WO2016143669A1 - Procédé de production de cellules agrégées et immobilisées à l'aide d'une solution de protéine plasma-aqueuse et cellules immobilisées et agrégées produites par ledit procédé de production - Google Patents

Procédé de production de cellules agrégées et immobilisées à l'aide d'une solution de protéine plasma-aqueuse et cellules immobilisées et agrégées produites par ledit procédé de production Download PDF

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WO2016143669A1
WO2016143669A1 PCT/JP2016/056632 JP2016056632W WO2016143669A1 WO 2016143669 A1 WO2016143669 A1 WO 2016143669A1 JP 2016056632 W JP2016056632 W JP 2016056632W WO 2016143669 A1 WO2016143669 A1 WO 2016143669A1
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protein
aqueous solution
cells
cell
creeping discharge
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PCT/JP2016/056632
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English (en)
Japanese (ja)
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佐藤　正明
大輔 吉野
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国立大学法人東北大学
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Priority to JP2017505284A priority Critical patent/JP6408689B2/ja
Publication of WO2016143669A1 publication Critical patent/WO2016143669A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • the present invention relates to a cell preparation obtained by applying artificial means. More specifically, the present invention relates to a method for producing an aggregated fixed product of cells using an aqueous protein solution prepared by low-temperature plasma discharge, and It is an invention relating to an assembly of cells produced by the production method.
  • Non-patent Document 1 a corneal regeneration treatment using a cell sheet for corneal epithelial stem cell exhaustion.
  • a single corneal epithelial cell sheet is simply attached to an affected area. It is possible to adhere to.
  • Non-patent Documents 2 and 3 In addition, clinical studies have been conducted on myocardium, periodontals, cartilage, etc. (Non-patent Documents 2 and 3), and in particular, a culture technique for highly efficient differentiation of human iPS cells into cardiomyocytes has recently been established. Basic research using human cardiomyocyte sheets has been conducted (Non-patent Document 4).
  • the object of the present invention is to find a means for producing a fixed product between cells by adhering cells or cell masses with a protein such as an extracellular matrix protein, and to contribute to various applications.
  • the inventors of the present invention have studied the above problems, and by using a protein aqueous solution obtained by performing plasma conversion of a protein-containing aqueous solution by low-temperature plasma discharge, using cells or cell masses as an adhesive solution, Surprisingly, the present invention was completed by finding that it is possible to produce a cell-fixed product in which cells or cell masses are adhered in a living state.
  • the present invention provides a protein aqueous solution (hereinafter also referred to as the aqueous solution of the present invention) that has been converted to plasma by low-temperature plasma discharge in a state where each cell is adhered to the surface of a plurality of cells or cell clusters.
  • a protein aqueous solution hereinafter also referred to as the aqueous solution of the present invention
  • the aqueous solution of the present invention provides a protein aqueous solution
  • the cell-fixed product of the present invention hereinafter, the cell-fixed product of the present invention. It is also an invention that provides.
  • the cell fixed thing of this invention can be used as the fixed thing of the "living cell” as mentioned above, in other cases, specifically, it is not alive (dead) It is of course possible to fix the “cells in a state”, and the “cells or cell masses” to be adhered by the production method of the present invention and the “cells” constituting the cell fixed product of the present invention are living cells. It may be a dead cell. Dead cells also include cell isolates such as isolated cell nuclei.
  • the cell or cell mass is not particularly limited, and may be a single-cell organism itself or may be isolated from a multicellular organism, for practical use or research in the medical field or food industry field described later. It is possible to select freely according to the purpose to contribute. It may be a eukaryotic unicellular organism or a eukaryotic multicellular organism cell or a prokaryotic cell.
  • the plurality of cells or cell masses may be the same type of cell, but two or more types of cells or the plurality of cell masses may be two or more types of cell masses having different constituent cells.
  • the present invention is excellent in that it can easily be combined with two or more types of cells or cell clusters.
  • epithelial cells or epidermal cells that are originally excellent in adhesion ability as cell types or cell clusters that are selected for adhesion, but it is also possible to use endothelial cells that do not have strong adhesion ability. It is possible to combine them.
  • epithelial cells or epidermal cells are contained in a plurality of cells or two or more cell clusters that are a plurality of cell clusters and different constituent cells is recognized.
  • endothelial cells are contained separately or in combination is recognized.
  • a “cell sheet” can be cited as a preferred embodiment of the cell mass.
  • a typical example of the assembly and fixing product of a plurality of cells is a laminated cell sheet in which a plurality of cell sheets are bonded to each other on the sheet surface.
  • the cell sheets as the unit to be laminated may be composed of the same cells or different cells. Further, the number of stacked layers is 2 or more and can be appropriately set as necessary.
  • the low temperature plasma discharge includes creeping discharge and dielectric barrier discharge, but creeping discharge is suitable in the present invention.
  • Creeping discharge is generally known as “a discharge phenomenon that occurs along the dielectric from the application electrode when the application electrode and the induction electrode are present across the dielectric”.
  • the protein contained in the aqueous solution of the present invention preferably includes an extracellular matrix protein.
  • the aqueous solution of the present invention can contain other functional substances.
  • the functional substance should be appropriately selected according to the specific purpose and is not particularly limited. Examples thereof include growth factors, antithrombotic factors, anticancer agents, immunosuppressants, antibodies, sugar chains, antibiotics. Substances and the like.
  • the aqueous solution of the present invention has two modes: (1) a “mist type” in which the aqueous solution is misted; and (2) a “droplet type” in which the aqueous solution is a droplet. Yes, the preparation method of each embodiment is different. Also, (3) a description of a creeping discharge device (device of the present invention) suitable for preparing the aqueous solution of the present invention will be given.
  • Mist type aqueous solution is a very fine particle (tens of nm) due to the repulsive force between charges due to the repulsive force between the protein molecules surrounded by water when the protein aqueous solution contacts the low temperature plasma discharge. It is thought that the mist is formed by bouncing to a diameter of about several hundred nm. An image of a mist type aqueous solution is shown in FIG.
