattern is formed on the substrate 9 in addition to the heating elements 12 (for example, electrothermal conversion elements). A discharge port plate 7, which is a structure made of a resin material and in which rows of discharge ports 3 and liquid supply channels 8 are formed, is formed by photolithography. A water-repellent layer 13 is formed on the ejection port plate 7 except for the ejection port 3 portion. As a result, the water-repellent layer 13 forms the outermost surface of the ejection head. Further, on the substrate 9, electric wiring, electrode portions 6, and the like are formed.

The electric wiring member 5 is a member for forming an electric signal path for supplying an electric signal to the electrode wiring on the substrate 9. The electric wiring member 5 is formed with an opening for incorporating the substrate 9. ing. An electrode terminal (not shown) connected to the electrode portion 6 of the chip is formed near the edge of the opening. The chip and electric wiring member 5 are adhered to a liquid supply/holding member 10 formed by resin molding. The electrical connection portion between the substrate 9 and the electrical wiring member 5 includes a first sealing material 2 for sealing the lower portion of the electrical connection portion and the portion where the electrode of the chip is not formed, and the upper portion of the electrical connection portion. is sealed by the second sealing material 4 that seals the . The sealing between the first sealing material 2 and the second sealing material 4 protects the electrical connection from corrosion due to the cell suspension and external impact.

<Flow channel height and channel width of liquid supply unit>
The height as the Z-direction (gravitational direction) length and the width as the Y-direction length of the liquid supply channel 8, which is the liquid supply portion, are each greater than 1.0 times the diameter of the cell, and the cell is less than 2.0 times the diameter of the When the height and width of the liquid supply channel 8 are more than 1.0 times the diameter of the cells, the liquid supply channel 8 is less likely to be clogged with cells. Thus, a cell suspension containing compounds and cells can be delivered to the foam (also called processing chamber) where the heating element 12 is located. Further, since the height of the liquid supply channel 8 is less than 2.0 times the diameter of the cell, when the cell is supplied to the foamed portion where the heating element 12 is located, the supplied cell generates heat. It will be located near the element 12 . Therefore, by adopting such a configuration, it becomes possible to efficiently perform the processing of opening a hole in the cell surface membrane in order to introduce a compound into the cell.

As for the height and width of the liquid supply channel described above, the shortest ones in the liquid supply channel are used. For example, the width of the liquid supply channel in FIG. 4(c) means the length indicated by the arrow of the supply channel immediately before the foaming portion on which the heating element 12 is located. Further, the shape of the liquid supply channel includes a linear shape, a meandering shape, and the like, but the shape is not limited to these, and any shape of the liquid supply channel may be adopted. Further, a structure may be employed in which the height and width of the liquid supply channel narrow from the liquid supply portion toward the bubbling portion, or the height and width of the liquid supply channel may each narrow from the liquid supply portion. Equal length structures may be employed towards the foam. Moreover, the height of the supply channel and the width of the supply channel are relative to the flow direction of the cell suspension flowing through the supply channel.

Moreover, the height and width of the liquid supply channel may be appropriately selected according to 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.

<Discharge port>
In this embodiment, as shown in FIG. 4(c), a circular ejection port 3 is employed, but any shape of ejection port may be appropriately selected and used. Examples of the shape of the ejection port include circular, elliptical, triangular, rectangular, square, polygonal, star-shaped, and shapes having projections. However, regarding the shape of the ejection port, the longest length is preferably 1.0 to 5.0 times the shortest length, more preferably 1.0 to 2.0 times.

Further, as the diameter of the ejection port in this embodiment, the length of the shortest part of the ejection port is used. The diameter of the outlet is more than 1.0 times and less than 2.0 times the diameter of the cell. When the diameter of the discharge port is larger than 1.0 times the diameter of the cells, the cell suspension to which stress is applied can be discharged without clogging the flow path by the cells due to the foaming by the heating element. Also, since the diameter of the outlet is less than 2.0 times the diameter of the cell, the stress received by the ejection causes efficient introduction of the compound.

Regarding the ratio between the diameter of the ejection port and the height of the liquid supply channel, any ratio can be adopted, but it is preferable that the diameter of the ejection port is larger than the height of the liquid supply channel. The reason for this is that by making the diameter of the ejection port larger than the height of the liquid supply channel, it becomes relatively easier for the liquid to be discharged in the direction of the ejection port. This reduces variations in the position of the cells in the flow path, making it possible to stably process the cells.

