Container for plasma-type cell processing device, plasma-type cell processing device, and molecule introduction method

Abstract

Provided is a container for a plasma-type cell processing device used in a plasma-type cell processing device that stimulates an object to be processed by plasma generated from a gas phase between electrodes separated by a gas phase, the container for a plasma-type cell processing device including a container body having an inner part as a housing chamber and an opening at the upper end and a lid body for closing the opening part. Either the container body or the lid body has an electrode receiving part that incorporates one of the separated electrodes E1 or that receives the electrode E1.

Description

Container for plasma cell treatment device, plasma cell treatment device and molecule introduction method

[0001] The present invention relates to a container for a plasma cell treatment device and a plasma cell treatment device. This application claims priority from Japanese Patent Application No. 2023-169637, filed on September 29, 2023, the contents of which are incorporated herein by reference.

[0002] In genetic engineering and pharmaceutical development, molecules are introduced into target cells or tissues (hereinafter collectively referred to as "target cells") to test the function and physiological activity of the molecules in the target cells. Examples of molecules include polynucleotides, signal transduction proteins, transcriptional regulators, small-molecule physiologically active substances, and drug candidates.

[0003] Methods for introducing molecules include electroporation, gene gun, liposome, cell fusion, viral vector, etc. However, the efficiency of introducing molecules into target cells and the like using these molecule introduction methods has not yet been satisfactory.

[0004] To address these problems, Patent Documents 1 and 2 propose a plasma cell treatment device that includes a capillary electrode, a counter electrode provided on the opposite side of the capillary electrode, and a power supply connected to the capillary electrode and the counter electrode. According to the inventions of Patent Documents 1 and 2, plasma generated between the electrodes stimulates target cells, which are the object of treatment, and introduces molecules into the target cells.

[0005] Patent No. 6189019 Patent No. 5737828

[0006] The inventions of Patent Documents 1 and 2 involve discharging a plasma treatment electrode placed on the upper surface of a cell culture surface, so that the target cells or other objects to be treated are exposed to a treatment space within the treatment device during treatment. If the target cells or other objects migrate and remain in the treatment space, there is a risk of contamination with subsequent target cells or other objects. The present invention aims to provide a container for a plasma cell treatment device that can treat objects to be treated without exposing them to the external atmosphere.

[0007] The present invention has the following aspects. <1> A container for a plasma cell processing device used in a plasma cell processing device that stimulates a processing target with plasma generated from a gas phase between electrodes spaced apart by a gas phase, the container comprising: a container having an interior containing a storage chamber and an opening at an upper end; and a lid that closes the opening, wherein one of the container and the lid has an electrode E1, one of the spaced apart electrodes, incorporated therein, or an electrode receiving portion that accepts the electrode E1. <2> The container for a plasma cell processing device according to claim 1, wherein the incorporated one electrode E1 has a conductive member that penetrates the container or the lid. <3> The container for a plasma cell processing device according to claim 2, wherein the electrode E1 incorporated in the lid has an irradiation surface that connects to a lower end of the conductive member, or the electrode E1 incorporated in the container has an irradiation surface that connects to a side surface of the conductive member, the irradiation surface being made of a patterned conductive material. <4> The container for a plasma cell processing device according to claim 3, wherein the irradiation surface is composed of a plurality of linear bodies, and the plurality of linear bodies include one or more first conductive wires extending in one direction of the irradiation surface and one or more second conductive wires intersecting the first conductive wires. <5> The container for a plasma cell processing device according to claim 4, wherein the width of the first conductive wire is 1 to 1000 μm, and the width of the second conductive wire is 1 to 1000 μm. <6> The container for a plasma cell processing device according to claim 1, wherein the electrode receiving portion has an insertion path for receiving the electrode E1, and when the lid is closing the opening, the insertion path extends from the upper surface to the lower surface of the lid or from the lower surface to the upper surface of the container, and the electrode receiving portion has a partition wall that closes the tip of the insertion path in the insertion direction. <7> The container for a plasma cell processing device according to claim 6, wherein a conductive member is included inside the insertion path. <8> The container for the plasma cell processing device according to any one of claims 1 to 7, wherein the container has a bottom wall at a lower end, and a cell culture scaffold on a surface of the bottom wall facing the storage chamber. <9> The container for the plasma cell processing device according to any one of claims 1 to 8, wherein the lid has one of the electrodes or the electrode receiving portion.<10> A container for a plasma cell processing device according to any one of claims 1 to 9, wherein the plasma cell processing device is for molecule introduction. <11> A plasma cell processing device having the container for a plasma cell processing device according to any one of claims 1 to 10. <12> The plasma cell processing device according to claim 11, wherein the plasma cell processing device is for molecule introduction. <13> A molecule introduction method for introducing a molecule into a target cell, comprising: providing a container having a container with an internal storage chamber and an opening at an upper end, and a lid that closes the opening, wherein either the container or the lid has an electrode E1 incorporated therein or an electrode receiving portion that receives the electrode E1; injecting target cells and molecules into the storage chamber of the container; if the container has the electrode receiving portion, inserting electrode E1 into the electrode receiving portion, and then arranging electrode E2 in a position facing electrode E1 across a gas phase; and applying a voltage between electrode E1 and electrode E2 with the opening closed by the lid and electrode E1 facing electrode E2 across a gas phase.

[0008] According to the container for the plasma cell treatment device of the present invention, the treatment object can be treated without being exposed to the outside.

[0009] Fig. 1 is a cross-sectional view of a plasma cell processing device according to a first embodiment. Fig. 2 is a cross-sectional view of a plasma cell processing device according to a second embodiment. Fig. 3 is a cross-sectional view of a plasma cell processing device according to a third embodiment. Fig. 4 is a cross-sectional view of a plasma cell processing device according to a first modified example. Fig. 5 is a cross-sectional view of a plasma cell processing device according to a second modified example. Fig. 6 is a cross-sectional view of a plasma cell processing device according to a third modified example. Fig. 7 is a cross-sectional view of a plasma cell processing device according to one embodiment.

[0010] In this specification and claims, the use of "to" indicating a range of values ​​means that the values ​​before and after it are included as the lower and upper limits.

