WO2018216568A1 - Récipient, feuille et corps cylindrique utilisé pour un récipient, et procédé pour fabriquer un récipient, une feuille et un corps cylindrique - Google Patents

Récipient, feuille et corps cylindrique utilisé pour un récipient, et procédé pour fabriquer un récipient, une feuille et un corps cylindrique Download PDF

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Publication number
WO2018216568A1
WO2018216568A1 PCT/JP2018/018926 JP2018018926W WO2018216568A1 WO 2018216568 A1 WO2018216568 A1 WO 2018216568A1 JP 2018018926 W JP2018018926 W JP 2018018926W WO 2018216568 A1 WO2018216568 A1 WO 2018216568A1
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liquid
container
wall
sheet
cylindrical body
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PCT/JP2018/018926
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English (en)
Japanese (ja)
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政彦 金岡
康明 金指
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株式会社ニコン
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a container, a sheet and a cylinder used for the container, and a method for manufacturing the container, the sheet, and the cylinder.
  • a microplate which is a flat plate provided with a number of depressions (wells), each well is used as a test tube or petri dish, biochemical analysis such as DNA analysis and immunoassay, clinical examination, drug schooling Used for cell culture and the like.
  • biochemical analysis such as DNA analysis and immunoassay
  • clinical examination drug schooling Used for cell culture and the like.
  • the inside of the well is also observed with an optical microscope.
  • the liquid level La of the culture solution L in the well 11 of the microplate 10 is higher in the liquid level height near the inner wall 11a of the well than the liquid level at the center of the liquid level.
  • a concave meniscus in which the liquid surface is bent concavely (convexly downward) may occur.
  • the concave meniscus is generated when the force attracting the culture solution and the inner wall 11a of the well is stronger than the influence of the surface tension of the culture solution.
  • This can be attributed to the fact that the inner walls of the wells of cell culture microplates are generally hydrophilized, and that amino acids and proteins in the culture medium are adsorbed on the inner walls of the wells during the culture period.
  • Patent Document 1 proposes a meniscus control device that suppresses the meniscus of the liquid in the well.
  • further meniscus suppression has been demanded.
  • the container is capable of holding a liquid, has at least one liquid holding portion that holds the liquid and includes an inner wall, and at least a part of the inner wall of the liquid holding portion.
  • a fine structure is formed, and the fine structure includes a plurality of protrusions, the height of the plurality of protrusions is in the range of 10 nm to 1000 ⁇ m, and the pitch between the plurality of protrusions is Containers are provided that are in the range of 10 nm to 1000 ⁇ m.
  • a cylinder used for a container including a container main body having a container for containing a liquid, and when the cylinder is disposed in the container, the outer wall of the cylinder is The inner wall of the cylinder is in contact with the inner wall of the container, the inner wall of the cylinder is in contact with the liquid, and a microstructure is formed on at least a part of the inner wall of the cylinder, and the microstructure has a plurality of convex portions.
  • a height of the plurality of protrusions is in the range of 10 nm to 1000 ⁇ m
  • a pitch between the plurality of protrusions is in the range of 10 nm to 1000 ⁇ m.
  • a sheet for use in a container including a container body having a container for containing a liquid, and a microstructure is formed on at least one surface of the sheet,
  • the cylindrical body is arranged in the housing portion by bending or bending the sheet so that one surface is the inner wall and the other surface is the outer wall, the outer wall of the tubular body is the inner wall of the housing portion
  • the microstructure is configured to include a plurality of convex portions, and the height of the plurality of convex portions is in the range of 10 nm to 1000 ⁇ m
  • a sheet having a pitch between the plurality of convex portions in the range of 10 nm to 1000 ⁇ m is provided.
  • a method for producing the container according to the first aspect, the cylindrical body according to the second aspect, or the sheet according to the third aspect includes forming a microstructure.
  • FIG. 1 It is a cross-sectional schematic diagram around the accommodating part (well) of the cell culture container of 1st Embodiment. It is a photograph of an example of a container main part (microplate) of a 1st embodiment.
  • (A) is a schematic diagram of the sheet
  • (b) is a schematic diagram which shows the state which curved the sheet
  • (b) is a schematic diagram which shows the meniscus of the culture solution accommodated in the cell culture container of 1st Embodiment
  • FIG. 1 It is a figure which shows the shape of the cross section of the liquid level of the culture solution accommodated in the microplate, and the inclination of this cross section. It is a figure explaining phase contrast observation using a phase contrast microscope.
  • (A) is a schematic diagram which shows a mode that the ring slit image of the direct light condensed with the objective lens is settled in the phase ring
  • (b) and (C) are the ring slit images settled in the phase ring.
  • FIG. 3 is a view showing the cross-sectional shape of the liquid surface of the culture solution accommodated in the cell culture containers of Examples 2 to 5 and Comparative Example 1.
  • (A) is a microscope picture which shows the observable area
  • (b) is a microscope picture which shows the observable area
  • the cell culture container 100 includes a plate body 12 and a container body 10 that is provided as a depression of the plate 12 and has a storage portion 11 that stores the culture solution L, and a cylindrical body 20 that is disposed in the storage portion 11.
  • the surface 20a constituting the inner wall of the cylindrical body 20 is provided with a region 20c in which a fine structure having liquid repellency is formed.
  • the entire surface 20a is the region 20c.
  • the surface 20a constituting the inner wall of the cylindrical body 20 defines a liquid holding unit that holds the culture solution L.