<Mechanism of compound introduction>
A presumed mechanism of compound introduction by the compound introduction device 100 according to the present embodiment will be described below with reference to FIGS. 4(b) and 5. FIG. FIG. 5 is an enlarged view of the vicinity of the heating element 12 shown in FIG. 4(b).

The cell suspension supplied from the liquid supply port 11 passes through the liquid supply channel 8 and fills up to the ejection port 3 . When the heating element 12 is energized while the cell suspension is filled, the water in the cell suspension is rapidly heated and foamed to form bubbles. At this time, the cell suspension present in the bubble 14 foamed by heating is rapidly extruded mainly toward the discharge port 3 direction (+Z direction in FIG. 5) with low flow resistance. At this time, the unprocessed cells 16 contained in the cell suspension supplied through the liquid supply channel 8 become cells 17 with holes in the cell membrane due to the strong shear flow generated by the 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, some of the compounds contained within the cell suspension are taken up into the cells. Also, the cell suspension extruded by the foaming is discharged through the ejection port 3 directly below the heating element 12 and the foaming section (processing chamber in which the heating element is arranged). In order to facilitate the discharge of the cell suspension containing the compound-introduced cells, it is preferable that the discharge port is arranged directly below the heating element and the processing chamber. If the liquid to be ejected has sufficient kinetic energy by extrusion of the liquid by foaming, the liquid is ejected in the form of flying droplets. In addition, by culturing the cells into which the compound contained in the discharged cell suspension has been introduced, the temporarily vacant holes in the cell membrane can be closed and, depending on the case, the cells can continue to survive. This allows continuous cell culture.

Thus, in this embodiment, the height 20 and width of the liquid feed channel are each greater than 1.0 times the diameter of the cell. As a result, the cell suspension can be supplied to the foamed portion where the heating element is located without clogging the channel with cells. In addition, since the height 20 of the liquid supply channel is less than 2.0 times the diameter of the cell, when the cell is supplied to the foam portion where the heating element 12 is located, the supplied cell generates heat. It will be located near the element 12 . Therefore, with such a configuration, efficient processing becomes possible.

Also, the outlet diameter 21 is greater than 1.0 times the cell diameter. As a result, the foaming of the heating element 12 can discharge the cell suspension to which stress is applied without clogging the flow path with the cells. Furthermore, the outlet diameter 21 is less than 2.0 times the cell diameter and is not too large. Therefore, the introduction of the compound occurs efficiently due to the stress experienced during ejection.

<Method of introducing compound>
The method for introducing a compound in this embodiment has at least the following three steps.

a step of supplying a cell suspension containing cells and a compound through a supply channel (first step); a step of introducing the compound into the cells (second step); A step of ejecting the cell suspension containing the cells from an ejection port (third step). Each of these steps will be described below, taking as an example the case where the introduction means for introducing the compound is a heating element.

In the first step, a compound and a cell suspension containing cells into which the compound is introduced are passed through a liquid supply channel included in a liquid supply section to form a foaming section (processing chamber) in which a heating element is present. ). The height of the liquid supply channel is more than 1.0 times and less than 2.0 times the diameter of the cell, and the width of the liquid supply channel is more than 1.0 times the diameter of the cell. Larger and less than 2.0 times. Note that the cell suspension may be introduced into the ejection head 101 from the cell suspension storage portion of the ejection head cartridge 1, or may be directly introduced into the ejection head 101 using a micropipette or the like. good. When the cell suspension is smoothly filled up to the ejection port of the ejection head 101 due to the wetting and spreading of the cell suspension due to the surface tension, the introduction operation is quickly performed as soon as the cell suspension is filled. When the ejection port of the ejection head 101 is not filled, the suction mechanism 104 or an external suction pump is used to suck from the ejection port, thereby enabling the filling. Alternatively, filling is made possible by pressurizing the reservoir storing the cell suspension using an external pressure pump.

In the second step, heat is generated by the heating element in the foamed portion where the heating element is located. As a result of the second step, the cell suspension foams, and the stress generated by the foaming applies a stress to the cell suspension, allowing the compound to be introduced into the cells.

In the third step, the cell suspension containing the compound-introduced cells is ejected from the ejection port. The diameter of the ejection port is more than 1.0 times and less than 2.0 times the diameter of the cell.