  • the droplet-type aqueous solution is considered to be in a state where an excessive charge is supplied to the aqueous solution containing protein molecules by low-temperature plasma discharge.
  • An image of a droplet type aqueous solution is shown in FIG.
  • both forms of the aqueous solution of the present invention can be used as the adhesive liquid, but the “droplet type” is generally easier to bond.
  • a mist type aqueous solution is performed by bringing the protein aqueous solution before being plasmatized into contact with low temperature plasma by creeping discharge at a specific feed rate, Alternatively, plasma mist formation for repelling the molecules by charging the protein and other functional substance molecules is performed according to the following (a) to (c).
  • the frequency of the AC voltage for performing the creeping discharge is 5 to 30 kHz, and the applied voltage is 2 to 4 kV.
  • the protein concentration of the protein aqueous solution before the above-described plasmatization is 25 ⁇ g / ml to 5 mg / ml.
  • C The specific feed rate of the aqueous protein solution before the above-mentioned plasmatization is 3.0 to 36 mm / min.
  • This feed rate unit “mm / min” is the “feed volume of protein aqueous solution per unit time ( ⁇ l or mm 3 / min)” and “the aqueous solution introduction site (described later) of the aqueous solution. It is a value calculated by dividing by “cross-sectional area (mm 2 ) perpendicular to the traveling direction”. Therefore, it is also possible to easily calculate the “feed volume of the protein aqueous solution per unit time” from the “feed speed” and “the cross-sectional area”.
  • the apparatus of the present invention described later As a means for creeping discharge in the preparation of the mist type aqueous solution, the apparatus of the present invention described later is suitable.
  • the cross-sectional area perpendicular to the traveling direction of the aqueous solution of the introduction field (described later) used as a charging field for the protein aqueous solution is preferable because the protein aqueous solution is charged with a high yield.
  • the upper limit is set.
  • the cross-sectional area is preferably 30 mm 2 or less.
  • the lower limit of the cross-sectional area in the case of this cylindrical state is not particularly limited, but about 1 mm 2 is preferable.
  • the distance between the application electrode plates of the opposite creeping discharge bodies is preferably 6 mm or less, and the lower limit is particularly limited. Although it is not, it is preferable that it is about 0.5 mm.
  • the surface area of the portion where the application electrode and the ground electrode of the creeping discharge body (described later), which is a creeping discharge generation mechanism, is overlapped is ensured to some extent because the protein aqueous solution is charged with a high yield.
  • the shape of the protein aqueous solution introduction field is cylindrical, the surface area is preferably 3 to 30 times, particularly preferably 3 to 10 times the cross-sectional area.
  • the distance between the application electrode plates is preferably multiplied by a distance of 3 to 30 times, particularly preferably 3 to 10 times.
  • the calculated surface area is preferred.
  • Preparation of a droplet-type aqueous solution involves contacting the protein aqueous solution before being plasmatized with low-temperature plasma by creeping discharge at a specific feed rate. Charging of a protein or a molecule of the protein and another functional substance is performed according to the following (a) to (c).
  • the frequency of the AC voltage for performing the creeping discharge is 5 to 30 kHz, and the applied voltage is 7.5 to 18 kV.
  • the protein concentration of the aqueous protein solution before the above-described plasmatization is 25 ⁇ g / ml to 255 mg / ml.
  • the specific feed rate of the aqueous protein solution before the above-mentioned plasmatization is 36 to 180 mm / min.
  • the device of the present invention described later is also suitable as a means for creeping discharge in the preparation of the droplet type aqueous solution.
  • the cross-sectional area perpendicular to the traveling direction of the aqueous solution of the introduction field (described later) used as a charging field for the protein aqueous solution is preferable because the protein aqueous solution is charged with a high yield.
  • the upper limit is set.
  • the shape of the introduction field is cylindrical
  • the cross-sectional area is preferably 30 mm 2 or less.
  • the lower limit of the cross-sectional area in the case of this cylindrical state is not particularly limited, but about 1 mm 2 is preferable.
  • the distance between the application electrode plates of the opposite creeping discharge bodies is preferably 6 mm or less, and the lower limit is particularly limited. Although it is not, it is preferable that it is about 0.5 mm.
  • the surface area of the portion where the application electrode and the ground electrode of the creeping discharge body (described later), which is a creeping discharge generation mechanism, is overlapped is ensured to some extent because the protein aqueous solution is charged with a high yield.
  • the shape of the protein aqueous solution introduction field is cylindrical, the surface area is preferably 3 to 30 times, particularly preferably 3 to 10 times the cross-sectional area.
  • the distance between the application electrode plates is preferably multiplied by a distance of 3 to 30 times, particularly preferably 3 to 10 times.
  • the calculated surface area is preferred.
  • Apparatus according to the present invention is provided with a field for introducing an aqueous protein solution, and in this introduction field, a low-temperature plasma discharge generated from a creeping discharge body is applied to the protein aqueous solution to produce plasma.
  • a creeping discharge device for producing a protein wherein the creeping discharge body includes an applied electrode on one of the front and back sides of a planar dielectric, and a ground electrode on the other in contact with the dielectric. The end of the application electrode on the surface of the dielectric and the end of the ground electrode are partly or completely aligned, or the end of the ground electrode is a part of the end of the application electrode.
  • the “low-temperature plasma discharge generated from the creeping discharge body” is a low-temperature plasma discharge (non-equilibrium plasma (described later)) having the same electron temperature at atmospheric pressure as the “dielectric barrier discharge” in nature.
  • the above-mentioned “protein aqueous solution introduction field” is a field for allowing the low temperature plasma discharge generated from the creeping discharge body to act on the protein aqueous solution before being plasmatized. As a premise for performing this action, it is necessary to introduce a protein solution before being plasmatized into the field. “Introduction” is an action of putting a protein solution before being plasmatized into the field, and includes, for example, entry and injection, regardless of specific means.
  • the structure of the “protein aqueous solution introduction field” is not particularly limited as long as it is a structure that allows low temperature plasma discharge by creeping discharge to act on the protein aqueous solution before being introduced into plasma.
  • an “introduction port” for introducing the protein aqueous solution before being converted into plasma and an “outlet port” for deriving the plasmad protein are preferably provided.