[0011] The plasma cell processing device of the present invention (hereinafter sometimes referred to as the "cell processing device") has electrodes separated by a gas phase and a container for the plasma cell processing device (hereinafter sometimes simply referred to as the "container") positioned between the electrodes. The cell processing device stimulates target cells, target tissue, etc. (collectively referred to as "target cells, etc.") that are the object to be processed. For example, the cell processing device generates plasma between the electrodes separated by a gas phase, irradiates an irradiation object in which target cells, etc. and molecules coexist, and introduces molecules into the target cells, etc. The introduction of molecules proceeds primarily by inducing endocytosis by the plasma. The cell processing device of the present invention will be described below with reference to an embodiment.

[0012] 1 has a first electrode 10, a second electrode 20, a power supply unit 30, and a container 100. In this specification, the "first electrode" corresponds to the electrode E1, and the "second electrode" corresponds to the electrode E2.

[0013] The second electrode 20 is spaced apart from the first electrode 10 and located below the lower end (tip) of the first electrode 10. That is, the second electrode 20 is located beyond the tip of the first electrode 10, and the first electrode 10 and the second electrode 20 are spaced apart. The power supply unit 30 is connected to the first electrode 10 and the second electrode 20 by wiring 12. The container 100 is located between the first electrode 10 and the second electrode 20. In this embodiment, the container 100 is placed on the second electrode 20 and is spaced apart from the first electrode 10.

[0014] In this embodiment, the first electrode 10 is a high-voltage electrode. The first electrode 10 in this embodiment is a rod-shaped body extending in one direction. The first electrode 10 may have a hollow structure such as a cylindrical or polygonal tube, or a solid structure such as a columnar or polygonal pillar. In particular, if the first electrode 10 has a solid structure, durability is increased and manufacturing is easy. Examples of materials for the first electrode 10 include metals such as stainless steel, copper, and tungsten, and carbon.

[0015] The thickness (outer diameter) of the first electrode 10 is, for example, 3 to 50 mm. When the first electrode 10 is in the shape of a polygonal cylinder or a polygonal column, the outer diameter is the diameter of a circumscribed circle of the cross section of the first electrode 10.

[0016] The second electrode 20 may be any electrode that functions as a ground electrode, such as a flat electrode. Examples of materials for the second electrode 20 include metals such as stainless steel, copper, and tungsten, and carbon.

[0017] In a plan view, the area A2 of the second electrode 20 is preferably larger than the area A1 of the first electrode 10, and more preferably larger than the area 111R of the bottom wall portion 112 of the container 110 on the side of the storage chamber 111. If the area of ​​the second electrode 20 is large, the plasma can be irradiated from the first electrode 10 to the irradiation target over a wider range. From this viewpoint, the area A2 is preferably at least one time the area 111R.

[0018] The power supply unit 30 is only required to apply a voltage between the first electrode 10 and the second electrode 20 and generate plasma between the two electrodes. The power supply unit 30 switches between starting and stopping the supply of electricity to the electrodes. The power supply unit 30 also adjusts the voltage and frequency applied to the electrodes. An example of the power supply unit 30 is a circuit that is connected to an external power source and includes an inverter. Another example of the power supply unit 30 is a secondary battery.

[0019] The container 100 includes a container body 110 and a lid 120. The container body 110 includes a bottom wall 112 and a container side wall 113 rising from the periphery of the bottom wall 112. The container body 110 has an opening 115 at its upper end, surrounded by the container side wall 113. The interior of the container 110, surrounded by the bottom wall 112 and the container side wall 113, forms a storage chamber 111. The container body 110 may be cylindrical or polygonal tubular. The bottom wall 112 of the container 100 is placed on the second electrode 20, but no electrode is present on the upper surface of the bottom wall 112 (the surface facing the storage chamber 111). The bottom wall 112 preferably has a cell culture scaffold on the surface facing the storage chamber 111. There are no particular limitations on the scaffold material for providing the cell culture scaffold, and any known scaffold material can be used. Examples of known scaffold materials include a scaffold material for cell culture described in JP 2024-111315 A, which contains a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion, and has a hydroxyl value of 1,100 mg KOH / g or less; a scaffold material for cell culture described in JP 2018-064542 A, which contains a protein having a repeating structure and a cell adhesion sequence such as an RGD sequence; a scaffold material for cell culture described in JP 2006-314285 A, which is composed of a molded product made of a polyvinyl acetal compound or a molded product made of the polyvinyl acetal compound and a water-soluble polysaccharide, and in which the degree of acetalization of the polyvinyl acetal compound is 20 to 60 mol %; and a scaffold material for cell culture described in WO 2007 / 090102 A1, which contains a first fibrous polymer scaffold material in which the fibers of the first fibrous polymer scaffold material are aligned.

[0020] The lid 120 has a lid main body 121 and an electrode receiving portion 124. The lid main body 121 has a top wall portion 122 and a lid side wall portion 123 that hangs down from the periphery of the top wall portion 122. The lid side wall portion 123 tapers downward. In this embodiment, when the lid 120 and the container 110 are combined (i.e., when the lid 120 covers the opening 115), the lid side wall portion 123 is located on the outer surface of the container side wall portion 113. In other words, the lid 120 of this embodiment is an externally fitted lid. Because the lid side wall portion 123 tapers downward, the lid 120 fits into the container 110 when the lid 120 and the container 110 are combined.

[0021] The electrode receiving portion 124 is a cylindrical or polygonal cylindrical member that hangs down from the underside of the top wall portion 122 toward the bottom wall portion 112. The electrode receiving portion 124 has an insertion path 125 therein. That is, the insertion path 125 extends from top to bottom. The electrode receiving portion 124 has a partition wall portion 126 that closes the lower end of the insertion path 125. The partition wall portion 126 is a portion of the electrode receiving portion 124 that separates the first electrode 10 from the storage chamber 111 at a location located in the region where the first electrode 10 and the second electrode 20 face each other, and is spaced apart from the inner bottom surface of the container 110. By including the partition wall portion 126 in the electrode receiving portion 124, the amount of exposure of target cells and the like in the storage chamber 111 to the environment (irradiation environment) in which the irradiation step described below is performed can be further reduced. The lid 120 only needs to close the opening 115 to such an extent that the target cells 44 in the storage chamber 111 are not directly exposed to the irradiation environment. Therefore, a gap may be formed between the lid 120 and the container 110.