The ejected cell suspension is ejected into the substrate or culture medium. In selecting the base material, it is necessary to consider the overall load on the cells, and the base material is selected according to the purpose. Specifically, in the case of extruding to a substrate for introduction processing, in the case of adhesive substrates such as glass or polystyrene, which have a high affinity for cells on the substrate surface, a medium containing serum or an extracellular matrix is prepared in advance. It is preferable to immerse the material in the solution and discharge the material after the treatment. Further, when a fluororesin such as PTFE, which has a low affinity for cells, is used as the base material, it is preferable to transfer the liquid to be ejected to a medium after ejection. When the introduction operation is performed on the culture dish, it is preferable to bring the cells into contact with a medium containing serum or extracellular matrix in advance on the culture dish. It is also possible to directly eject the cells from the ejection head 101 onto the medium filled in the culture dish, which is selected according to the purpose.

The expelled cell suspension can be immediately transferred to a dish containing the optimal medium for cells, but it can also be retained in a saturated steam environment, and when the introduction amount is increased. is preferred to be left alone.

When the cells are ejected onto the substrate, the cell culture solution on the substrate can be removed with a pipette or the like and seeded onto a dish. Furthermore, the compound introduction operation can be repeated by injecting the cell suspension once ejected into the ejection head 101 and ejecting it. In this case, it is preferable to use a substrate having a low affinity between cells and the surface of the substrate.

In the following description, a compound to be introduced into cells is also referred to as a "compound to be introduced".

<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 with an optical microscope or the like equipped with an image sensor. Using an image recorded using an image sensor, the cell diameter can be obtained based on the distance information corresponding to the pre-stored image. Images are preferably recorded and measured after the cells have been focused to maximize diameter.

<Compounds to be introduced>
The compound to be introduced can be appropriately selected depending on the purpose. Compounds that can be introduced include, for example, nucleic acids, proteins, labeling substances, and the like. It should be noted that the compounds are not limited to these examples as long as they are of a size that can be included in the transfected cells. 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 cells.

The cell size can be measured using a laser diffraction particle size distribution meter or the like after treating the cells with an enzyme or the like and then making a cell suspension. Furthermore, it can be placed in a hemocytometer or the like and measured using an optical microscope. Similarly, the size of the introduced compound can also be measured. In particular, a dynamic light scattering type particle size distribution analyzer can be used for compounds having a particle size of 1 μm or less. Nucleic acids are examples of typical compounds that can be applied in this embodiment.

<Nucleic acid>
For the purposes of transient and stable nucleic acid expression or gene interference, exogenous RNA (ribonucleic acid) or DNA (deoxyribonucleic acid) different from that derived from the transfected cell can be used as the introduction compound. A secondary structure such as a single-stranded primary structure, a hairpin-like stem-loop structure, or a helix structure can be used as the higher-order structure of the nucleic acid. Also, tertiary structures such as A-Form, B-Form, or Z-Form can be used. A quaternary structure such as a supercoil can also be used. These higher-order structures can be suitably used depending on the purpose. Moreover, these nucleic acids may be labeled with a fluorescent compound or a radioactive isotope, and can be used depending on the purpose.

<RNA>
Examples of RNAs handled in this embodiment include messenger RNAs that play a role in copying and transporting sequences from DNA to ribosomes, which are sites of protein synthesis. Also included are ribosomal RNA, which is a constituent of ribosomes, and transfer RNA that carries amino acids with corresponding sequences to ribosomes. Other examples include nuclear low-molecular-weight RNA, nucleolar low-molecular-weight RNA, microRNA, or siRNA having an interfering action. .

<DNA>
Any of single-stranded DNA, double-stranded DNA, triple-stranded DNA and quadruple-stranded DNA can be selected as the DNA used in this embodiment. As for the shape, a linear shape, a circular shape, or the like is generally used, but in recent years, as typified by DNA origami, any shape may be used, and the shape does not matter. Double-stranded DNA is preferred from the standpoint of material stability, and circular plasmid DNA is more preferred from the standpoint of ease of culturing in E. coli or yeast. Furthermore, in order to be introduced into cells, it is necessary to be introduced into the cells from the cell membrane, so the surface area is preferably as small as possible. For example, even if the DNA has the same sequence, a circular shape is preferable to a chain shape, and a supercoiled DNA that is guided by the twist of the DNA is more preferable.