  • the shape include a cylindrical shape, a hollow flat plate shape (the introduction field is formed by a gap between facing plate-shaped creeping discharge bodies), a honeycomb shape, and a hollow laminated plate shape. is there.
  • the cylindrical shape includes a cylindrical shape and a polygonal cylindrical shape, and may be an aggregated tube (honeycomb shape or the like) in which the length directions of a plurality of cylindrical units are aligned, but a cylindrical shape is particularly preferable.
  • the “creeping discharge body” is a unit for performing low-temperature plasma discharge by the creeping discharge action in the atmosphere of “introduction site of protein aqueous solution”.
  • the creeping discharge body includes an application electrode on one of the front and back sides of the planar dielectric, and a ground electrode on the other side in contact with the dielectric. Either the end of the application electrode and part or all of the end of the ground electrode are aligned, or the end of the ground electrode is provided so as to protrude from part or all of the end of the application electrode. It is a unit that performs creeping discharge. The creeping discharge is generated on the “applied electrode side of the creeping edge portion that is aligned or protrudes”.
  • a region where creeping discharge to the application electrode side is performed is also referred to as a “creeping discharge portion”.
  • the “planar shape” indicating the shape of the dielectric may be planar or curved.
  • the curved surface is not particularly limited, such as a columnar curved surface such as a cylindrical curved surface, a developable surface such as a conical surface, a spherical curved surface, and a rotating surface.
  • An application electrode and a ground electrode are provided in contact with each other on the front and back surfaces of such a dielectric to constitute a “creeping discharge body”.
  • the application electrode is provided on one of the front and back sides of the planar dielectric, and the ground electrode is provided in contact with the dielectric on the other side.
  • the end portion of the application electrode and the end portion of the ground electrode are partly or entirely aligned, or the end portion of the ground electrode is provided so as to protrude from a part or all of the end portion of the application electrode. ing.
  • the ground electrode end portion and the application electrode end portion are aligned, or in the “creeping discharge body end portion” where the ground electrode end portion protrudes, “the end portion is aligned, or
  • the ratio of “the end portion of the ground electrode protrudes” is most preferably 100%, but depending on the design and the like, about 80% is practical and within the preferable range.
  • the applied electrode surface of one or two or more “creeping discharge bodies” is a surface in contact with the atmosphere of “introduction site of protein aqueous solution”.
  • the number of “creeping discharge bodies” provided so as to be in contact with the atmosphere of the introduction site can be freely designed. By increasing / decreasing the number of “creeping discharge bodies”, it is possible to increase / decrease the number of places where low-temperature plasma is generated by creeping discharge within the “introduction site of protein aqueous solution”.
  • the protein aqueous solution introduction site is inside the cylindrical body, and one or two or more of the “creeping discharge bodies” constitute all or part of the cylindrical body.
  • a mode in which the “creeping discharge body” is one will be described later.
  • the application electrode (inside of the cylinder) and the ground electrode (outside of the cylinder) are in contact with each other at two or more locations on the cylindrical side surface of the cylindrical dielectric, while satisfying the above conditions at the end.
  • a plurality of short creeping discharge bodies having a short length are provided by providing an interval in the length direction of the tubular body (the direction connecting both openings).
  • a part constituting the “introduction field of the protein aqueous solution” is a dielectric
  • the structural part other than the dielectric is an insulating material, or an insulating treatment is performed, Insulation must be maintained.
  • a liquid feeding mechanism is provided toward the inner surface of the dielectric of the device of the present invention or the inner space of the cylindrical structure formed by the application electrode.
  • the apparatus of the present invention is used as a creeping discharge means, and before being plasmatized toward the “introduction site of the protein aqueous solution” of the creeping discharge device.
  • the aqueous solution of the present invention can be efficiently prepared by bringing the aqueous solution of protein at a specific feed rate into contact with the low-temperature plasma of the creeping discharge generated inside the introduction field. . It is preferable that the protein aqueous solution before the treatment is fed through the liquid feeding mechanism.
  • a means for efficiently bonding cells or cell masses with high yield is provided.
  • the production method of the present invention can maintain the same or different types of cells easily and alive, regardless of the cell culture environment, even for endothelial cells with inherently poor adhesion.
  • cell sheets made of the same or different types of cells can be easily adhered to each other and laminated.
  • it is easy to combine with a simple cell sheet preparation method for example, Kikuchi, T. et al .: Biomaterials, 35, 2428 (2014)
  • Kikuchi, T. et al .: Biomaterials, 35, 2428 (2014) shown in the examples described later, It is possible to perform the adhesion process in a shorter time than a conventional three-dimensional culture method or the like that requires dimensional co-culture.
  • the present invention since cell-cell adhesion is easy, until a cell is adhered, a single type of cell may be cultured in a sheet shape or a lump shape, and co-culture of different cells with potential for competition may be performed. There is no need to carry out, and the culture work as a premise can be simply completed.
  • the present invention provides a way to freely produce cell-fixed materials such as cell sheets composed of the same or different types of cells, and contributes greatly in the field of regenerative medicine and the food industry.
  • cell scaffolds can be easily combined by placing a cell sheet or a cell mass on an aqueous solution containing an extracellular matrix protein such as collagen that is excessively charged.
  • Cells are attached to extracellular matrix proteins through the focal adhesion plaque-constituting protein integrin (transmembrane protein), which is a scaffold, and this integrin and extracellular matrix are electrostatically bound to each other.
  • the aqueous protein solution has an effect.
  • the adhesion is improved by electrostatically attracting the charged protein in the protein aqueous solution to the negatively charged cells.
  • the aqueous solution of the present invention is a plasma aqueous solution obtained by converting a protein aqueous solution into plasma by low temperature plasma discharge.
  • Low temperature plasma means that the electron temperature (T e ) is clearly larger than the gas temperature (T g ) (T e / T g > 100), and T e and T g Is a plasma that has not reached equilibrium, also known as non-equilibrium plasma.
  • the low temperature plasma discharge is a discharge for obtaining this low temperature plasma, and corresponds to glow discharge, corona discharge, dielectric barrier discharge, creeping discharge, and the like. Among these, it is preferable to use creeping discharge or dielectric barrier discharge.