[0022] The material of the container 110 may be a conductive material or an insulating material. However, from the viewpoint of preventing localized discharge due to high voltage, an insulating material is preferable. Examples of insulating materials include resin, ceramics, glass, etc. Examples of resins include polystyrene, polyolefin, polyester, acrylic, etc. For example, if the container 100 is to be disposable, the material of the container 110 is preferably resin. Examples of conductive materials include the same materials as those of the first electrode 10.

[0023] The material of the lid body 121 is the same as the material of the container 110. The material of the lid body 121 may be the same as or different from the material of the container 110. From the viewpoint of generating plasma between the electrodes, at least one of the container 110 and the lid body 121 is made of an insulating material.

[0024] The material of the electrode receiving portion 124 may be a conductive material, an insulating material, or a composite of a conductive material and an insulating material. For example, the material of the partition wall 126 may be a conductive material, and the material of the other members of the electrode receiving portion 124 may be an insulating material. Alternatively, for example, the material of the entire electrode receiving portion 124 may be an insulating material, and the material of the lid main body 121 may be an insulating material. Alternatively, for example, the lid main body 121 and the electrode receiving portion 124 may be integrally molded from an insulating material. When the partition wall 126 is made of an insulating material, the thickness of the partition wall 126 is, for example, 0.1 to 10 mm.

[0025] The inner diameter R111 of the container 110 is not particularly limited and is, for example, 1 to 150 mm. When the container 110 is polygonal cylindrical, the inner diameter R111 is the diameter of a circumscribing circle of the container 110 in a planar view. When the container 110 is polygonal cylindrical, the number of corners of the container 110 in a planar view is not particularly limited and is, for example, 4 to 20. Note that, in a planar view, the area of ​​the inner bottom surface of the container 110 is preferably smaller than the area of ​​the second electrode 20. When the area of ​​the inner bottom surface of the container 110 is smaller than the area of ​​the second electrode 20, plasma can be irradiated over a wider area and more uniformly. The internal height H111 of the container 110 is not particularly limited and is, for example, 5 to 50 mm.

[0026] The length L125 of the insertion path 125 in the electrode receiving portion 124 may be, for example, 1 to 50 mm, as long as it is large enough to receive the first electrode 10 and determine the position of the first electrode 10. The thickness of the insertion path 125 can be determined appropriately depending on the thickness of the first electrode 10.

[0027] <Method of Using the Cell Processing Device> The method of using the cell processing device will be described using an example in which the cell processing device is used for molecule introduction. An example of a molecule introduction method is an introduction method including a pre-incubation step, a contact step, a sealing step, and an irradiation step.

[0028] In the pre-culture step, target cells, etc. 44, which are the object to be treated, are cultured. A conventionally known culture method can be used as the culture method. An example of the culture method is a method in which target cells, etc. 44 and a culture medium are injected into the container 110 (i.e., the storage chamber 111) and the target cells, etc. 44 are cultured. After the pre-culture, the culture medium is removed from the container 110, and the pre-cultured target cells, etc. 44 are obtained.

[0029] Examples of target cells etc. 44 include animal cells including human cells, non-human animal individuals, cells collected from human individuals, cells within human individuals, human individuals, plant cells, and microbial cells. Note that cells collected from human individuals include cells that are not intended to be returned to human individuals, and cells intended to be returned to human individuals for use in regenerative medicine, etc. Furthermore, the above-mentioned cells collected from human individuals also include cells cultured from cells collected from human individuals.

[0030] The culture medium used for the pre-culture is not particularly limited and may be appropriately selected depending on the purpose. The culture conditions for the pre-culture step are, for example, 30 to 40°C and 24 to 72 hours.

[0031] The number of target cells etc. 44 after pre-culture is determined appropriately taking into consideration the capacity of the container 100. For example, when the container 110 has a size equivalent to one well of a 96-well culture plate, the number of target cells etc. 44 after pre-culture is 1 x 10 ３ ~1 x 10 ６ For example, if the container 110 has a size equivalent to one well of a six-well container, the number of target cells 44 after pre-culture is 1×10 ４ ~1 x 10 ７ It is considered to be an individual.

[0032] The contacting step is a step in which the target cells 44 and the molecules 42 are brought into contact with each other by coexisting them. The molecules 42 are added to the container 110 from which the culture medium has been removed. At this time, the molecules may be made into an aqueous solution or dispersion (collectively sometimes referred to as an "aqueous liquid"), and this aqueous liquid may be added to the container.

[0033] The molecule 42 is a molecule selected for introduction into a target cell or the like 44. Examples of the molecule 42 include a polymeric compound, a low-molecular-weight physiologically active substance, and a drug candidate. Examples of the polymeric compound include polynucleotides such as DNA and RNA or derivatives thereof, proteins or peptides such as signal transduction proteins and transcriptional regulatory factors, and derivatives thereof. As the DNA or RNA, constructs, plasmids, phagemids, and the like that are capable of expressing genes in the target cell or the like are preferred. The molecule 42 may be one type alone or a combination of two or more types.

[0034] The sealing step is a step of combining the container 110 and the lid 120 and closing the opening 115 with the lid 120. The method for introducing molecules according to the present embodiment includes the sealing step, which makes it possible to prevent contamination of the target cells 44 and the like in the environment in which the irradiation step described below is performed.

[0035] The irradiation step is a step of irradiating plasma into the storage chamber 111 while the target cells, etc. 44 and the molecules 42 coexist and are in contact with each other. Through the irradiation step, the molecules 42 are introduced into the target cells, etc. 44. At this time, because the opening 115 is closed by the lid 120, the target cells, etc. 44 are not exposed to the internal space of the cell processing device 1 (e.g., the space for placing a container inside the device) or the space where the irradiation step is performed (e.g., the inside of a cell culture chamber or a clean bench). This prevents contamination by bacteria, such as target cells from other lots.