  • the culture medium that can be accommodated in the cell culture vessel 100 of the present embodiment is not particularly limited.
  • an aqueous culture medium that is, an aqueous solution is mainly used.
  • the aqueous culture solution (aqueous solution) has a polarity almost equal to that of water, but tends to increase in viscosity when it contains serum, fatty acids, and amino acids.
  • an organic solvent culture solution such as alcohol, ether, hexane, or a water-organic solvent mixed culture solution is also used.
  • a culture solution containing an organic solvent is different from water in polarity and viscosity.
  • the cell culture container 100 of the present embodiment can cope with culture solutions having various polarities and viscosities by appropriately changing the liquid repellency of the surface 20a constituting the inner wall in contact with the culture solution L.
  • Specific examples of the culture liquid L include, for example, physiological saline, Dulbecco's modified Eagle medium (DMEM), Eagle's medium (MEM), ⁇ -modified Eagle's MEM medium, RPMI 1640 medium, Click medium, and Iskov modified Dulbecco medium (IMDM). , L-15 medium, McCoy's 5A medium, MCDB medium, 199 medium, and other general-purpose culture media used for cell culture.
  • the container body 10 is not particularly limited as long as it has a storage portion 11 capable of storing the culture medium L, and examples thereof include a petri dish (petri dish), a test tube, a Spitz tube, and the like. Although the container main body 10 should just have at least one accommodating part 11, it is preferable to have multiple.
  • a microplate (well plate) 10 ⁇ / b> W having a plurality of accommodating portions as shown in FIG. 2 is used as the container body 10.
  • the microplate 10W is an experimental / inspection instrument provided with a plurality of wells (depressions or holes) 11W that function as a container for the culture medium L, and each well 11W can be used as a test tube or a petri dish.
  • the wells 11W are arranged in an array of 8 vertical and 12 horizontal.
  • the capacity of the well 11W is generally several ⁇ L to several mL.
  • the accommodating portion 11 of the container body 10 (hereinafter referred to as “well 11 of the microplate 10” in the present embodiment as appropriate) is partitioned by a substantially cylindrical inner wall 11a and a bottom 11b.
  • the bottom 11b may be a flat flat bottom, a U-shaped round bottom, a V-shaped conical bottom, or the like, depending on the use of the microplate 10.
  • the bottom 11b is preferably a flat bottom so that observation can be performed without being affected by the shape of the bottom.
  • the shape of the well 11 is a substantially cylindrical shape.
  • the well 11 may have a shape in which the inner diameter ⁇ 2 of the bottom is smaller than the inner diameter ⁇ 1 of the opening, and the inner wall 11a is tapered from the opening toward the bottom.
  • the material constituting the microplate 10 is not particularly limited, and plastics such as polystyrene, polypropylene, polyethylene, acrylic resin, glass, or the like can be used depending on the application. Among them, polystyrene is excellent as a material for a microplate for microscopic observation because it has high transparency and excellent optical properties.
  • the microplate 10 may be a black plate, a white plate, a transparent plate, or the like depending on the application. When performing microscopic observation, the microplate is preferably a transparent plate, and at least the bottom 11b of the well 11 is preferably transparent.
  • the meniscus of the culture solution L in the well 11 can be reduced, and a region in the culture solution that can be observed with a microscope can be widely used. For this reason, in this embodiment, even a microplate with a large number of wells, a microplate with a small well capacity, or a microplate with a small well diameter ( ⁇ 1), which is strongly influenced by the meniscus of the culture solution, is used as a container body. Can be efficiently used for microscopic observation. For example, in this embodiment, a microplate having 6 to 384 wells, preferably 6 to 96, more preferably 6, 24, or 96 wells is used. it can.
  • a microplate or petri dish having a well volume of 20 to 16000 ⁇ L, preferably 100 to 16000 ⁇ L, and a microplate or petri dish having a well diameter ( ⁇ 1) of 2 to 50 mm, preferably 6 to 35 mm can be used.
  • the cylindrical body 20 shown in FIG. 3B is used as the cylindrical body 20 disposed in the well 11 by bending the flexible sheet 20A shown in FIG.
  • One surface 20a of the sheet 20A has a region 20c in which a fine structure having liquid repellency is formed.
  • the surface 20a is curved and disposed in the well 11 so that the surface 20a becomes the inner wall of the cylindrical body 20.
  • one surface 20 a of the sheet 20 ⁇ / b> A serves as an inner wall of the cylindrical body 20 and contacts the culture medium L
  • the other surface 20 b serves as an outer wall of the cylindrical body 20. It contacts the inner wall 11 a of the well 11.
  • a fine structure is formed on the entire surface 20a of the sheet 20A, and the entire surface 20a is a liquid-repellent region 20c, but the liquid-repellent region 20c is the liquid-repellent region 20c. Since the area
  • the liquid-repellent region 20c is the entire surface 20a, that is, when the fine structure is formed on the entire surface 20a, the position adjustment of the liquid surface La of the culture solution is not necessary, and the sheet 20A is manufactured. There are advantages such as being easy.
  • the liquid repellent region 20c is limited to a minimum region in contact with the liquid surface La of the culture solution L, so that the liquid repellent fine The effect of structure on culture formation is minimized.
  • the microstructure is not formed on the other surface 20b, but the present embodiment is not limited to this. If a fine structure is formed on one surface 20a, a fine structure may or may not be formed on the other surface 20b.
  • the region 20c where the fine structure is formed exhibits liquid repellency due to the so-called lotus effect (lotus leaf effect) and has a low affinity with the culture medium L, and therefore the force to attract the culture medium L is weak.