<Proteins>
Proteins handled in this embodiment include proteins that are dissolved, dispersed, or carried on and dispersed on a substrate for the purpose of introduction into a cell suspension. Its structure consists of an amino acid sequence, and has a primary structure including a polypeptide, a secondary structure such as α-helix and β-sheet, a tertiary structure including these secondary structures, and a quaternary structure such as hemoglobin. A higher-order structure can 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. In addition, protective proteins such as globulin, regulatory proteins such as calmodulin, other various membrane proteins, zinc finger nuclease for genome editing, Cas9 protein used for CRISPR / Cas9, and the like.

<Labeling substance>
As the labeling substance handled in this embodiment, the labeling can be confirmed from the outside of the cell at the time of introduction into the cell, and the above-mentioned nucleic acids or proteins may be chemically or physically modified to introduce the labeling substance. . The labeling substance may have a recognizable absorption or emission wavelength different from that of the transfected cells. Moreover, for the purpose of introduction into a cell suspension, it may exist in a state of being dissolved, dispersed, or supported and dispersed on a base material. Specific examples include deuterium, stable isotope substances such as C13 and N15, radioactive substances, dyes, fluorescent dyes, pigments, fluorescent pigments, quantum dots, nanodiamonds, fullerenes, carbon nanosheets, and carbon nanotubes.

<Cell type>
Cells used in the present embodiment include adherent cells, planktonic cells, spheroids (aggregate cells), and the like. Specific examples thereof include human cervical cancer cell lines, nerve cells, hepatocytes, fibroblasts, myoblasts, smooth muscle cells, cardiomyocytes, skeletal muscle cells, stem cells, mesodermal stem cells, Embryonic stem cells, glial cells, fetal stem cells, and hematopoietic stem cells are included. Also included are mast cells, adipocytes, neural stem cells, blood cells and the like.

Also, microbial cells and plant cells can be exemplified. More specific 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 them, the cells are preferably eukaryotic cells. The cell diameter (average value of all cells) is a size that allows ejection from the ejection port, and is preferably 1 μm or more and 100 μm or less, for example.

<Cell suspension>
A cell suspension has at least a compound and cells into which the compound has been introduced. Moreover, in the present invention, the cell suspension is obtained by dispersing cells in a liquid. The cells in the cell suspension may be in a state in which they can be dispersed in the liquid by stirring, and the cells may settle in the liquid when left standing. In addition, it is preferable to contain other components as appropriate in order to allow survival of the cells during and after introduction processing. Other ingredients include salts, sugars, ribonucleotides, growth factors or hormones, pH buffers, surfactants, chelating agents, water-soluble organic solvents, proteins and amino acids, antibacterial agents, moisturizers, or thickeners. etc.

<Salts>
Examples of salts handled in this embodiment may be inorganic salts or organic salts used in cell culture. Specific examples include sodium chloride, potassium chloride, sodium citrate, and the like.

<Saccharides>
Examples of saccharides used in this embodiment include saccharides for the purpose of providing nutrients to cells or adjusting osmotic pressure. Specific examples include glucose, sucrose, fructose, and the like.

<Ribonucleotides>
As an example of ribonucleotides handled in this embodiment, ribonucleotides can be used for the purpose of assisting cell metabolism. Specific examples include adenosine triphosphate, guanosine triphosphate, and the like.

<Growth factors or hormones>
Examples of growth factors or hormones treated in this embodiment include, for example, human growth hormone. Also other animal growth hormones (such as bovine, porcine or chicken growth factors), insulin, oxytocin, angioteocin, methionine enkephalins, substance P. ET-1, FGF, KGF, EGF, IGF, PDGF, LHRH, GHRH, FSH, DDAVP, PTH, vasopressin, glucagon, somatostatin and the like.

<pH buffer>
Examples of pH buffers used in this embodiment include citrate buffers, phosphate buffers, Tris buffers, HEPES buffers, and the like.

<Surfactant>
Examples of surfactants used in this embodiment include water-soluble anionic, cationic, amphoteric or nonionic surfactants, and one or more of them can be added.

<Chelating agent>
Specific examples of chelating agents handled in this embodiment include ethylenediaminetetraacetic acid (EDTA), glycol etherdiaminetetraacetic acid (EGTA), and the like.

<Water and water-soluble organic solvent>
An aqueous liquid medium containing water or a mixture of water and a water-soluble organic solvent can be used for the cell suspension treated in this embodiment. A cell suspension can be obtained by adding cells and a compound to be introduced to an aqueous liquid medium.