[0036] The container 100 containing the molecules 42 and the target cells, etc. 44 is placed on the second electrode 20. Next, the first electrode 10 is inserted into the insertion path 125 of the lid 120, and the position of the first electrode 10 is determined. In the irradiation step, power is supplied to the first electrode 10 from the power supply unit 30, and a voltage is applied between the first electrode 10 and the second electrode 20. When a voltage is applied between the electrodes, plasma is generated from the first electrode 10 toward the second electrode 20 (applying a voltage between the first electrode 10 and the second electrode 20 to generate plasma between the electrodes is also referred to as plasma irradiation). When plasma irradiation is performed, the molecules 42 are introduced into the target cells, etc. 44.

[0037] The distance from the lower end of the first electrode 10 to the irradiation target (the surface of the liquid where the molecules 42 and the target cells 44 etc. coexist) is preferably 0.1 to 5 mm. When the distance from the lower end of the first electrode 10 to the irradiation target is within the above range, plasma can be generated more stably.

[0038] The AC voltage applied between the first electrode 10 and the second electrode 20 is preferably 1 to 30 kVpp, and more preferably 5 to 20 kVpp. Setting the AC voltage to the above-mentioned lower limit or higher can further increase the introduction efficiency. Setting the AC voltage to the above-mentioned upper limit or lower can suppress the temperature rise of the electrodes, thereby suppressing the temperature rise inside the container 100. The unit "Vpp (Volt peak to peak)" that represents the AC voltage is the potential difference between the maximum value and the minimum value of the AC voltage waveform.

[0039] The frequency of the alternating current applied between the first electrode 10 and the second electrode 20 is preferably 0.1 to 500 kHz, more preferably 10 to 200 kHz. Setting the frequency to the above lower limit or higher can further increase the introduction efficiency. Setting the frequency to the above upper limit or lower can further reduce damage to target cells, etc.

[0040] The plasma irradiation time in the irradiation step is, for example, preferably 10 nanoseconds to 1 second, and more preferably 100 nanoseconds to 0.1 second. If the irradiation time is equal to or greater than the above-mentioned lower limit, the amount of molecules 42 introduced into target cells, etc. 44 can be further increased. If the irradiation time is equal to or less than the above-mentioned upper limit, damage to target cells, etc. can be further reduced.

[0041] In the irradiation step, the environment in the storage chamber 111 is not particularly limited, but may be, for example, CO ２ It may be in the presence of CO ２ The concentration is, for example, 1 to 10% by volume.

[0042] The method for introducing a molecule may include a post-culture step after the irradiation step. The post-culture step is a step of culturing target cells, etc. into which the molecule has been introduced (sometimes referred to as "introduced cells, etc."). The culture conditions for the post-culture step are the same as those for the pre-culture step and can be appropriately determined depending on the type of introduced cells, etc., the intended use of the introduced cells, etc., etc. The culture conditions for the post-culture step may be the same as or different from those for the pre-culture step.

[0043] The resulting transfected cells are used, for example, for substance production, gene therapy, and the like.

[0044] Second Embodiment <Cell Processing Device> A cell processing device according to a second embodiment will be described. The configurations different from the first embodiment will be mainly described, and descriptions of other configurations will be omitted. This embodiment differs from the first embodiment in whether the lid has a conductive member or not.

[0045] The cell processing device 1a in FIG. 2 includes a container 100a having a container body 110 and a lid 120a. The lid 120a includes a lid main body 121 and an electrode joint 124a. The electrode joint 124a includes a cylindrical or polygonal guide member 128 that hangs down from the underside of the top wall 122 toward the bottom wall 112, a partition wall 126 that closes the lower end of an insertion passage 125 within the guide member 128, and a conductive member 127 located within the insertion passage 125. That is, the electrode joint 124a includes the conductive member 127 that penetrates the lid main body 121. Note that, although the electrode joint 124a includes the partition wall 126 in this embodiment, it may also be configured without the partition wall 126 (i.e., the conductive member 127 is exposed to the storage chamber 111) as long as the guide member 128 and the conductive member 127 are hermetically joined. Furthermore, the electrode junction 124a may have an irradiation surface portion (hereinafter also referred to as "irradiation portion") on the lower surface of the partition wall portion 126 (i.e., the surface facing the second electrode 20). Examples of the irradiation portion include a flat plate with a mesh-like, circular, or polygonal hole shape (e.g., a punched metal-like), a thin film made of a conductive material patterned with a mesh-like, circular, or polygonal hole shape, and the like. In this case, the irradiation portion is connected to the conductive member 127 in the insertion path. The conductive member connected to the irradiation portion may be cylindrical, rectangular, columnar, or prismatic, may be a conductive wire such as a copper wire, or may be a pattern printed on the lid 120a or the like. Alternatively, the above-mentioned irradiation portion may be provided instead of the partition wall portion 126. A preferred example of the mesh-shaped irradiation surface portion is an irradiation surface portion that is composed of a plurality of linear bodies, each of which includes one or more first conducting wires extending in one direction of the surface of the irradiation surface portion and one or more second conducting wires intersecting the first conducting wires. The width of the first conducting wire and the width of the second conducting wire are each independently preferably 1 to 1000 μm, more preferably 1 to 200 μm, and even more preferably 1 to 100 μm.

[0046] The conductive member 127 is electrically connected to the power supply unit 30 to function as the first electrode 10a. The first electrode 10a may be, for example, an electrode having the same shape as the first electrode 10 in the first embodiment. Furthermore, the first electrode 10a may be, for example, a flat electrode. The material of the first electrode 10a is the same as that of the first electrode 10.

[0047] The material of the guide portion 128 may be a conductive material or an insulating material. For example, the guide portion 128 may be made of the same material as the lid main body 121 and may be molded integrally with the lid main body 121. The material of the partition portion 126 is the same as the material of the guide portion 128. The material of the partition portion 126 may be the same as or different from the material of the guide portion 128. Note that when the electrode joint portion 124a has an irradiation portion on the lower surface of the partition portion 126 and the partition portion 126 is made of a conductive material, the conductive member 127 is connected to the irradiation portion via the partition portion 126 made of a conductive material. When the electrode joint portion 124a has an irradiation portion on the lower surface of the partition portion 126 and the partition portion 126 is made of an insulating material, the partition portion 126 has another conductive member that connects the conductive member 127 and the irradiation portion. In other words, the irradiation portion is electrically connected to the conductive member 127.