  • lotus effect lotus leaf effect
  • the meniscus of the liquid level of the culture solution L in the cell culture container 100 of this embodiment shown in FIG. 4A is compared with the meniscus of the culture solution L in the microplate 10 shown in FIG. Become smaller.
  • the liquid level La of the culture solution L approaches a flat surface, and the range (field of view) in which the optical microscope can be observed can be expanded.
  • the fine structure having liquid repellency of the present embodiment is formed from a plurality of irregularities, and if the liquid repellency is increased compared to before providing the structure by providing the structure, the shape and size of the irregularities are particularly It is not limited.
  • the expression of liquid repellency due to the fine unevenness is presumed to be due to the fact that the liquid cannot enter the recessed portions of the unevenness and an air layer exists there. Due to the presence of this air layer, the number of contact points between the surface on which the irregularities are formed and the liquid is reduced, and the apparent contact angle is larger than the contact angle with respect to the liquid originally possessed by the material with the irregularities. .
  • the liquid repellency phenomenon when the surface is provided with unevenness can be explained by the Cassie equation represented by the following equation (1).
  • the cell culture container of this embodiment can change liquid repellency suitably by designing a microstructure based on Cassie's formula.
  • ⁇ f is the apparent contact angle of the surface on which the unevenness to the liquid is formed
  • ⁇ m is the original contact angle of the material on which the unevenness to the liquid is formed
  • a is the unevenness of the unevenness shown in FIG.
  • the width of the portion, L indicates the width (periodic width) that combines the concave and convex portions of the concave and convex portions shown in FIG.
  • the fine structure of the present embodiment may include a plurality of convex portions.
  • a moth-eye structure composed of a large number of cone-shaped protrusions (convex portions) can be mentioned, but the shape of the convex portions is not limited thereto.
  • seat 20A may be circular, an ellipse, a triangle, a square, a rectangle, a polygon, and these may be arbitrary combinations.
  • the fine structure may be constituted by a combination of very complicated uneven shapes, or may be a fractal structure.
  • the size of the protrusions for example, the height, diameter, width, etc. of the protrusions can be selected preferably in the range of 10 nm to 1000 ⁇ m, more preferably 10 nm to 100 ⁇ m, and the pitch between the protrusions is preferably It can be selected from the range of 10 nm to 1000 ⁇ m, more preferably 1 ⁇ m to 1000 ⁇ m, and still more preferably 10 ⁇ m to 1000 ⁇ m.
  • the contact angle with respect to the culture solution L accommodated in the well 11 is preferably 120 ° or more. If the contact angle with respect to the culture solution L is 120 ° or more, the concave meniscus of the culture solution L can be sufficiently reduced, and a region that can be observed with a microscope can be widely used. Moreover, it is preferable that the contact angle with respect to the culture solution L of the area
  • the contact angle of the region 20c with respect to the culture solution L is 130 ° or less, the convex meniscus of the culture solution L can be sufficiently reduced.
  • the contact angle of the region 20c with respect to the culture medium L is more preferably 120 ° to 130 °, and further preferably 125 ° to 130 °.
  • the contact angle in this embodiment means the value in room temperature (23 degreeC).
  • the contact angle of the region 20c where the fine structure is formed with respect to the culture solution L can be adjusted by changing the shape, size, period, etc. of the fine structure according to the type of the culture solution L.
  • the method of forming a fine structure having liquid repellency on the sheet 20A is not particularly limited.
  • the microstructure may be directly cut into the sheet 20A using a tool or the like, or the sheet 20A is hot-pressed using a mold in which the microstructure is formed, thereby converting the microstructure into the sheet 20A.
  • the fine structure may be formed by the attached fine particles by attaching the fine particles to the surface of the sheet 20A.
  • the microstructure may be formed on the sheet 20 by dry etching such as reactive ion etching (RIE) or sputter etching.
  • RIE reactive ion etching
  • a fine structure may be formed on the sheet 20 by shot blasting described later in the second embodiment.
  • the material constituting the sheet 20A is not particularly limited and can be selected according to the use, but it is necessary to have flexibility that can be bent into a cylindrical shape, so that polystyrene, polypropylene, polyethylene, acrylic resin, cycloolefin-based A plastic such as a resin, polytetrafluoroethylene (PTFE), or amorphous fluororesin is preferred. Further, it is preferable that the fine structure of the sheet 20A is formed of the same material as the portion other than the fine structure of the sheet 20A.
  • the region 20c of the sheet 20A is provided with a liquid-repellent fluorine coat or the like, the liquid-repellent property can be ensured, but the coating material dissolves into the culture solution in the well, and is applied to cells and bacteria. There are concerns about adverse effects.
  • the fine structure and the portion other than the fine structure of the sheet 20A are made of the same material without providing such a liquid repellant coating. Can be formed.
  • the sheet 20A is preferably formed of the same material as the microplate 10.
  • the microplate 10 is made of polystyrene
  • the sheet 20A is also preferably made of polystyrene.
  • the sheet 20A is formed of a material different from that of the microplate 10, it is necessary to check whether the material used in the sheet 20A has an adverse effect on cells or the like accommodated in the microplate 10, This is because if the sheet 20A is formed of the same material as the microplate 10, such a confirmation experiment or the like becomes unnecessary.
  • the cylindrical body 20 made of the sheet 20A may have a cylindrical shape by curving the sheet 20A, or the sheet 20A unless the field of view for microscopic observation is abnormally narrow.