  • (1) -1 Creeping discharge
  • creeping discharge by applying an alternating voltage of an appropriate frequency from an alternating current power source to the application electrode, a strong electric field is formed in the application electrode in synchronization with the alternating current cycle, A glow-like low-temperature plasma discharge can be obtained by repeating the phenomenon in which the emitted electrons are imparted to the surface of the dielectric and the phenomenon in which they return to the applied electrode again.
  • An apparatus for preparing the aqueous solution of the present invention by generating a creeping discharge is an essential equipment for generating the creeping discharge, specifically, an AC power source, a ground electrode, a dielectric, and an applied electrode. If there is, there is no particular limitation, and it can be freely designed and manufactured according to the specific processing purpose.
  • the ground electrode, the dielectric, and the application electrode are in contact with each other, and power is supplied from the AC power source to the application electrode, and a ground wire is connected to the ground electrode for grounding.
  • FIG. 2 is an explanatory sectional view of the principle of creeping discharge used in the present invention.
  • an application electrode 3 having a smaller surface area and a ground electrode 4 on the other surface are maintained in contact with each other on one surface of a plate-like dielectric 2 having the largest surface area.
  • the application electrode 3 is electrically connected to an AC power source 5 and can supply an AC voltage to these application electrodes.
  • the ground electrode 4 is grounded by connecting a ground wire 6.
  • the contact area of the ground electrode 4 with the dielectric 2 is larger than the contact area of the application electrode 3 with the dielectric 2. This is the “creeping discharge body” described above.
  • the creeping discharge body shown in FIG. 2 generates a low-temperature plasma discharge 7 at the edge of the application electrode 3, and the surface area of the ground electrode 4 is clearly larger than the surface area of the application electrode 3.
  • the low-temperature plasma discharge 7 is only generated at the edge of the application electrode 3.
  • the low-temperature plasma discharge 7 is applied to the edge of the edge at the protruding portion. It occurs on the electrode 3 side.
  • the low temperature plasma discharge 7 is generated on the side of the ground electrode 4 at the edge of the end.
  • This low-temperature plasma discharge on the ground side cannot be sufficiently charged because the electric charge flows to the ground, and the plasma effect of the aqueous protein solution is diminished, so it is not used as a plasma means for the aqueous protein solution in the present invention.
  • the applied voltage is preferably 2 to 18 kV, and the frequency is preferably 1 to 30 kHz, and particularly preferably 3 to 10 kHz. If the frequency is less than 1 kHz, pulsed micro-discharge in the order of nanoseconds cannot be sustained, and the low-temperature plasma discharge is not stable. As a result, it is necessary to apply an extremely high voltage and it is difficult to generate the low-temperature plasma itself. Is also envisaged. On the other hand, if the frequency exceeds 30 kHz, it is difficult to supply a voltage necessary for generating a polarization electric field, and as a result, it is difficult to generate low-temperature plasma.
  • these applied voltage and frequency conditions are conditions that do not take into account specific application to the present invention, and the conditions for proper implementation of the present invention are slightly different from these conditions. For example, if the frequency is too low, the voltage required for generating low-temperature plasma increases, but in the case of a mist type aqueous solution, protein combustion becomes apparent when the applied voltage becomes high.
  • the atmospheric pressure for performing creeping discharge is most preferably atmospheric pressure, but it can also be lower than that.
  • a special hermetic container is required, which causes problems such as a decrease in the generation efficiency of chemical species and emission of ultraviolet rays.
  • a pressure vessel is required, and the generation of low-temperature plasma tends to be difficult. Therefore, it can be said that the actual benefit of carrying out the present invention in an environment other than atmospheric pressure is poor. If the range of atmospheric pressure is enumerated, it is about 0.02 MPa to atmospheric pressure.
  • the thickness of the dielectric is determined according to common technical knowledge, taking into account the frequency and voltage of the AC voltage used, as well as atmospheric pressure and dielectric materials. Although it is possible to select, approximately 0.5 to 2.5 mm is preferable. In particular, when this thickness is too thin, the tendency of the dielectric to burn out is recognized. For glass, about 0.2 mm and for polymers, about 0.5 mm are exemplified as the lower limit of the thickness. On the other hand, if this thickness is too thick, the voltage required for generating low-temperature plasma increases in accordance with Paschen's law.
  • the dielectric in the creeping discharge corresponds to a paraelectric having a relative dielectric constant of about 100 or less
  • the materials include glass, forsterite, aluminum oxide (alumina), barium niobate, barium titanate, Examples thereof include polyvinylidene fluoride (PVDF), vinylidene fluoride-trifluoride ethylene copolymer (VDF / TrFE), vinylidene cyanide vinyl acetate copolymer (VDCN / Vac), etc., but are not limited thereto. Absent.
  • the application electrode and the ground electrode may be the same or different, and metal electrodes such as an aluminum electrode, a stainless steel electrode, a copper electrode, a magnesium electrode, an iron electrode, a tungsten electrode, and a platinum electrode can be used. However, it is not limited to these. It is also possible to use an electrode in which materials are appropriately combined.
  • Dielectric Barrier Discharge is a discharge form called silent discharge, ozonizer discharge, barrier discharge, dielectric barrier AC discharge, or the like.
  • the dielectric barrier discharge is a discharge generated in a gap when an AC voltage is applied with a dielectric interposed between electrodes. Even if a discharge occurs, the electric charge cannot flow into the electrode due to the presence of the dielectric, and a reverse electric field is generated because the electric charge accumulates on the dielectric, so that the discharge stops in the form of glow discharge and the final discharge There is no transition to arc discharge, which is a stage. Therefore, the dielectric barrier discharge can maintain a stable and continuous discharge such as a glow discharge. In particular, the dielectric barrier discharge can stably obtain a glow-like discharge diffused even at atmospheric pressure or higher due to the presence of the dielectric.