[0048] The material of the conductive member 127 is the same as the material of the first electrode 10a. The material of the conductive member 127 and the material of the first electrode 10a may be the same or different.

[0049] <Method of Using the Cell Processing Device> An example of a method of using the cell processing device of this embodiment will be described using a method of introducing molecules as an example. An example of the method of introducing molecules is an introduction method having a pre-incubation step, a contact step, a sealing step, and an irradiation step. The pre-incubation step, the contact step, and the sealing step are the same as the pre-incubation step, the contact step, and the sealing step of the first embodiment.

[0050] The irradiation step of this embodiment is the same as that of the first embodiment, except that the conductive member 127 of the container 100a serves as the first electrode 10a.

[0051] (Third embodiment) <Cell processing device> A cell processing device according to a third embodiment will be described. The configuration that differs from the first and second embodiments will be mainly described, and a description of other configurations will be omitted. This embodiment differs from the first and second embodiments in that the lid and the container are integrated. An example of the container 100b of this embodiment is a so-called microchannel device.

[0052] The cell processing device 1b in Fig. 3 has a container 100b. The container 100b has a container body 110b and a lid body 120b, and the container body 110b and the lid body 120b are inseparably integrated. "Inseparably integrated" means that they are joined to such an extent that they cannot be easily separated.

[0053] The container 110b has a bottom wall 112 and a container side wall 113b rising from the periphery of the bottom wall 112. The lid 120b has a flat lid main body 121b and an electrode receiving portion 124. Note that this embodiment may have an electrode joint portion 124a (second embodiment) instead of the electrode receiving portion 124. The lid 120b closes the opening 115 of the container 110b. The lid 120b has a flow path 114 that penetrates from the top surface into the storage chamber 111. That is, the lid 120b has a flow path 114 that communicates between the storage chamber 111 and the outside of the container 100b. The number of flow paths 114 is not particularly limited and may be, for example, 2 to 4.

[0054] <Method of Using the Cell Processing Device> An example of a method of using the cell processing device of this embodiment will be described using a method of introducing molecules as an example. The example of the method of introducing molecules includes a cell filling step, a pre-incubation step, a contact step, and an irradiation step. The irradiation step is the same as in the first embodiment.

[0055] The cell filling step in this embodiment will be described. Target cells 44 and a culture medium are poured into the flow channel 114 of the lid 120b. The poured target cells 44 pass through the flow channel 114 and fill the storage chamber 111.

[0056] After the cell filling step, the target cells, etc. 44 in the storage chamber 111 are cultured (pre-culture step) in the same manner as in the first embodiment. After the culture, the culture medium is removed from the container 110b to obtain the pre-cultured target cells, etc. 44. Examples of means for injecting the culture medium, etc. into the container 110 through the flow path 114 or discharging the culture medium, etc. from the container 110 include a pump, a syringe, etc.

[0057] After removing the culture medium from the pre-culture step, the molecules 42 are injected into the flow channel 114 of the lid 120b. The injected molecules 42 pass through the flow channel 114 and fill the storage chamber 111 (for example, a well in a microchannel device). In this way, the molecules 42 are filled into the container 110b, and the molecules 42 are brought into contact with the target cells, etc., in the storage chamber 111 (contact step). Next, with the molecules 42 and the target cells, etc., 44 in contact with each other, an irradiation step is performed, thereby introducing the molecules 42 into the target cells, etc., 44.

[0058] According to this embodiment, the container 110b and the lid 120b are integrated to create a cleaner environment inside the storage chamber 111, thereby more effectively preventing contamination.

[0059] In the cell processing device described in the first to third embodiments, the electrode 10a is incorporated into the lid, or the lid has an electrode receiving portion that receives the electrode 10, but the present invention is not limited to this. The cell processing device of the present invention may have the electrode 10c incorporated into the container, or the container may have an electrode receiving portion that receives the electrode 10c.

[0060] (First Modification) <Cell Processing Apparatus> A cell processing apparatus according to a first modification will be described. The configuration that differs from the first embodiment will be mainly described, and a description of other configurations will be omitted. This modification differs from the first embodiment in that the container has an electrode receiving portion.

[0061] The cell processing device 1c of FIG. 4 includes a first electrode 10c, a second electrode 20c, a power supply unit 30, and a container 100c.

[0062] The second electrode 20c may be an electrode spaced apart from the first electrode 10c and extending around the container side wall 113c near the bottom wall 112c. The lower end 21c of the second electrode 20c is preferably positioned below the upper surface 41 of the irradiation target. The container may also include a conductive member corresponding to the second electrode. The power supply unit 30 is connected to the first electrode 10c and the second electrode 20c via wiring 12.

[0063] In this embodiment, the first electrode 10c is a high-voltage electrode. The first electrode 10c in this embodiment is a rod-shaped body extending in one direction. The first electrode 10c may have a hollow structure such as a cylindrical or polygonal tube, or a solid structure such as a columnar or polygonal pillar. In particular, if the first electrode 10c has a solid structure, durability is increased and manufacturing is easy. Examples of materials for the first electrode 10c include metals such as stainless steel, copper, and tungsten, and carbon.

[0064] The thickness (outer diameter) of the first electrode 10c is, for example, 3 to 50 mm. When the first electrode 10c has a polygonal cylindrical or polygonal prism shape, the outer diameter is the diameter of a circumscribed circle of the cross section of the first electrode 10c.

[0065] The second electrode 20c may be any electrode that functions as a ground electrode, and may be, for example, a cylindrical electrode that covers the surface of the container side wall 113c opposite to the storage chamber 111c. Examples of materials for the second electrode 20c include metals such as stainless steel, copper, and tungsten, and carbon.

[0066] The surface area of ​​the second electrode 20c is preferably larger than the surface area of ​​the first electrode 10c. If the surface area of ​​the second electrode 20c is larger, the plasma can be irradiated from the first electrode 10c to the irradiation target over a wider range.