  • 20A may be bent into a polygonal cylinder.
  • the thickness D20 of the sheet 20A is preferably 300 ⁇ m or less, and more preferably 200 ⁇ m or less so that processing into a cylindrical shape is easy.
  • the thickness of the sheet 20A is preferably 100 ⁇ m or more, and more preferably 150 ⁇ m or more so as to have rigidity capable of maintaining the shape of the cylindrical body.
  • the sheet 20A of the present embodiment that is arranged in the well 11 by being bent or the like always has a force to return to the plane body due to its rigidity. With this force, the sheet 20A can be brought into close contact with the inner wall 11a of the well 11 without using an adhesive or the like. Therefore, there is no risk of contamination of the culture solution by an adhesive or the like.
  • the vertical length 20d of the sheet 20A is substantially the same as the depth 11d of the well. It is also preferable that the size is 90% to 110% of the cell depth 11d.
  • the lateral length 20e of the sheet 20A is preferably substantially the same as the perimeter of the well opening with the inner diameter ⁇ 1, or is 90% to 110% of the perimeter of the cell opening with the inner diameter ⁇ 1. It is preferable.
  • the cylindrical body 20 made of the sheet 20 ⁇ / b> A may be arranged in any manner in the well 11 as long as the region 20 c where the fine structure is formed is arranged so as to come into contact with the liquid level La of the culture solution L. .
  • the cylindrical body 20 may be completely stored in the well 11.
  • the cylindrical body 20 may protrude from the opening of the well 11 to the extent that it does not interfere with microscopic observation or other experimental work.
  • the cylindrical body 20 made of the sheet 20A may be disposed in all the wells 11 included in the microplate 10, or may be disposed only in some of the wells.
  • the inclination of the liquid level with respect to the horizontal plane is ⁇ 0.02 to 0 in a cross section passing through the liquid level center of the liquid level La of the culture liquid.
  • the liquid level region of 0.02 is preferably 50% or more of the entire liquid level, and more preferably 70% or more.
  • the liquid level center is the center of the circle.
  • FIG. 6 shows a cross-sectional shape passing through the center of the liquid level of the culture solution contained in the well of the microplate.
  • FIG. 6 also shows the primary differential coefficient (dh / dx) of the liquid level as the inclination of the liquid level.
  • the region that can be observed with a microscope is a circular region having a diameter of 13.2 mm centering on the center of the liquid surface.
  • the inclination (dh / dx) of the liquid level in the region that can be observed with a microscope is 0 to 0.02.
  • the cell culture container of the present embodiment can be obtained by setting the region where the inclination of the liquid level is ⁇ 0.02 to 0.02 to 50% or more, more preferably 70% or more of the entire liquid surface area of the culture solution. A field of view sufficient as an observation container can be obtained.
  • a concave meniscus is generated, and the inclination of the liquid level when the liquid level is higher than the height at the center of the liquid level is indicated by a positive value.
  • a convex meniscus is generated, and the inclination of the liquid level when the liquid level descends from the height at the center of the liquid level is indicated by a negative value.
  • the inclination of the liquid level of the culture solution in this embodiment means a value at room temperature (23 ° C.).
  • the inclination of the liquid level of the culture solution can be adjusted by changing the shape, size, period, etc. of the microstructure provided in the cell culture vessel according to the type of the culture solution.
  • the allowable inclination of the liquid surface that is, the inclination (dh / dx) of the liquid surface in the region where the microscope can be observed varies depending on the numerical aperture NA and magnification of the eyepiece of the microscope used for observation. For example, the larger the numerical aperture NA and the magnification, the smaller the allowable range of the liquid level inclination. Even if the numerical aperture NA and the magnification are relatively large, a sufficient microscope can be used if the region where the inclination of the liquid level is ⁇ 0.02 to 0.02 is 50% or more of the entire liquid surface area of the culture medium. Observations can be made.
  • the sample to be observed is, for example, a transparent microorganism such as a cell or a bacteria cultured in the culture solution L in the storage unit 11.
  • These samples are phase objects that give a phase change to light entering the sample.
  • the human eye cannot perceive light differences that differ only in phase.
  • the phase contrast microscope visualizes this phase difference and enables observation (phase difference observation).
  • FIG. 7 shows a phase difference observation optical system 50 of a phase contrast microscope.
  • a ring diaphragm (ring slit) 52 having a ring-shaped opening, a condenser lens 53, a stage 54, an objective, along the optical path of light from the light source 51 toward the sample image plane 57.
  • the lens 55 and the phase plate 58 are arranged in this order.
  • the ring diaphragm 52 is disposed at the front focal position of the condenser lens 53, and the phase plate 58 is disposed at the exit pupil position of the objective lens 55 (the rear focal position of the objective lens 55 or its conjugate position).
  • the cell culture container 100 of this embodiment is disposed on the stage 54, and the observation target sample in the cell culture container 100 is disposed at the rear focal position of the condenser lens 53.
  • the phase plate 58 has a phase film (for example, a phase film that shifts the phase of the light by a quarter wavelength) and a filter that reduces the light by absorbing the incident light on the transparent plate.
  • a phase ring 56 (for example, an ND filter) is formed around the optical axis of the phase difference observation optical system 50.
  • the observation light incident on the phase plate 58 the observation light incident on the phase ring 56 is attenuated and the phase shifts.
  • the observation light that has come off the phase ring 56 and entered the transparent plate passes through the phase plate 58 without being subjected to a phase shift action or a dimming action.