The solvent used in the present embodiment is not particularly limited, and examples thereof include water and physiological saline. Phosphate buffer (hereinafter referred to as PBS), buffers such as Tris, and Dulbecco's Modified Eagle Medium (hereinafter referred to as D-MEM) may also be used. Also, Iscove's Modified Dulbecco's Medium (hereinafter referred to as IMDM) and Hanks' Balanced Salt Solutions (hereinafter referred to as HBSS) can be used. Also, Minimun Essential Medium-Eagle, Earle's Salts Base, with Non-Essential Amino Acid (hereinafter referred to as MEM-NEAA). Cell culture media such as RPMI (Roswell Park Memorial Institute Medium) 1640 are also included. In addition, commercially available buffers for electroporation, commercially available buffers for FACS analysis, etc., or infusion solutions such as lactated Ringer's solution can be used. These solvents preferably contain water in an amount of 50% or more. Also, two or more of these solvents can be mixed and used. The water is preferably deionized by ion exchange or the like and heat sterilized by an autoclave or the like. Moreover, the content of water in the cell suspension medium is preferably 30% by mass or more and 99% by mass or less with respect to the mass of the cell suspension.

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, dimethylsulfoxide, and the like. 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 with respect to the total mass of the cell suspension.

<Protein and amino acids>
Examples of proteins and amino acids handled in this embodiment include serum such as fetal bovine serum (hereinafter referred to as FBS) or horse serum.

<Antibacterial agent>
Examples of antibacterial agents handled in this embodiment include antibiotics such as sodium azide or penicillin streptomycin, which can be used by adding to the cell suspension. In particular, saline, buffers such as PBS or Tris, cell culture media such as D-MEM, IMDM, and HBSS, commercially available FACS analysis buffers, etc., and infusion solutions such as lactated Ringer's solution are suitable salts for cells. It is preferably used for controlling concentration or pH.

<Moisturizer>
Examples of moisturizing agents handled in this embodiment include polyhydric alcohols such as glycerin, propylene glycol, butylene glycol, or sorbitol. Also included are mucopolysaccharides such as hyaluronic acid and chondroitin sulfate, protein hydrolysates such as soluble collagen, Yulastin and keratin. These can be used singly or in combination.

<Thickener>
Examples of thickeners used in the present embodiment include starches such as oxidatively modified starch, enzyme modified starch, thermochemically modified starch, cationic starch, amphoteric starch, and esterified starch. Also included are cellulose derivatives such as carboxymethyl cellulose, hydroxyethyl cellulose, and ethyl cellulose, and natural or semisynthetic polymers such as casein, gelatin, and soybean protein. In addition, completely or partially saponified water-soluble polymer compounds are also included. Examples of water-soluble polymer compounds include polyvinyl alcohol, acetoacetylated polyvinyl alcohol, and carboxy-modified polyvinyl alcohol. Polyvinyl alcohols such as olefin-modified polyvinyl alcohol and silyl-modified polyvinyl alcohol are also included. At least one water-soluble polymer compound can be appropriately selected and used from among these. In this embodiment, the liquid to be used preferably has a viscosity of 0.1 Pa·s (pascal second) or more, and the above thickening agent may be added as necessary.

<Method for preparing cell suspension>
In the cell suspension of the present embodiment, cells cultured by adhesion culture, suspension culture, etc. are separated into single cells or small cells by the action of enzymes, etc., and then centrifuged. Using the difference in specific gravity, only the cells are sedimented. Thereafter, after removing the supernatant medium other than the cells, a medium containing the compound to be introduced is added and stirred using a pipette or a stirrer to prepare a cell suspension.

The concentration of the cell suspension to be produced may be adjusted according to the purpose, but from the viewpoint of production efficiency, it is preferably 1000 cells/mL or more and 100 million cells/mL or less, and 100,000 cells/mL or more and 10 million/mL or less is more preferable.

Moreover, before introducing the cell suspension into the ejection 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 channel in the ejection head 101 to be used. This allows removal of large cell clumps from the cell suspension.

<Culture operation method>
The processed cells can be cultured in the desired culture system. Specifically, animal cells are preferably cultured in an incubator maintained at a culture temperature of 37 degrees Celsius and saturated steam with a carbon dioxide concentration of 5%. During culturing, it is preferable to confirm the growth state of the cells and perform medium exchange or passage.