[0067] The power supply unit 30 only needs to apply a voltage between the first electrode 10c and the second electrode 20c and generate plasma between the two electrodes. The power supply unit 30 switches between starting and stopping the supply of electricity to the electrodes. The power supply unit 30 also adjusts the voltage and frequency applied to the electrodes. An example of the power supply unit 30 is a circuit connected to an external power source and including an inverter. Another example of the power supply unit 30 is a secondary battery.

[0068] The container 100c includes a container 110c and a lid 120c. The container 110c includes a bottom wall 112c, a container side wall 113c rising from the periphery of the bottom wall 112c, and an electrode receiving portion 124c. The container 110c has an opening 115c at its upper end, surrounded by the container side wall 113c. The interior of the container 110c, surrounded by the bottom wall 112c, the container side wall 113c, and the electrode receiving portion 124c, forms a storage chamber 111c. The container 110c may be cylindrical or polygonal tubular. The bottom wall 112c preferably has a cell culture scaffold on the surface facing the storage chamber 111c. The cell scaffold has the same configuration as the cell scaffold in the first embodiment.

[0069] The lid 120c has a top wall 122c and a lid side wall 123c that hangs down from the periphery of the top wall 122c. The lid side wall 123c tapers downward. In this embodiment, when the lid 120c and the container 110c are combined (i.e., when the lid 120c covers the opening 115c), the lid side wall 123c is located on the outer surface of the container side wall 113c. In other words, the lid 120c in this embodiment is an external lid. Because the lid side wall 123c tapers downward, the lid 120c fits into the container 110c when the lid 120c and the container 110c are combined.

[0070] The electrode receiving portion 124c is a cylindrical or polygonal cylindrical member extending vertically from the bottom wall portion 112c toward the underside of the top wall portion 122c. The electrode receiving portion 124c has an insertion path 125c therein. That is, the insertion path 125c extends from bottom to top. The electrode receiving portion 124c has a partition wall portion 126c separating the first electrode 10c from the storage chamber 111c at a location located in the region where the first electrode 10c and the second electrode 20c face each other. The partition wall portion 126c is spaced from the inner wall surface of the container side wall portion 113. By including the partition wall portion 126c in the electrode receiving portion 124c, the amount of exposure of target cells and the like in the storage chamber 111c to the irradiation environment can be further reduced. The lid 120c only needs to close the opening 115c to such an extent that the target cells and the like in the storage chamber 111c are not directly exposed to the irradiation environment (the atmosphere outside the container). Therefore, a gap may be formed between the lid 120c and the container 110c.

[0071] The material of the container 110c may be a conductive material or an insulating material. However, from the viewpoint of preventing localized discharge due to high voltage, an insulating material is preferable. Examples of insulating materials include resin, ceramics, glass, etc. Examples of resins include polystyrene, polyolefin, polyester, acrylic, etc. For example, if the container 100c is to be disposable, the material of the container 110c is preferably resin. Examples of conductive materials include the same materials as those of the first electrode 10c.

[0072] The material of the lid 120c is the same as the material of the container 110c. The material of the lid 120c may be the same as or different from the material of the container 110c. From the viewpoint of generating plasma between the electrodes, at least one of the container 110c and the lid 120c is made of an insulating material.

[0073] The material of the electrode receiving portion 124c may be a conductive material, an insulating material, or a composite of a conductive material and an insulating material. For example, the material of the partition wall 126c may be a conductive material, and the material of the other components of the electrode receiving portion 124c may be an insulating material. Alternatively, for example, the material of the entire electrode receiving portion 124c may be an insulating material, and the material of the container 110c may also be an insulating material. Alternatively, for example, the container 110c and the electrode receiving portion 124c may be integrally molded from an insulating material. When the partition wall 126c is made of an insulating material, the thickness of the partition wall 126c is, for example, 0.1 to 10 mm.

[0074] The inner diameter R111c of the container 110c is not particularly limited and is, for example, 1 to 150 mm. When the container 110c is polygonal cylindrical, the inner diameter R111c is the diameter of a circumscribing circle of the container 110c in a planar view. When the container 110c is polygonal cylindrical, the number of corners of the container 110c in a planar view is not particularly limited and is, for example, 4 to 20. The internal height H111c of the container 110c is not particularly limited and is, for example, 5 to 50 mm.

[0075] The length L125c of the insertion path 125c in the electrode receiving portion 124c is sufficient to receive the first electrode 10c and determine the position of the first electrode 10c, and is, for example, 1 to 50 mm. The thickness of the insertion path 125c can be determined appropriately depending on the thickness of the first electrode 10c.

[0076] <Method of Using the Cell Processing Device> The method of introducing a molecule, which is an example of a method of using the cell processing device of this modified example, is the same as the method of introducing a molecule using the cell processing device of the first modified example.

[0077] (Second Modification) <Cell Processing Device> A cell processing device of a second modification will be described. The configuration that differs from the first modification will be mainly described, and a description of other configurations will be omitted. This modification differs from the first modification in whether the container has a conductive member or not.

[0078] The cell processing device 1d of Figure 5 includes a container 100d having a container 110d and a lid 120c. The container 110d has a bottom wall 112d, a container side wall 113d rising from the periphery of the bottom wall 112d, and an electrode joint 124d. The container 110d has an opening 115c at its upper end surrounded by the container side wall 113d. In the container 110d, the interior surrounded by the bottom wall 112d, the container side wall 113d, and the electrode joint 124d forms a storage chamber 111c. The container 110d may be cylindrical or polygonal. The bottom wall 112d preferably has a cell culture scaffold on the surface facing the storage chamber 111d.