  • the light beam emitted from the light source 51 is narrowed into a ring shape by the opening of the ring diaphragm 52.
  • the light beam that has passed through the opening of the ring diaphragm 52 becomes parallel light by the condenser lens 53 and enters the sample on the stage 54 as uniform illumination light.
  • the light that has entered the sample is divided into direct light that passes through the sample or medium portion, and diffracted light that is delayed in phase by the sample (phase object) and diffracted and bent.
  • the diffracted light includes structural information inside the sample, such as a boundary portion between the sample (cell or the like) and the solution, as a phase change.
  • the diffracted light and the direct light that have passed through the sample reach the phase plate 58 through the objective lens 55, respectively.
  • the direct light incident on the phase plate 58 through the cell culture vessel 100 forms an image of the opening of the phase stop 52 (that is, a pupil stop image) on the phase ring 56 on the phase plate 58. For this reason, the brightness of the direct light is reduced at the same time as the phase is shifted by a predetermined amount.
  • the diffracted light is diffracted by the phase object, only a small amount of diffracted light passes through the phase ring 56, and most of the diffracted light passes through the transparent plate off the phase ring 56. For this reason, most of the diffracted light travels without being subjected to a phase shift effect or a dimming effect.
  • the direct light that has passed through the phase plate 58 and the diffracted light reach the image plane 57 and interfere with each other to form an image.
  • the phase ring 56 shifts the phase of the direct light so that the phase difference between the diffracted light and the direct light becomes 1 / 2 ⁇ or 0 (zero), thereby causing a phase difference between the diffracted light and the direct light. Becomes 1 / 2 ⁇ or 0 (zero), and the phase difference caused by the sample (phase object) is observed as the contrast of light.
  • the direct light is condensed by the objective lens 55 so as to fall within the ring width of the phase ring 56.
  • the observation optical system including the illumination optical system and the objective optical system of the phase contrast microscope is adjusted so that these are realized.
  • the problem described below occurs.
  • the culture solution L acts as an optical lens, and the magnification, focal length, and aberration of the phase difference observation optical system 50 are reduced. affect.
  • the ring slit image 60 of the direct light condensed on the phase plate 58 by the objective lens 55 becomes a blurred image as shown in FIG. , It will not fit in the phase ring 56.
  • the straight light (leakage light) passing through the transparent plate off the phase ring 56 is mixed with diffracted light having sample information, and the contrast and resolution of the observation image on the image plane 57 are reduced.
  • the area where the ring slit image 60 and the phase ring 58 do not overlap that is, the amount of leakage light, tends to increase as the radius of curvature of the meniscus of the liquid surface La decreases.
  • the optical axis of the lens formed by the culture solution L in addition to the above-mentioned image blur is generated.
  • the ring slit image 60 is shifted in the direction perpendicular to the optical axis of the objective lens 55 due to the eccentricity of the lens formed by the culture medium L, and is distorted (deformed) into the shape of the image. Occurs.
  • the image shift direction tends to spread radially around the optical axis of the phase difference observation optical system 50 in the eccentric direction of the culture solution lens with respect to the phase difference observation optical system 50.
  • the cell culture container 100 of this embodiment solves the above-described problems in phase difference observation by reducing the meniscus of the culture solution L in the well 11.
  • the coefficient a of the quadratic term that is a measure of the curvature of the liquid surface shape is preferably ⁇ 0.013 to 0.013.
  • the liquid level center is the center of the circle.
  • the coefficient a of the secondary term is ⁇ 0.013 to 0.013, the above-described displacement and deformation of the ring slit image 60 can be suppressed.
  • the ring slit image 60 formed by the direct light passing thereover overlaps the phase ring 56 as shown in FIG. Thereby, an observation image with high contrast and high resolution can be obtained.
  • the ring slit image 60 formed by the direct light passing therethrough is not displaced or deformed at all.
  • the coefficient a of the quadratic term is more preferably ⁇ 0.005 to 0.005 because the above-described displacement and deformation of the ring slit image 60 can be further suppressed.
  • the coefficient a of the secondary term is ⁇ 0.005 to 0.005
  • the ring slit image 60 formed by the direct light that has passed therethrough is 50% or more of the total liquid surface area of the culture medium L. Overlap. Thereby, in 50% or more of the whole liquid surface area of the culture solution L, an observation image with high contrast and resolution can be obtained from the light passing therethrough.
  • the cell culture container 100 of the present embodiment described above can reduce the meniscus generated on the liquid surface La of the culture medium L, and can widen the range that can be observed with a microscope. Further, the region 20c of the cylindrical body 20 that comes into contact with the liquid surface La is not coated with a liquid repellent material, but has liquid repellency due to the Lotus effect due to the fine structure. For this reason, there is no possibility that the coating material dissolves into the culture medium L and adversely affects cells and the like. Furthermore, since the liquid repellency of the region 20c can be designed by the shape, size, pitch, and the like of the fine structure, a cell culture vessel that can handle various types of culture solutions L can be obtained.
  • the microplate (container body) 10 and the cylindrical body 20 are separate members. For this reason, it is possible to configure the cell culture container 100 by selecting the optimal cylinder 20 according to the type of the culture solution L.
  • the cylindrical body 20 may be always arranged in the well 11 during cell culture. However, the cylindrical body 20 is placed in the well 11 only when it is desired to reduce the meniscus of the liquid level of the culture solution such as microscopic observation. It is also possible to use the method of arranging When cells or the like adhere to the cylinder 20 and are contaminated, the effect of reducing the meniscus of the culture solution L is weakened.