<Method for confirming introduction of compound into cells>
The method for confirming the introduction of a compound into a cell differs depending on the compound to be introduced, so a confirmation method suitable for each case may be used. For example, when a plasmid DNA that expresses a fluorescent protein, GFP, is introduced into the same cell, the sample after a certain period of time is examined for the presence or absence of GFP emission using a fluorescence microscope. You can check the amount introduced. In addition, introduction can be quantitatively confirmed by using flow cytometry to count the number of luminous cells after dispersing the cultured cells into single cells using an enzyme or the like. Furthermore, it can also be disintegrated and measured using a fluorescence spectrophotometer, luminometer, or the like. Also, ELISA or immunostaining using antigen-antibody reaction can be used. It is also possible to measure the introduced DNA and the 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.

[Example]
Examples of the present embodiment will be described below. It should be noted that the following examples are merely examples, and are not limited to these examples. In the text, "%" is based on mass unless otherwise specified.

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

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

(Liquid composition 1 containing compound to be introduced)
As shown in Table 1 below, 500 μl of a trypan blue solution containing trypan blue, which is the compound to be introduced, and 500 μl of Gene Pulser electroporation buffer (manufactured by Lonza) were mixed in a microtube to obtain liquid composition 1. rice field.

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

First, 0.5 mol/L-EDTA Solution (pH 8.0) (manufactured by Nacalai Tesque) 0.2 mL and 1 mol/L-Tris-HCl Buffer Solution (pH 8.0) (manufactured by Nacalai Tesque) 1 mL were mixed to obtain a mixture.

Next, 98.8 mL of sterilized pure water was added to this mixture to prepare a TE buffer (10 mM Tris 1 mM EDTA (pH 8)). Freeze-dried CMV-Fresno RFP (manufactured by ATUM, base pair number 5.5 kbp) and 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, filled in a quartz cell, and the concentration was identified by a DNA concentration measuring device (GeneQuant 1300, Biochrom) to determine the concentration of the DNA solution. DNA concentration was 2.0 μg/μL.

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.

A cell suspension was prepared by the following procedure.

RAW264.7, a cell line established from mouse monocytic leukemia purchased from the American Type Culture Collection, was subjected to the following procedure so as to have a concentration of 2.0 million cells/mL.

Cell line RAW264.7 was dispersed at 2.0 million cells/mL in 20 mL of D-MEM medium containing 10% FBS, 1% penicillin-streptomycin, and 1% MEM-NEAA. After that, about 2.0 million seeds were sown in each 100 mm dish made of polystyrene.

The 10% FBS used was manufactured by Global Life Science Technologies Japan. 1% penicillin streptomycin was used from Sigma-Aldrich. 1% MEM-NEAA manufactured by Thermo Fisher Scientific was used. D-MEM manufactured by Thermo Fisher Scientific was used. A 100 mm polystyrene dish manufactured by Corning was used.

Cells were grown by incubating dishes with D-MEM medium containing seeded cells at 37° C. in the presence of 5% CO ₂ . After 2 to 4 days, it was confirmed that about 70% of the bottom surface of the dish was covered with cells, and after removing the supernatant medium, the dish was rinsed with PBS. Cells were detached from the dish using PBS containing 0.25% trypsin and 1 mM EDTA (manufactured by Thermo Fisher Scientific). A cell suspension containing RAW264.7 cells was then harvested from the dish.

In a sterilized centrifuge tube, the above D-MEM medium was added to the cell suspension collected from the dish so that the total volume was 50 mL, and then centrifuged at a temperature of 4 degrees Celsius and a centrifugal force of 90 G. The cells were treated with a machine (CF16RX II, manufactured by Hitachi Koki Co., Ltd.) for 5 minutes to sediment the cells. After gently removing the supernatant of the precipitated cell pellet, the above D-MEM medium was added to the cell pellet to obtain a cell suspension. This cell suspension was again subjected to the above cell culture procedure twice.

During the third run, the number of cells required for the desired cell concentration was set aside into another centrifuge tube. Thereafter, the mixture was centrifuged, and the above liquid composition 1 was added instead of the above D-MEM medium and stirred using a micropipette. Furthermore, it was passed through a cell strainer (manufactured by Corning, mesh 40 μm) to obtain a cell suspension 1 (2.0 million cells/μL) used for introducing the compound into the cells. A cell suspension 2 was obtained in the same manner as the cell suspension 1, except that the liquid composition 2 was used instead of the liquid composition 1.