[0079] The electrode junction 124d includes a cylindrical or polygonal guide portion 128d that extends vertically from the bottom wall portion 112d toward the upper surface of the top wall portion 122c, a partition wall portion 126c that closes the upper end of the insertion passage 125c in the guide portion 128d, and a conductive member 127d that is located within the insertion passage 125c. That is, the electrode junction 124d includes the conductive member 127d that penetrates the bottom wall portion 112d of the container 110d. Note that, although the electrode junction 124d includes the partition wall portion 126c in this modification, it may also be configured without the partition wall portion 126c (i.e., the conductive member 127d is exposed to the accommodation chamber 111d) as long as the guide portion 128d and the conductive member 127d are hermetically joined. The electrode joint 124d may also have an irradiation unit on the outer surface of the partition 126c (i.e., the surface facing the container side wall 113d). The irradiation unit may be, for example, a flat plate with a mesh-like, circular, or polygonal hole shape (e.g., a punched metal-like plate), or a thin film made of a conductive material patterned with a mesh-like, circular, or polygonal hole shape. In this case, the irradiation unit is connected to the conductive member 127d in the insertion path. The conductive member connected to the irradiation unit may be cylindrical, rectangular, columnar, or prismatic, or may be a conductive wire such as a copper wire, or a pattern printed on the container 110d or the like. Alternatively, the above-described irradiation unit may be provided instead of the partition 126c. Note that preferred aspects of the mesh-like irradiation surface unit are the same as those of the mesh-like irradiation surface unit in the second embodiment.

[0080] The conductive member 127d is electrically connected to the power supply unit 30 to function as the first electrode 10d. The first electrode 10d may have a shape similar to that of the first electrode 10c in the first modified example. Alternatively, the first electrode 10d may be a flat electrode. The material of the first electrode 10d is the same as that of the first electrode 10c.

[0081] The material of the guide portion 128d may be a conductive material or an insulating material. For example, the guide portion 128d may be made of the same material as the bottom wall portion 112d and may be molded integrally with the bottom wall portion 112d. The material of the partition portion 126c is the same as the material of the guide portion 128d. The material of the partition portion 126c may be the same as or different from the material of the guide portion 128d. Note that when the electrode joint portion 124d has an irradiation portion on the upper surface of the partition portion 126c and the partition portion 126c is made of a conductive material, the conductive member 127d is connected to the irradiation portion via the partition portion 126c made of a conductive material. When the electrode joint portion 124d has an irradiation portion on the upper surface of the partition portion 126c and the partition portion 126c is made of an insulating material, the partition portion 126c has another conductive member connecting the conductive member 127d to the irradiation portion. That is, the irradiation unit is electrically connected to the conductive member 127d.

[0082] The material of the conductive member 127d is the same as the material of the first electrode 10d. The material of the conductive member 127d and the material of the first electrode 10d may be the same or different.

[0083] <Method of Using the Cell Processing Device> An example of a method of using the cell processing device of this modified example will be described using a method of introducing molecules as an example. An example of a method of introducing molecules is an introduction method having a pre-incubation step, a contact step, a sealing step, and an irradiation step. The pre-incubation step, the contact step, and the sealing step are the same as the pre-incubation step, the contact step, and the sealing step of the first modified example.

[0084] The irradiation step of this modified example is the same as that of the first modified example, except that the conductive member 127d of the container 100d is used as the first electrode 10d.

[0085] (Third Modification) <Cell Processing Device> A cell processing device of a third modification will be described. The configuration that differs from the first and second modifications will be mainly described, and a description of other configurations will be omitted. This modification differs from the first and second modifications in that the lid and the container are integrated. An example of the container 100e of this modification is a so-called microchannel device.

[0086] The cell processing device 1e in Fig. 6 includes a container 100e. The container 100e includes a container body 110e and a lid body 120e, which are inseparably integrated. "Inseparably integrated" means that the container body 110e and the lid body 120e are joined together to such an extent that they cannot be easily separated.

[0087] The container 110e has a bottom wall 112c and a container side wall 113e rising from the periphery of the bottom wall 112c. The lid 120e closes an opening 115c of the container 110e. The lid 120e has a flow path 114e that penetrates from the top surface into the storage chamber 111c. That is, the lid 120e has a flow path 114e that connects the storage chamber 111c with the outside of the container 100e. The number of flow paths 114e is not particularly limited and may be, for example, 2 to 4.

[0088] <Method of Using the Cell Processing Device> An example of a method of using the cell processing device of this modified example will be described using a method of introducing molecules as an example. The example of the method of introducing molecules includes a cell filling step, a pre-incubation step, a contact step, and an irradiation step. The irradiation step is the same as in the first modified example.

[0089] The cell filling process in this modified example will be described. Target cells 44 and a culture medium are poured into the flow channel 114e of the lid 120e. The poured target cells 44 pass through the flow channel 114e and fill the storage chamber 111c.

[0090] After the cell filling step, the target cells, etc. 44 in the storage chamber 111c are cultured (pre-culture step) in the same manner as in the first modified example. After the culture, the culture medium is removed from the container 110e to obtain the pre-cultured target cells, etc. 44. Examples of means for injecting the culture medium, etc. into the container 110e through the flow path 114e or discharging the culture medium, etc. from the container 110e include a pump, a syringe, etc.

[0091] After removing the culture medium from the pre-culture step, the molecules 42 are injected into the flow channel 114e of the lid 120e. The injected molecules 42 pass through the flow channel 114e and fill the storage chamber 111c (e.g., a well in a microchannel device). In this manner, the molecules 42 are filled into the container 110e, and the molecules 42 are brought into contact with target cells, etc., in the storage chamber 111c (contact step). Next, with the molecules 42 and the target cells, etc., 44 in contact, an irradiation step is performed, thereby introducing the molecules 42 into the target cells, etc., 44.

[0092] According to this modification, the container 110e and the lid 120e are integrated to create a cleaner environment inside the storage chamber 111c, thereby more effectively preventing contamination.

[0093] (Uses) The plasma cell treatment device of the present invention can stimulate target cells, etc., which are the treatment objects, while mitigating damage to them. Therefore, the plasma cell treatment device of the present invention is suitable for cell treatment such as cell proliferation and molecule introduction. In particular, the plasma cell treatment device of the present invention is more suitable for molecule introduction.

[0094] Other Embodiments In the first embodiment, the electrode receiving portion hangs down from the lower surface of the top wall portion, but the present invention is not limited to this. The lower surface of the top wall portion may be flush.