  • the contact time between the culture solution L and the cylinder 20 is shortened, so that the cylinder 20 is contaminated. Can reduce the possibility.
  • the cell culture container 100 of this embodiment is not connected with the some cylinders 20, but is independent. For this reason, for example, even when different types of culture solutions are used in one well plate, different types of cylinders suitable for each culture solution can be arranged in each well. In addition, it is possible to selectively place cylinders only in the wells where the meniscus needs to be lowered, and even when the sheet is contaminated by cell adhesion or the like, only the contaminated sheet can be replaced. Is economical.
  • the cell culture container 100 of the present embodiment uses a cylindrical body of a flexible sheet 20A.
  • the size of the cylinder 20 formed from the sheet 20A can be adjusted to some extent, and one size sheet 20 can correspond to a plurality of wells 11 having a plurality of sizes and shapes.
  • FIG. 9 a cell culture container using the cylinder 30 shown in FIG. 9 will be described.
  • the cell culture container of this embodiment is the same as the cell culture container 100 of the first embodiment shown in FIG. 1 except that the cylinder 30 shown in FIG. 9 is used instead of the cylinder 20 shown in FIG. It is the same composition as. Therefore, description of configurations other than the cylindrical body 30 shown in FIG. 9 is omitted.
  • a cylinder 30 formed in a cylindrical shape is used.
  • the inner wall 30a of the cylindrical body 30 has a region 30c in which a fine structure having liquid repellency is formed.
  • the entire inner wall 30a is a region 30c where a fine structure is formed.
  • the material, size, and structure of the fine structure having liquid repellency of the cylindrical body 30 of the present embodiment are the same as those of the cylindrical body 20 of the first embodiment.
  • the cylindrical body 30 of the present embodiment does not need to be curved unlike the cylindrical body 20 of the first embodiment, so it is not necessary to have flexibility, and the upper limit value of the thickness D30 of the cylindrical body 30 is also There is no particular limitation as long as the field of view that can be observed with a microscope is not extremely narrowed.
  • the thickness D30 of the cylindrical body 30 can be set to 200 ⁇ m to 1600 ⁇ m, for example.
  • a method for forming a fine structure having liquid repellency on the inner wall 30a of the cylindrical body 30 is not particularly limited.
  • the microstructure can be transferred to the inner wall 30a of the cylindrical body 30.
  • the mold in which the microstructure is formed is preferably made of a material having a large volume expansion coefficient among ordinary mold materials and having a specific heat smaller than that of the resin material of the cylindrical body 30 to be molded.
  • a mold having a large volume expansion coefficient and a small specific heat is disposed in a space defined by the inner wall 30a of the cylindrical body 30 and expands sufficiently during hot pressing to contact the inner wall 30a of the cylindrical body 30.
  • the microstructure can be transferred to. Thereafter, when the resin material of the cylinder 30 having a large specific heat relative to the mold and a sufficiently large volume expansion coefficient is heated, the cylinder 30 is sufficiently expanded and separated from the mold, and the mold is removed from the mold. The body 30 can be easily taken out from the space.
  • the volume expansion coefficient of the material constituting such a mold is preferably 5 ⁇ 10 ⁇ 6 / K to 20 ⁇ 10 ⁇ 6 / K.
  • the material of the mold is preferably a metal, and examples thereof include steel containing iron as a main component and containing a metal such as carbon, chromium, molybdenum, and tungsten.
  • a shot blasting process in which a particle collides with a workpiece.
  • a shot blasting process for example, a sandblasting process
  • no mold is required.
  • the inventor of the present application has found that the degree of liquid repellency depends on the particle size of blast particles in sandblasting.
  • a fine structure having liquid repellency when a fine structure having liquid repellency is formed on a substrate of a resin material such as polystyrene (PS), the contact angle of water tends to increase as the particle size of the blast particle increases.
  • PS polystyrene
  • the inventor of the present application has found that a fine structure having hydrophobicity with a contact angle of 120 ° or more can be formed by blasting the surface of a polystyrene substrate using blast particles having a particle diameter of 20 ⁇ m or more.
  • the mold 30 may be shot and blasted to give the mold a fine structure having liquid repellency, and the cylinder 30 may be hot pressed using the mold.
  • the smaller the blast particle size the larger the water contact angle.
  • the contact angle can be measured using, for example, a contact angle meter (manufactured by Kyowa Interface Science, DropMaster DM700).
  • the cell culture container of the present embodiment described above can reduce the meniscus generated on the liquid surface of the culture solution, can widen the range that can be observed with a microscope, and has no risk of adversely affecting cells or the like in the culture solution.
  • the same effects as those of the cell culture container 100 of the first embodiment described above can be obtained.
  • the cell culture container of this embodiment does not need to process a sheet
  • the plurality of cylinders 30 may be independent from each other, or provided with a connecting portion that connects the cylinders 30 arranged in the adjacent wells 11. The plurality of cylinders 30 may be connected to each other. By doing so, the trouble of arranging the cylindrical body 30 in each of the plurality of wells 11 can be saved.
  • a cell culture container 300 having at least one liquid holding unit that holds the culture solution L shown in FIG. 10 will be described.
  • the cell culture container 300 of this embodiment is comprised from the container main body 40, and unlike a 1st and 2nd embodiment, it does not have a cylinder. Instead, a fine structure having liquid repellency is formed directly on the container body 40.