  • An apparatus for generating a dielectric barrier discharge to prepare the aqueous solution of the present invention includes an essential facility for generating a dielectric barrier discharge, specifically, an AC power source, a feeding electrode, a dielectric, And if a ground electrode is provided, a restriction
  • limiting will not be recognized in particular, but it can design and produce freely according to the specific process objective.
  • an AC voltage of an appropriate frequency is applied from an AC power source through a dielectric between a power supply electrode and a ground electrode, so that a glow is formed in a gap formed between these electrodes.
  • the low temperature plasma discharge can be obtained.
  • the frequency of the AC voltage is preferably in the order of 10 to 100 Hz to about 30 kHz, and more preferably 1 to 10 kHz. If it is smaller than the order of 10 to 100 Hz, an extremely high voltage is required for generating the low temperature plasma, and it may be difficult to generate the low temperature plasma itself. If it exceeds 30 kHz, it is difficult to generate low-temperature plasma.
  • the applied voltage of the dielectric barrier discharge is several kV to several tens kV, and preferably 5 to 30 kV under atmospheric pressure.
  • the atmospheric pressure in the dielectric barrier discharge is most preferably atmospheric pressure, but it can also be lower than that.
  • a special hermetic container is required, which causes a problem in that the generation efficiency of chemical species is reduced and ultraviolet rays are emitted.
  • dielectric barrier discharge is performed at a pressure higher than atmospheric pressure, a pressure vessel is required, and the generation of low-temperature plasma tends to be difficult. Therefore, it can be said that the actual benefit of carrying out the present invention in an environment other than atmospheric pressure is poor.
  • the range of atmospheric pressure is 0.02 MPa to about atmospheric pressure.
  • the “feed electrode” and the “ground electrode” may be the same or different. Specifically, the “ground electrode” of the above-described creeping discharge is used. And “applied electrode”. Further, the dielectric is the same as the creeping discharge described above.
  • Aqueous solution The aqueous solution of the present invention can be prepared by bringing the aqueous solution into contact with the low-temperature plasma generated as described above and charging the solution molecules.
  • the “aqueous solution” that is brought into contact with the low-temperature plasma is a solution in which protein is dissolved as a solute in an aqueous solvent mainly composed of water.
  • the aqueous solvent may be water as long as the protein is easily dissolved, but depending on the type of protein, it may be slightly soluble in water. In such a case, an appropriate salt or the like should be added. In some cases, the solubility of the target protein can be improved. About what kind of salt etc. and what amount are added, it can carry out by following the conventional method according to the kind of protein to melt
  • the lower limit of the protein concentration in the aqueous solution before being plasmatized is preferably about 25 ⁇ g / ml, and more preferably about 50 ⁇ g / ml. If the protein concentration is lower than 25 ⁇ g / ml, the density of protein fixation on the surface of the object will also be reduced, and it will be difficult to sufficiently exhibit the desired biocompatibility. For example, if the protein is poorly soluble in an aqueous solvent and it is difficult to ensure a large protein concentration, it is possible to compensate for the thinness of the single fixing density by performing the fixing process repeatedly. .
  • the upper limit depends on the desired form of the aqueous solution of the present invention.
  • the form when the form is a mist type, if the protein concentration is too high, mist formation becomes difficult, and the upper limit is about 3 mg / ml.
  • the droplet type is not particularly limited, and the protein concentration can be set up to the limit of dissolution, and in many cases, it is preferably about 255 mg / ml or less.
  • Proteins include peptides and also glycoproteins. Although the kind of protein to be used is not particularly limited, it is preferable to include an extracellular matrix protein. In addition, other than extracellular matrix proteins, naturally derived polyamino acids such as polylysine are also suitable.
  • extracellular matrix proteins examples include proteins such as collagen, elastin, proteoglycan and glucosaminoglycan; glycoproteins such as fibronectin, laminin and vitronectin; and processed proteins such as gelatin, but are not limited thereto. Absent.
  • aqueous solution of the present invention “other functional substances” can be contained.
  • the functional substances include growth factors, antithrombotic factors, anticancer agents, and immunosuppressive agents.
  • growth factors include growth factors, antithrombotic factors, anticancer agents, and immunosuppressive agents.
  • antibodies, sugar chains, antibiotics and the like can be mentioned.
  • the aqueous solution of the present invention has two modes of (1) mist type and (2) droplet type, and these modes include application conditions such as applied voltage, By controlling the protein concentration of the aqueous solution and the feed rate, it can be prepared by distinction.
  • an embodiment of the apparatus of the present invention that performs creeping discharge will be disclosed with reference to the drawings, and a method for preparing an aqueous solution of the present invention using the apparatus will be described for each of these two types of embodiments.
  • FIG. 3 is an explanatory diagram in the case of preparing a mist type aqueous solution mainly composed of a longitudinal section in the apparatus 1 of the present invention that performs creeping discharge.
  • 1) is the main body portion 10 thereof.
  • FIG. 4B is a cross-sectional view taken along the solid line AA ′ in FIG.
  • FIG. 5 is an enlarged explanatory view of a wall section in the vicinity of the outlet 108 of the main body portion 10 where creeping discharge of the device 1 of the present invention is performed.
  • the main body portion 10 includes a dielectric tube 101, an application electrode 102, and a ground electrode 103, and the application electrode 102 is in close contact with the entire inner surface except for the vicinity of the lowermost portion of the inner surface of the dielectric tube 101.
  • the application electrode 102 is formed on the outer surface of the outer surface of the dielectric tube 101 so that its end does not protrude from the overlapping portion of the ground electrode 103 and the dielectric tube 101. A part of the side surface of the lower region is surrounded and brought into close contact, and is electrically grounded through the ground wire 105.
  • the “protein aqueous solution introduction field” 107 is inside the cylindrical main body portion 10.
  • the application electrode 102 is in contact with the atmosphere of the introduction field 107.
  • the introduction place 107 is provided with a lead-out port 108 through which an atmosphere outside the introduction place 107 can be circulated.
  • the outlet 108 is a cylinder port of the introduction place 107.
  • the applied electrode 102 is electrically connected to an AC power source via a conductive wire 14 'or the like.
  • An insulator 104 surrounds and is in close contact with the remaining upper portion of the outer surface between the dielectrics 101, and electrical insulation of the ground electrode 103 is directed toward the upper portion of the main body portion 10.