[0095] In the second embodiment and the second modified example, the conductive member is rod-shaped, but the present invention is not limited to this. When the lid body has a conductive member, the conductive member may be plate-shaped.

[0096] In the first and second embodiments and the first and second modifications, the lid body is an outer fitting lid, but the lid body of the present invention may also be a so-called inner fitting lid.

[0097] In the first to third embodiments and the first to third modified examples, the connection portion has an insertion path, but the present invention is not limited to this. For example, the connection portion may be a metal plate, metal foil, or the like located on the upper surface of the lid body. However, from the viewpoint of optimizing the discharge distance, it is preferable to provide an insertion path to bring the first electrode or the conductive member closer to the irradiation target.

[0098] In the first to third embodiments, the container is placed on the second electrode (i.e., the second electrode is located on the lower surface of the bottom wall of the container), but the present invention is not limited to this. For example, as shown in FIG. 7, the second electrode 20c may be an electrode that runs around the container side wall 113 near the bottom wall 112. When using the second electrode 20c, it is preferable that the lower end 21 of the second electrode 20c is located below the upper surface 41 of the irradiation target. The container may also have a conductive member corresponding to the second electrode.

[0099] In the first to third embodiments and the first to third variations, the container is a combination of one container having one storage chamber and one lid, but the present invention is not limited to this. For example, the container of the present invention may be a combination of one container having two or more storage chambers and one lid that closes the opening of the container. Alternatively, the container of the present invention may be a combination of one container having two or more storage chambers and two or more lids that close each of the storage chambers.

[0100] In the first to third embodiments and the first to third modified examples, one container has one electrode receiving portion or electrode junction portion, but two or more electrode receiving portions or electrode junction portions may be provided on one lid so that two or more first electrodes act on one container.

[0101] In one preferred embodiment of the present invention, one of the first and second electrodes is positioned so that a portion thereof contacts or penetrates into the inner or outer surface of the bottom wall of the container. For example, the variations shown in Figures 4 to 6 above are included in this embodiment. However, as a variation of the embodiment shown in Figures 1 to 3, a configuration in which the tip of the first electrode contacts or penetrates into the inner surface of the bottom wall of the container may also be used. In this preferred embodiment, if cells are disposed on the inner surface of the bottom wall, the current reaching the cells and the plasma generated between the electrodes can be efficiently supplied to the cells.

[0102] According to the container for the plasma cell treatment device of the present invention, the treatment object can be treated without being exposed to the outside.

[0103] 1, 1a, 1b, 1c, 1d, 1e cell processing device 10, 10a, 10c first electrode 20, 20c second electrode 42 molecule 44 target cell etc. 100, 100a, 100b, 100c, 100d, 100e container 111, 111c storage chamber 110, 110b, 110c, 110d, 110e container 112, 112c, 112d bottom wall 113, 113b, 113c, 113d, 113e side wall 115, 115c opening 120, 120a, 120b, 120c, 120d, 120e cover 121, 121b cover body 122, 122c top wall 123, 123c Cover sidewalls 124, 124c, 124e Electrode receiving portions 124a, 124d Electrode bonding portions 125, 125c Insertion paths 126, 126c Partition walls 127, 127d Conductive members 128, 128d Gaide portion

Claims

1. A container for a plasma cell processing device used in a plasma cell processing device that stimulates an object to be processed with plasma generated from the gas phase between electrodes spaced apart by the gas phase, the container having a container with a storage chamber inside and an opening at the upper end, and a lid that covers the opening, wherein either the container or the lid has one of the spaced apart electrodes, electrode E1, built in, or has an electrode receiving portion that receives electrode E1.

2. A container for a plasma cell processing device as described in claim 1, wherein the incorporated electrode E1 has a conductive member penetrating the container or the lid.

3. A container for a plasma-type cell processing device as described in claim 2, wherein the electrode E1 incorporated in the lid has an irradiation surface portion connected to the lower end of the conductive member, or the electrode E1 incorporated in the container has an irradiation surface portion connected to the side of the conductive member, and the irradiation surface portion is made of a patterned conductive material.

4. A container for a plasma-type cell processing device as described in claim 3, wherein the irradiation surface portion is composed of a plurality of linear bodies, and the plurality of linear bodies include one or more first conductive wires extending in one direction of the surface of the irradiation surface portion and one or more second conductive wires intersecting the first conductive wires.

5. The container for a plasma cell processing device according to claim 4, wherein the width of the first conducting wire is 1 to 1000 μm, and the width of the second conducting wire is 1 to 1000 μm.

6. A container for a plasma-type cell processing device as described in claim 1, wherein the electrode receiving portion has an insertion path for receiving the electrode E1, and when the opening is covered with the lid, the insertion path extends from the upper surface to the lower surface of the lid, or from the lower surface to the upper surface of the container, and the electrode receiving portion has a partition portion that covers the end of the insertion path in the insertion direction.

7. A container for a plasma cell processing device according to claim 6, having a conductive member inside the insertion path.

8. A container for a plasma cell processing device as described in any one of claims 1 to 7, wherein the container has a bottom wall portion at a lower end and a cell culture scaffold on the surface of the bottom wall portion facing the storage chamber.

9. A container for a plasma cell processing device according to any one of claims 1 to 8, wherein the lid body has one of the electrodes or the electrode receiving portion.

10. A container for a plasma cell processing device according to any one of claims 1 to 9, wherein the plasma cell processing device is for introducing molecules.

11. A plasma cell processing device comprising a container for the plasma cell processing device according to any one of claims 1 to 10.

12. The plasma cell treatment device according to claim 11, which is used for introducing molecules.

13. A method for introducing a molecule into a target cell, comprising: providing a container having a container with an opening at an upper end and a lid for covering the opening, wherein either the container or the lid has an electrode E1 built in or an electrode receiving portion for receiving the electrode E1; injecting target cells and molecules into the container's container; if the container has an electrode receiving portion, inserting electrode E1 into the electrode receiving portion and then arranging electrode E2 in a position opposite electrode E1 via the gas phase; and applying a voltage between electrode E1 and electrode E2 with the opening covered by the lid and electrode E1 and electrode E2 facing each other via the gas phase.

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