  • a container body 40 shown in FIG. 10 includes a plate 42 and a storage portion 41 that is provided as a depression of the plate 42 and stores the culture medium L.
  • the storage portion 41 is partitioned by a substantially cylindrical inner wall 41a and a bottom 41b.
  • the And the substantially cylindrical inner wall 41a has the area
  • a fine structure is formed on the entire inner wall 41a of the container body 40, and the entire inner wall 41a is a region 40c, but the region 40c is in contact with the liquid surface La of the culture medium L. If there is, it may be a part of the inner wall 40a.
  • the configuration of the container body 40 of the present embodiment is the same as that of the container body 10 of the first embodiment, except that a fine structure is provided on the inner wall 41a.
  • the structure of the fine structure provided in the inner wall 40a of this embodiment is the same as the structure of the fine structure provided in the cylinder 20 of the first embodiment.
  • a method for forming a fine structure having liquid repellency on the inner wall 41a of the container body 40 is not particularly limited.
  • the inner wall 41a is formed using a mold having a fine structure.
  • the microstructure may be transferred to the inner wall 41a by hot pressing, or the microstructure may be formed on the inner wall 41a by shot blasting.
  • the bottom part 41b of the accommodating part 41, the part except the accommodating part (well) 41 in the plate 42, ie, the connection part which connects the some accommodating part 41, are mentioned, for example. .
  • the cell culture container 300 of the present embodiment described above can reduce meniscus generated on the liquid surface of the culture solution, widen the range that can be observed with a microscope, and has no risk of adversely affecting cells or the like in the culture solution.
  • the same effects as those of the cell culture containers of the first and second embodiments described above can be obtained.
  • the cell culture container 300 of this embodiment does not need to arrange
  • Example 1 In Example 1, a sheet 20A (see FIG. 3) constituting the cylindrical body 20 used in the cell culture container 100 shown in FIG. 1 was produced.
  • a fine structure having liquid repellency was formed on one surface of the polystyrene sheet by reactive ion etching (RIE) to produce a sheet having a fine structure.
  • RIE reactive ion etching
  • aluminum oxide particles are generated from the aluminum oxide substrate and deposited on the polystyrene sheet.
  • the polystyrene sheet is etched with the aluminum oxide particles functioning as a mask, and as a result, a fine structure is formed on the polystyrene sheet.
  • the polystyrene sheet was ultrasonically washed with pure water to obtain a sheet on which a fine structure was formed.
  • FIGS. 11A and 11B show SEM photographs of microstructures formed by reactive ion etching (RIE) for 15 seconds.
  • ⁇ Measurement of sheet contact angle> The contact angle of the surface of the obtained sheet subjected to the RIE treatment, that is, the surface on which the fine structure was formed, was measured using a contact angle meter (DropMaster DM700, manufactured by Kyowa Interface Science). As the culture solution, DMEM (Dulbecco's modified Eagle medium) was used, and the contact angle was measured at room temperature (23 ° C.). The results are shown in Table 1.
  • Example 2 to 5 In Examples 2 to 5, as shown in Table 1, the sheet 20A shown in FIG. 3 was formed by the same method as in Example 1 except that the etching time was 60 seconds, 70 seconds, 80 seconds, and 90 seconds, respectively. Produced. Furthermore, the contact angle with respect to the culture solution of the surface on which the microstructure was formed was measured by the same method as in Example 1. The results are shown in Table 1.
  • the produced sheet 20A was curved to form a cylindrical body 20 and placed in the well 11 of the microplate 10 to produce the cell culture container 100 shown in FIG.
  • a polystyrene microplate having 24 wells 11 (well diameter: 16 mm) was used (BD, BD Falcon).
  • FIG. 12 shows a cross-sectional shape passing through the center of the liquid level of the culture liquid in Examples 2 to 5.
  • the value of coefficient a was determined.
  • Table 1 The liquid level shape was measured at room temperature (23 ° C.).
  • Comparative Example 1 In Comparative Example 1, a polystyrene sheet not subjected to RIE treatment was used. The contact angle with respect to the culture solution of the sheet not subjected to RIE treatment was measured by the same method as in Example 1. The results are shown in Table 1. Further, the cell culture vessel 100 shown in FIG. 1 was prepared by the same method as in Examples 2 to 5 except that a sheet not subjected to RIE treatment was used, and the liquid level shape of the liquid level La of the culture solution was measured. did. FIG. 12 shows a cross-sectional shape passing through the center of the liquid level of the culture solution. Similarly to Examples 2 to 5, the liquid surface shape in the diametric direction of the cell from the liquid surface center shown in FIG.
  • the region in which the fine structure of the sheet produced in Examples 2 to 5 was formed has a high contact angle of 120 ° or more with respect to the culture solution, and the fine structure
  • the liquid repellency was improved as compared with the sheet of Comparative Example 1 in which no was formed.
  • the meniscus of the culture solution was suppressed. This is presumably because, in Examples 2 to 5, the liquid repellency of the region in contact with the liquid level of the culture medium of the sheet was improved by forming the fine structure.
  • Example 3 the liquid level was able to be suppressed flat over the whole surface. Further, from the results of Examples 2 to 5, it was confirmed that there was a boundary between the downwardly convex meniscus and the upwardly convex meniscus when the contact angle was around 125 °.
  • Example 6 In the well 11 of the cell culture vessel 100 having a well diameter ( ⁇ 1) of 16 mm (24 wells), the cells are cultured in the culture medium L, and then a sheet prepared under the same conditions as the sheet used in Example 3 is prepared in the well 11. The microscopic observation in the well was performed.