  • the AC power supply is provided with a function generator 13 and an amplifier 14 and a monitor oscilloscope 15 in order to make it possible to adjust the applied voltage, frequency, etc., and each of them has conductive lines 13 ', 14', and 15 '. And are electrically connected to the application electrode 102.
  • An AC voltage based on the AC power source is applied toward the application electrode 102.
  • a strong electric field is formed in synchronization with the AC cycle at the edge of the portion of the application electrode 102 facing the ground electrode 103, and electrons emitted from the portion are applied to the dielectric 101.
  • the opposite end 1032 of the ground electrode 103 is greatly recessed from the other end 1021 of the application electrode 102 that is clearly longer than the ground electrode 102 in the length direction of the cylinder.
  • the low temperature plasma 106 is greatly generated at the edge of the end portion 1021 of the application electrode in the vicinity of the outlet 108, and a part of the low temperature plasma 106 is located on the side of the outlet 108 on the surface of the application electrode 102. Part is covered.
  • the low temperature plasma 106 ' is generated at the edge of the opposite end 1032, as described above, this is not used for the plasma conversion of the protein aqueous solution.
  • the sealing plug 17 is inserted on the side where the insulator 104 is provided, that is, the upper portion, and the applied electrode 102 on the inner side surface of the main body portion 10 is pressed. Sealed. Further, in the sealing plug 17, one of the arms (162) in the T-shaped hollow conduit 16 is fixed by being penetrated in a state where the pressure from the material of the sealing plug 17 is received. Of the other two arms of the conduit 16, one 161 is connected to the outer cylinder 111 of the syringe 110 of the syringe pump 11 via the conduit 114 in an airtight state, and the other is connected to the air pump 12 in an airtight state. Yes.
  • the syringe pump 11 includes a syringe 110 and a support base 115.
  • the syringe 110 has one end opened with a collar portion 1112 and the other end provided with a liquid lead-out portion 1111.
  • the other end of the syringe 110 is kept airtight at the other end.
  • An aqueous rod 1121 is provided which is slidable in the vertical direction and preferably has an inner rod 112 provided with a pressing portion 1122 at the other end.
  • One end of the inner rod 112 is inserted with an appropriate amount of the solution 113.
  • fixing portions 1153 and 1153 ′ of the guide rod 1152 are provided, and the push-in portion 1154 is penetrated by the guide rod 1152 and guided and supported while being movable on the rod.
  • the fixing portion 1153 is provided with a recess 1155 in which the outer cylinder 111 of the syringe 110 can be placed in a stable state.
  • the outer cylinder 111 of the syringe 110 is placed in the concave portion, the pressing portion 1122 at the other end of the inner rod 112 is directed to the pushing portion 1154, and the pushing portion 1154 is guided by the guide rod 1152 in the direction of arrow B.
  • the aqueous solution 113 By pushing, the aqueous solution 113 can be stably led out to the conduit 114.
  • the aqueous protein solution 113 before being converted into plasma enclosed in the syringe pump 11 is pushed out toward the T-shaped conduit 16 by the pressure in the direction of arrow B due to the pressing of the inner rod 112, and further The air pump 12 is pushed toward the opening 162 of the conduit 16 by the pressure in the direction of arrow C by the air pump 12.
  • the aqueous protein solution 113 ′ that has been pushed into the main body 10 and has not yet been converted into plasma is fed toward the inner space of the cylindrical structure formed by the application electrode 102, and from the exposed surface of the application electrode 102.
  • a desired mist-like aqueous solution 18 of the present invention is generated in contact with the low-temperature plasma 106 formed in a circular shape toward the inner space, and is discharged from the outlet 108 of the introduction field 107 to the outside.
  • the preparation method of the mist type aqueous solution is performed according to the following (a) to (c) as described above.
  • the frequency of the AC voltage for performing creeping discharge is 5 to 30 kHz, preferably 5 to 10 kHz. If this frequency is less than 5 kHz, the voltage value necessary for low-temperature plasma generation becomes excessively large, and the tendency of the protein itself to burn becomes strong, and if it exceeds 30 kHz, low-temperature plasma generation becomes difficult.
  • the applied voltage is 2 to 8 V, preferably 2 to 4 kV. When the applied voltage is less than 2 kV, the charge becomes insufficient, and it becomes difficult to make the protein solution mist. If it exceeds 8 kV, the ratio of protein burning etc. increases as described above, and mist formation becomes inefficient.
  • the protein concentration of the aqueous protein solution before being plasmatized is 25 ⁇ g / ml to 5 mg / ml, preferably 50 ⁇ g / ml to 3 mg / ml as described above.
  • the pressure of the field is preferably atmospheric pressure, and the gas can be selected from air, an inert gas such as helium and argon, etc., but air is preferred.
  • the thickness of the dielectric is as described above.
  • the droplet type aqueous solution can be prepared by using the apparatus used for the preparation of the mist type aqueous solution described above.
  • the condition (a) regarding the frequency and the applied voltage is that the range of the applied voltage is mist. Unlike the mold, it is 7.5 to 18 kV, preferably 7.5 to 16 kV.
  • the reason why the lower limit value and the upper limit value of the applied voltage are specified is that the range of the applied voltage becomes higher than that of the mist type because the feed rate of (c) is larger than that of the mist type.
  • the upper limit of the concentration of the protein aqueous solution before being plasmatized is not recognized as in the mist type, and the upper limit is about 255 mg / ml. .
  • the feed rate of the aqueous protein solution before the plasma conversion of (c) is 36 to 180 mm / min.
  • Cells or cell masses that are subject to cell production by carrying out the production method of the present invention are not particularly limited, and examples include prokaryotic cells, eukaryotic cells, and modified cells derived from these cells. Stem cells Even that is possible. Not only one type but also a combination of two or more types of cells may be used.
  • the method of “adhesion of the aqueous solution of the present invention to the surface of a plurality of cells or cell clusters” described above is not particularly limited, and can be freely selected according to the mode and purpose of immobilizing a plurality of cells. it can.