The resulting cell suspensions 1 and 2 were filled in 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). did. After that, the diameter of the cells was obtained by measuring the diameter of the longest part of 1000 cells using the recorded images. The diameter of the cells was obtained as the diameter of 50% of the cells on the number basis in the cell diameter distribution obtained by the above measurement.

<Example 1>
In this example, a compound was introduced into cells using the compound introduction device 100 of FIG. 1 in the following manner.

First, 200 μL of the cell suspension 1 prepared by the above operation was injected into the ejection head 101 using a micropipetter. After that, the ejection head 101 was conveyed to the suction mechanism 104 and grounded, and then the suction motor 105 was operated to fill the cell suspension 1 into each channel and the ejection port 3 of the ejection head 101 . After filling, the introduction operation program was run. By executing the introduction operation program, the ejection head 101 is moved away from the suction mechanism 104, and then transported to the upper part of the glass bottom dish (manufactured by Iwaki Co., Ltd.) having the inner bottom surface wetted with 100 μL of the D-MEM medium set in advance. rice field. After that, all the ejection ports 3 are used to perform a liquid ejection operation for outputting an image with a processing duty of 100% (specifically, a solid image of 1.5 cm×1.5 cm), into a glass bottom dish. , the cell suspension 1 was discharged to obtain a sample. This liquid ejection operation was repeated 40 times to confirm continuous workability (the number of times of continuous printing possible in Table 2). In the compound introduction device used in this example, "processing duty is 100%" means that 23.0 n (nano) g cells are placed in a unit area of 1/600 inch x 1/600 inch at a resolution of 600 dpi x 600 dpi. It means to apply one drop of suspension. Incidentally, during the liquid ejection operation, the ventilation fan motor 210 was driven to operate the intake fan of the intake port 109 .

The sample thus obtained is covered with a top plate of a glass bottom dish, taken out of the apparatus, and 2.5 mL of the D-MEM medium warmed to 37° C. is gently poured using a micropipette. rice field. Then, the cells were observed using a phase-contrast microscope (manufactured by Olympus, model number: CKX41) to determine the processing rate.

<<Evaluation of processing rate>>
As described above, the processing rate of the compound-introduced cells using the cell suspension 1 was measured. Specifically, using a phase-contrast microscope, the cells were observed in bright field mode with a 10x objective lens, and the number of cells stained purple with trypan blue and the total number of cells were counted. Then, the processing rate was calculated according to the formula of processing rate=purple cell number/total cell number×100, and the calculated processing rate was evaluated according to the following five-level criteria. The results of such evaluation are set forth in Table 2. Incidentally, in the present embodiment, C-rank or lower is defined as an unacceptable level from the viewpoint of production efficiency.
AA: processing rate of 50% or more A: processing rate of 40% or more and less than 50% B: processing rate of 20% or more and less than 40% C: processing rate of 10% or more and less than 20% D: processing rate of less than 10%

<Examples 2 and 5 and Comparative Examples 1 and 2>
As shown in Table 2, the same operation as in Example 1 was performed except that the diameter of the ejection port, the height and width of the liquid supply channel, and the thickness of the nozzle were changed. Also, samples of Comparative Examples 1 and 2 were produced. Then, the processing rates of the obtained samples were evaluated in the same manner as in Example 1. The results of such evaluation are shown in Table 2. In Comparative Example 1, the liquid ejection operation could not be repeated 40 times.

<Example 3>
A sample was prepared in the same manner as in Example 1 except that Cell Suspension 2 was used instead of Cell Suspension 1. Then, the obtained samples are incubated at 37°C in the presence of 5% CO ₂ to grow the cells, and after 24 hours, the gene expression is evaluated using a fluorescence microscope to determine the presence or absence of DNA introduction into the cells. did.

<<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. Therefore, the expression of this fluorescent protein was used to evaluate DNA transfer.