  • Eagle's medium (MEM) was used as the culture solution, and Hela cells were used as the cells to be cultured.
  • a cell observation device capable of phase difference observation (Nikon Engineering Co., Ltd., BioStudio-T) is used, and a phase difference observation objective lens (Nikon Corporation CFI plan Fluor DL 4 ⁇ ) is used. The phase difference was observed.
  • the magnification of the objective lens is 4 times and the NA is 0.13.
  • a photograph of the observation result is shown in FIG. In the photograph of FIG. 13 (a), a region where the microscopic observation of the cell in the central portion of the well is indicated by a dotted line.
  • Example 2 Microscopic observation in the well was performed in the same manner as in Example 6 using a cell culture container having the same configuration as in Example 6 except that the sheet on which the microstructure was formed was not used. That is, in Comparative Example 2, the microplate itself was used as the cell culture container. A photograph of the observation result is shown in FIG. Also in the photograph of FIG. 13 (b), similarly to the photograph of FIG. 13 (a), a region where the cells in the central portion of the well can be observed with a microscope is indicated by a dotted line.
  • Example 6 was wider than the microscopic observable region in Comparative Example 2.
  • Example 6 as shown in FIG. 13A, a good observation image with high contrast and high resolution was obtained over a wide range from the center of the well to the periphery.
  • Comparative Example 2 as shown in FIG. 13B, the contrast of the observation image obtained in the center portion of the well is low, and the resolution is significantly lowered in the region slightly deviated from the center portion in the outer peripheral direction. The image could not be obtained.
  • Example 6 it is presumed that the meniscus generated on the liquid surface of the culture solution is reduced by arranging the sheet in which the fine structure is formed in the well, and the culture solution surface in the well has become flat (horizontal). . Therefore, in Example 6, it is presumed that in the phase difference observation, the displacement and deformation of the ring slit image 60 shown in FIG. 8 can be suppressed, thereby obtaining an observation image with high contrast and high resolution.
  • the cell culture container of this embodiment can reduce the meniscus generated on the liquid surface of the culture solution accommodated therein, and can expand the range that can be observed with a microscope.
  • the cell culture container of the present embodiment can be appropriately changed by designing the shape, size, pitch, etc. of the fine structure having liquid repellency, so that it is compatible with various types of culture liquids. Is possible.

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Abstract

L'invention concerne un récipient qui est apte à contenir un liquide et qui comporte au moins un réservoir de liquide contenant le liquide et pourvu d'une paroi interne, une structure fine étant formée sur au moins une partie de la paroi interne du réservoir de liquide, et qui est pourvu d'une pluralité de sections de saillie comportant une hauteur comprise dans la plage de 10 nm à 1000 µm, et un pas entre ces dernières compris dans la plage de 10 nm à 1000 µm.
PCT/JP2018/018926 2017-05-24 2018-05-16 Récipient, feuille et corps cylindrique utilisé pour un récipient, et procédé pour fabriquer un récipient, une feuille et un corps cylindrique WO2018216568A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6269979A (ja) * 1985-09-20 1987-03-31 Terumo Corp メニスカス制御用器具およびこれを備えた光透過型検査用装置
JPH11153523A (ja) * 1997-11-19 1999-06-08 Advance Co Ltd 液体保持容器
JP2005249399A (ja) * 2004-03-01 2005-09-15 Kawamura Inst Of Chem Res マイクロ流体素子及びその製造方法
US20100047845A1 (en) * 2007-02-26 2010-02-25 Stemcell Technologies Inc. Method of reducing curvature in a meniscus of liquid medium
JP2016067322A (ja) * 2014-09-30 2016-05-09 富士フイルム株式会社 プラスチック製容器
JP2016116539A (ja) * 2016-03-31 2016-06-30 大日本印刷株式会社 ボトル型の細胞培養容器及びその製造方法
JP2017001166A (ja) * 2015-06-15 2017-01-05 学校法人関東学院 撥水性面の形成方法及びその方法を用いて形成された撥水性面を備えた撥水性物品
WO2017030196A1 (fr) * 2015-08-20 2017-02-23 東京エレクトロン株式会社 Récipient de culture et procédé de culture cellulaire et procédé d'observation de cellules utilisant le récipient de culture

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6269979A (ja) * 1985-09-20 1987-03-31 Terumo Corp メニスカス制御用器具およびこれを備えた光透過型検査用装置
JPH11153523A (ja) * 1997-11-19 1999-06-08 Advance Co Ltd 液体保持容器
JP2005249399A (ja) * 2004-03-01 2005-09-15 Kawamura Inst Of Chem Res マイクロ流体素子及びその製造方法
US20100047845A1 (en) * 2007-02-26 2010-02-25 Stemcell Technologies Inc. Method of reducing curvature in a meniscus of liquid medium
JP2016067322A (ja) * 2014-09-30 2016-05-09 富士フイルム株式会社 プラスチック製容器
JP2017001166A (ja) * 2015-06-15 2017-01-05 学校法人関東学院 撥水性面の形成方法及びその方法を用いて形成された撥水性面を備えた撥水性物品
WO2017030196A1 (fr) * 2015-08-20 2017-02-23 東京エレクトロン株式会社 Récipient de culture et procédé de culture cellulaire et procédé d'observation de cellules utilisant le récipient de culture
JP2016116539A (ja) * 2016-03-31 2016-06-30 大日本印刷株式会社 ボトル型の細胞培養容器及びその製造方法

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