  • the aqueous solution is a mist type
  • spraying on the surface of a cell or a cell mass, contact with a mist atmosphere, etc. may be mentioned.
  • contact with the mist atmosphere accompanied by the movement of the cell or cell mass can also be performed.
  • examples include pouring onto the surface of a cell or cell mass, immersion of the cell or cell mass in an aqueous solution, application of an aqueous solution to the cell or cell mass surface, and the like.
  • the aqueous solution of the present invention has extremely low surface tension due to the charging of the constituent molecules by low-temperature plasma, and the aqueous solution can be easily and evenly attached to the cell surface by contact. .
  • the standard of the amount of contact per time depends on the protein concentration of the aqueous solution of the present invention, but in the case of the mist type, it is set so that the remaining amount of the aqueous solution per unit area of the contact surface is 2 ⁇ l / cm 2 or more. It is preferable to do. In the case of the droplet type, it is preferable to set the residual amount of the aqueous solution per unit area of the contact surface of 48 ⁇ l / cm 2 or more.
  • these processes are preferably performed in an environment where the cells continue to be utilized. Therefore, these processes are performed in a short time without drying the cells, and then the produced cell fixed product is subjected to cell survival conditions, preferably cell culture conditions, or cryopreservation conditions. It is preferable to maintain the viability of the cells underneath.
  • the use of the cell fixed product obtained by the production method of the present invention is not particularly limited.
  • it can be used for practical use and research in the medical field, the food industry field, and the like.
  • the present invention can be applied to specific cells and used as a cell fixed product.
  • cardiomyocytes, blood vessel-derived cells, muscle-derived cells, epithelial cells, epidermal cells, endothelial cells, mesothelial cells, hepatocytes, cornea-derived cells, etc. using the production method of the present invention, in sheet form, After immobilization so as to form a lump or the like, it can be used for a desired use as the cell fixed product of the present invention.
  • endothelial cells the following examples are given with a focus on vascular endothelial cells, but are not limited thereto.
  • the cells or cells constituting the cell mass used in the production method of the present invention are not particularly limited, and may be unicellular organisms themselves or separated from multicellular organisms. It is possible to select freely according to the purpose that contributes to practical use and research in the medical field and the food industry field described later. It may be a eukaryotic unicellular organism or a eukaryotic multicellular organism cell or a prokaryotic cell. In addition, for example, even cells and cell lines directly isolated from living organisms, stem cells [ES cells (embryonic stem cells) and iPS cells (induced pluripotent stem cells)], are induced after the stem cells are induced. It may be a differentiated cell or a cell transformed by gene transfer.
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • the human cells to which the present invention is applied are not particularly limited. For example, if only epithelial cells or epidermal cells originally having strong adhesion ability are used. In addition, endothelial cells having weak adhesive strength can also be applied.
  • endothelial cells examples include umbilical vein endothelial cells, coronary artery endothelial cells, saphenous vein endothelial cells, pulmonary vein endothelial cells, pulmonary artery endothelial cells, aortic endothelial cells, skin vascular endothelial cells, bladder microvascular endothelial cells, Cardiac microvascular endothelial cells, cutaneous microlymphatic endothelial cells, mesenteric vascular endothelial cells, kidney glomerular endothelial cells, lymphatic endothelial cells, breast microvascular endothelial cells, ovarian microvascular endothelial cells, placental microvascular endothelial cells, prostate Examples thereof include vascular endothelial cells such as microvascular endothelial cells, retinal microvascular endothelial cells, small intestine microvascular endothelial cells, thyroid vascular endothelial cells, and sinusoidal endothelial cells;
  • both the application electrode 102 and the ground electrode 103 are made of aluminum sheet tape (thickness 90 ⁇ m) on the glass tube wall surface with a metal rod. It was extended and used for rubbing. Therefore, the inner diameter of the tube considering the thickness of the application electrode 103 is also approximated to 6 mm (the same applies hereinafter).
  • the main body 10 was constructed using an insulating tape (Kapton (registered trademark) / polyimide tape: Toray DuPont) as the insulator 104.
  • a glass tube was used as the T-shaped conduit 16, and a rubber plug was used as the sealing plug 17.
  • a low-temperature plasma generator was produced.
  • a peak voltage of 8 kV and a frequency of 5 kHz are applied to the application electrode of the apparatus described above, and the ground electrode 103 of the main body portion of the low-temperature plasma generator is in contact with the dielectric 101.
  • a creeping discharge on the inner peripheral surface of the applied electrode 102 corresponding to the area is performed to generate a low-temperature plasma, and a collagen-containing aqueous solution [3 mg / ml collagen aqueous solution (Thermo Scientific) per 1 ml of the aqueous solution] 0.17 ml of 10-fold concentration Medium 199 (Thermo Scientific), 0.017 ml of 1 mol / l sodium hydroxide aqueous solution (Wako Pure Chemical Industries), and 0.213 ml of ultrapure water
  • To the main body via a T-shaped conduit by pressing with a syringe pump Min was flowed by feeding, in contact with the low-temperature plasma, were prepared of the protein aqueous solution of the present invention the droplet type.
  • the surface area of the creeping discharge body that generates low-temperature plasma was approximated as the contact area of the ground electrode 103 with respect to the dielectric 101 and was 94 mm 2 .
  • the upper layer is (i) Madin-Darby canine kidney cells; MDCK cell, dog kidney renal tubular epithelial cell line (Tohoku University Institute of Aging Medicine, Medical Cell Resource Center, Cell (Bank) and the lower layer were constructed as (ii) human umbilical vein endothelial cells (HUVECs (Cell Applications)).
  • FIG. 7 shows a photomicrograph image representing the state of a system using a collagen aqueous solution that has been subjected to the droplet-type plasma treatment after incubation at 37 ° C. for 30 minutes on a gelatin-coated plastic dish. It is the photograph of the space
  • the other part was entirely covered with MDCK cells.
  • the upper two photos (4x on the left and 10x on the right) focus on HUVECs cells, and the two lower photos (4x on the left and 10x on the right) focus on MDCK cells. . It can be seen that both cells are firmly adhered to each other.