Specifically, from samples incubated for 24 hours, medium was removed, rinsed with 1×PBS, and then 2 mL of 1×PBS was added. After replacing the medium with 1 × PBS, using a fluorescence microscope (manufactured by Keyence, model number: BZ-8000), 10 × objective lens, bright field mode, fluorescence mode (TRITC: excitation 540 ± 12.5 nm, fluorescence 605 ±27.5 nm, cut 565 nm). Then, the number of cells emitting fluorescence and the number of all cells were counted, and the introduction rate was calculated according to the formula of introduction rate = (number of cells emitting fluorescence/total number of cells) x 100, and the calculated introduction rate was calculated. Evaluation was made according to the following criteria. The results of such evaluation are set forth in Table 2.
Yes: Introduction rate 1% or more No: Introduction rate less than 1%

<Example 4 and Example 6>
As shown in Table 2, the same operation as in Example 3 was performed except that the diameter of the ejection port, the height and width of the liquid supply channel, and the thickness of the nozzle were changed. A sample was made. Cells of the obtained samples were grown in the same manner as in Example 3, and gene expression was evaluated in the same manner as in Example 3. The results of such evaluation are set forth in Table 2.

<Comparative Example 3 and Comparative Example 4>
Comparative Examples 3 and 4 will be described below as comparative examples of Examples 1 and 3. FIG. Specifically, in Comparative Examples 3 and 4, in order to confirm the effect of foaming, the cell suspension was taken out by external suction through the ejection port instead of the ejection operation without foaming by the heating element. , was added in a glass bottom dish. Except for this, the same operations as in Examples 1 and 3 were performed to prepare samples that were not subjected to the foaming treatment. Using the prepared samples, the processing rate was evaluated in Comparative Example 3, and the gene expression was evaluated in Comparative Example 4. The results of such evaluation are set forth in Table 2.

<Other embodiments>
Further, the present invention supplies a program that implements one or more functions of the above-described embodiments to a system or device via a network or a storage medium, and one or more processors in the computer of the system or device executes the program. It can also be realized by a process of reading and executing. It can also be implemented by a circuit (for example, ASIC) that implements one or more functions.

Claims (14)

A compound introduction device for introducing a compound into cells,
a supply channel for supplying a cell suspension containing the cells and the compound;
an introduction means for introducing the compound into the cell;
an ejection port for ejecting a cell suspension containing the cells into which the compound has been introduced;
has
The height of the supply channel with respect to the flow direction of the cell suspension flowing in the supply channel is more than 1.0 times and less than 2.0 times the diameter of the cells, and the supply channel the width is greater than 1.0 and less than 2.0 times the diameter of the cell;
The diameter of the outlet is more than 1.0 times and less than 2.0 times the diameter of the cell.
A compound introduction device characterized by: The introduction means is an energy generating element that generates energy in the height direction of the supply channel,
The compound introduction device according to claim 1, characterized in that: The introducing means is a heating element,
The compound introduction device according to claim 1, characterized in that: Further having a processing chamber in which the heating element is arranged,
4. The compound introduction device according to claim 3, characterized in that: The discharge port is arranged directly below the heating element and the processing chamber,
5. The compound introduction device according to claim 4, characterized in that: When heat is generated by the heating element, stress due to foaming generated by the heat generation is applied to the cell suspension inside the processing chamber,
6. The compound introduction device according to claim 4 or 5, characterized in that: As a result of the stress acting for 1 μs or more and 10 μs or less, droplets of the cell suspension are ejected from the ejection port 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, characterized by: The heat generation time of the heating element is 0.1 μs or more and 5 μs or less,
8. The compound introduction device according to any one of claims 3 to 7, characterized in that: the diameter of the outlet is greater than the height of the supply channel;
9. The compound introduction device according to any one of claims 1 to 8, characterized in that: the cell has a diameter of 1 μm or more and 100 μm or less;
10. The compound introduction device according to any one of claims 1 to 9, characterized in that: 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 any one of claims 1 to 10, characterized in that: said cell is a eukaryotic cell,
12. The compound introduction device according to any one of claims 1 to 11, characterized in that: The height of the supply channel means the length in the direction of gravity,
13. The compound introduction device according to any one of claims 1 to 12, characterized in that: A compound introduction method for introducing a compound into a cell, comprising:
supplying a cell suspension containing the cells and the compound through a supply channel;
introducing the compound into the cell;
a step of ejecting a cell suspension containing the cells into which the compound has been introduced from an ejection port;
has
The height of the supply channel with respect to the flow direction of the cell suspension flowing in the supply channel is more than 1.0 times and less than 2.0 times the diameter of the cells, and the supply channel the width is greater than 1.0 and less than 2.0 times the diameter of the cell;
The diameter of the outlet is more than 1.0 times and less than 2.0 times the diameter of the cell.
A method for introducing a compound, characterized by:

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