  • FIG. 8 (1) shows a microscopic image of a four-fold gap after the above-described cultivation with droplet-type plasma-treated collagen. The left figure is the microscopic image on the gelatin-coated plastic dish, and the right figure is the microscopic image on the collagen gel.
  • FIG. 8 (2) shows a microscopic image of a four-fold gap after the above culture with untreated collagen.
  • the left figure is the microscopic image on the gelatin-coated plastic dish, and the right figure is the microscopic image on the collagen gel.
  • HUVECs cells which are originally endothelial cells with weak adhesive strength, are used.
  • stable cell adhesion with a good yield is recognized by the present invention.
  • the usefulness of the present invention has been clarified.
  • an untreated collagen solution or a collagen solution that has undergone a droplet-type plasma treatment is applied as an adhesive between each cell sheet layer. 250 ⁇ l was applied from the top of each cell sheet.
  • the four-layer heterogeneous cell sheet is cultured for 24 hours in the above-described culture medium for HUVECs cells, and then using 4% paraformaldehyde / phosphate buffer (Wako Pure Chemical Industries, Ltd.). Fixing treatment (30 minutes standing, room temperature) was performed.
  • FIG. 9 shows the results when a collagen aqueous solution subjected to a droplet type plasma treatment is used, and the results using untreated collagen.
  • FIG. 9 (1) shows the result in the system using plasma collagen
  • FIG. 9 (2) shows the result in the system using untreated collagen.
  • the plasma-treated collagen of the present invention as a cell adhesion liquid, cells of any combination can be easily adhered to each other even if cells with inherently low adhesion such as endothelial cells are included. It turns out that it will be possible. Further, it is obvious that this is not only the sheet type but also other forms such as hetero-type cell clusters.
  • Dielectric tube 102 Application electrode 1021: End portion on the outlet side of the application electrode 1022: End portion on the non-outlet port side of the application electrode 103: Ground electrode 1031: Conduction of the ground electrode End portion 1032 on the outlet side: End portion 104 on the non-outlet side of the ground electrode 104: Insulator 105: Ground wire 106: Low temperature plasma discharge 106 ′: Low temperature plasma discharge 107 on the ground electrode side 107: Introduction site 108 of the protein aqueous solution: Outlet 11: Syringe pump 110: Syringe 111: Outer tube 1111: Liquid outlet 1111: Collar 112: Internal rod 1121: Sliding body 1 22: pressing portion 113, 113 ': an aqueous solution before

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Abstract

La présente invention concerne un moyen permettant de faire adhérer des cellules ou des masses cellulaires l'une à l'autre à l'aide d'une protéine telle qu'une protéine de matrice extracellulaire afin de produire des cellules agrégées et immobilisées. L'objet de la présente invention est d'appliquer le moyen à des fins diverses. Plus particulièrement, il a été découvert que des cellules agrégées et immobilisées peuvent être produites à l'aide d'une solution de protéine plasma-aqueuse sous la forme de brouillards ou de gouttelettes de liquide en tant que solution d'adhérence de cellules ou de masses cellulaires, la solution de protéine plasma-aqueuse étant produite par la mise en œuvre de la plasmarisation d'une protéine de matrice extracellulaire au moyen d'une décharge plasmatique à basse température, de préférence au moyen d'un dispositif de décharge de plasma à basse température induite par décharge de surface. La découverte de la description conduit à l'accomplissement de la présente invention.
PCT/JP2016/056632 2015-03-06 2016-03-03 Procédé de production de cellules agrégées et immobilisées à l'aide d'une solution de protéine plasma-aqueuse et cellules immobilisées et agrégées produites par ledit procédé de production WO2016143669A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
JP2020195411A (ja) * 2017-01-31 2020-12-10 凸版印刷株式会社 三次元組織体及びその製造方法、並びに、三次元組織体の形成剤
JP7361324B1 (ja) * 2022-11-04 2023-10-16 ナルックス株式会社 噴霧装置及び噴霧方法

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JP2003210156A (ja) * 2002-01-24 2003-07-29 Sumitomo Bakelite Co Ltd 細胞培養基材、その細胞培養法及びその生物試験法
WO2006093151A1 (fr) * 2005-02-28 2006-09-08 Cellseed Inc. Feuille de cellules cultivees, son procede de production et son procede d’application
WO2012118099A1 (fr) * 2011-02-28 2012-09-07 学校法人 東京女子医科大学 Feuillet cellulaire produisant des cytokines et son procédé d'utilisation
WO2015079759A1 (fr) * 2013-11-27 2015-06-04 国立大学法人大阪大学 Corps de tissu tridimensionnel et son procédé de production

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JP2003210156A (ja) * 2002-01-24 2003-07-29 Sumitomo Bakelite Co Ltd 細胞培養基材、その細胞培養法及びその生物試験法
WO2006093151A1 (fr) * 2005-02-28 2006-09-08 Cellseed Inc. Feuille de cellules cultivees, son procede de production et son procede d’application
WO2012118099A1 (fr) * 2011-02-28 2012-09-07 学校法人 東京女子医科大学 Feuillet cellulaire produisant des cytokines et son procédé d'utilisation
WO2015079759A1 (fr) * 2013-11-27 2015-06-04 国立大学法人大阪大学 Corps de tissu tridimensionnel et son procédé de production

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020195411A (ja) * 2017-01-31 2020-12-10 凸版印刷株式会社 三次元組織体及びその製造方法、並びに、三次元組織体の形成剤
JP2023030074A (ja) * 2017-01-31 2023-03-07 凸版印刷株式会社 三次元組織体及びその製造方法、並びに、三次元組織体の形成剤
JP7560050B2 (ja) 2017-01-31 2024-10-02 Ｔｏｐｐａｎホールディングス株式会社 三次元組織体及びその製造方法、並びに、三次元組織体の形成剤
JP7361324B1 (ja) * 2022-11-04 2023-10-16 ナルックス株式会社 噴霧装置及び噴霧方法
WO2024095447A1 (fr) * 2022-11-04 2024-05-10 ナルックス株式会社 Dispositif d'atomisation et procédé d'atomisation

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