Cell support substrate

Abstract

To provide a cell support substrate that prevents cells from unintentionally migrating on a main surface.SOLUTION: We have found that by adjusting the arithmetic mean roughness Ra on one of the main surfaces of a nonwoven fabric to be greater than 0.10 μm and less than 3.02 μm, a cell support substrate in which cells are prevented from unintentionally migrating on the main surface can be realized. In addition, by providing a support on another main surface on opposite to the main surface of the nonwoven fabric having an arithmetic mean roughness Ra of greater than 0.10 μm and less than 3.02 μm, a cell support substrate in which the nonwoven fabric is reinforced by the support can be provided. In particular, when the nonwoven fabric and the support are laminated and integrated, a cell support substrate in which the nonwoven fabric is more effectively reinforced by the support can be provided. As a result, a cell support substrate in which cells are further prevented from unintentionally migrating on the main surface due to deformation of the nonwoven fabric can be provided.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a support substrate for cells used when transporting or observing cells.

Background Art

[0002] For pathological diagnoses such as cytodiagnosis and confirmation of cell shapes, cells are observed using an optical microscope, an electron microscope, or the like. At this time, in order to improve the observability, the cells to be observed may be transported while placed on a support such as a non-woven fabric and then subjected to an observation process. In addition, when analyzing or analyzing the cells after collecting the observed cells, or when using them for cell differentiation, regenerative medicine, treatment, etc., they may be transported and observed while immersed in a liquid such as physiological saline so that the cells can remain alive.

[0003] As a non-woven fabric used for a cell holding substrate suitable for such cell observation, for example, as disclosed in JP 2021-96139 (Patent Document 1), a cell holding substrate for preparing a cell observation specimen containing an inorganic fiber sheet is known. The cell holding substrate according to Patent Document 1 is characterized in that the maximum cross-sectional height Wt of the undulation curve on the main surface of the inorganic fiber sheet is adjusted to less than 35 μm so that a clear cell image can be obtained. Further, as the inorganic fiber sheet in a more preferable embodiment in Patent Document 1, an inorganic fiber sheet prepared by an electrospinning method is mentioned.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The applicant of the present application has studied the cell transport performance and ease of cell observation exhibited by a cell support substrate as disclosed in the prior art such as Patent Document 1. That is, a cell support substrate comprising a nonwoven fabric, in which the maximum cross-sectional height of the undulation curve on one main surface of the nonwoven fabric is less than 35 μm, was prepared. Next, cells were placed on the main surface of the prepared cell support substrate, and attempts were made to transport and observe the cells.

[0006] As a result of the investigation, it was found that during the transportation and observation of cells, cells sometimes moved unintentionally on the main surface of the cell support substrate. As a result, cells sometimes fell off the main surface of the cell support substrate during transportation, which sometimes interfered with the next process, such as the cell observation process. In addition, problems occurred in which cells fell out of the field of view during observation, and cells adhered to each other, making it difficult to observe the cells, making pathological diagnosis such as cytology and confirmation of cell shape difficult.

[0007] This problem was particularly evident when the cells to be transported or observed were cell masses with a shape close to a sphere, and also when the cells were transported and observed while immersed in a liquid such as physiological saline so that they could remain alive.

[0008] An object of the present invention is to provide a cell support substrate in which unintended migration of cells on a main surface is prevented. [Means for solving the problem]

[0009] The present invention relates to "(Claim 1) A cell support substrate comprising a nonwoven fabric, The arithmetic average roughness Ra on one main surface of the nonwoven fabric is greater than 0.10 μm and less than 3.02 μm; Supporting substrate for cells. (Claim 2) The nonwoven fabric has a support on a main surface opposite to the one main surface. The cell support substrate according to claim 1. The cell support substrate according to claim 2, wherein the nonwoven fabric and the support are laminated and integrated. It is.

Advantages of the Invention

[0010] As a result of continuous study on a cell support substrate provided with a nonwoven fabric by the applicant of the present application, by adjusting the arithmetic mean roughness Ra on one main surface of the nonwoven fabric to be greater than 0.10 μm and less than 3.02 μm, it has been found that a cell support substrate can be realized in which cells are prevented from unintentionally moving on the main surface.

[0011] In addition, by providing a support on the main surface opposite to the main surface of the nonwoven fabric where the arithmetic mean roughness Ra is greater than 0.10 μm and less than 3.02 μm, a cell support substrate in which the nonwoven fabric is reinforced by the support can be provided.

[0012] In particular, when the nonwoven fabric and the support are laminated and integrated, a cell support substrate in which the nonwoven fabric is more effectively reinforced by the support can be provided.

[0013] That is, even when an external force acts on the nonwoven fabric of the cell support substrate, it is prevented that the nonwoven fabric is unintentionally deformed, such as wrinkles occurring on the main surface of the nonwoven fabric. As a result, it is possible to provide a cell support substrate in which the unintentional movement of cells on the main surface due to the deformation of the nonwoven fabric is further prevented.

Embodiments for Carrying Out the Invention

[0014] In the present invention, various configurations can be appropriately selected, such as the following configurations. In addition, unless otherwise specified, various measurements described in the present invention are carried out under atmospheric pressure. Further, the measurements are carried out under the temperature condition of 25°C. And, unless otherwise specified, various measurement results described in the present invention are obtained by measuring up to a value one digit smaller than the required value, and the required value is calculated by rounding off the said value. As a specific example, when the value up to the second decimal place is the required value, the value up to the third decimal place is obtained by measurement, and the value up to the second decimal place is calculated by rounding off the obtained value of the third decimal place, and this value is taken as the required value. And, each upper limit value and each lower limit value exemplified in the present invention can be arbitrarily combined.

[0015] The support substrate for cells according to the present invention includes a nonwoven fabric, and can be, for example, a support substrate for cells composed of a nonwoven fabric alone, or a support substrate for cells formed by laminating a nonwoven fabric and a support.

[0016] As the constituent fibers of the nonwoven fabric, for example, inorganic fibers composed of inorganic components such as glass, silica, or alumina, or organic fibers composed of resin can be adopted. Note that the organic fibers are, for example, polyolefin resins (e.g., polyethylene, polypropylene, polyolefin resins with a structure in which a part of hydrocarbons is substituted with a halogen such as a cyano group, fluorine, or chlorine), polymethylpentene, styrene resins, polyvinyl alcohol resins, polyether resins (e.g., polyether ether ketone, polyacetal, modified polyphenylene ether, aromatic polyether ketone, etc.), polyester resins (e.g., polyethylene terephthalate, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene naphthalate, polybutylene naphthalate, polycarbonate, polyarylate, wholly aromatic polyester resin, etc.), polyimide resins, polyamideimide resins, polyamide resins (e.g., aromatic polyamide resins, aromatic polyetheramide resins, nylon resins, etc.), resins having a nitrile group (e.g., polyacrylonitrile, polyacrylonitrile copolymer, etc.), urethane resins, epoxy resins, polysulfone resins (e.g., polysulfone, polyether sulfone, etc.), fluorine resins (e.g., polytetrafluoroethylene, polyvinylidene fluoride, etc.), cellulose resins, polybenzimidazole resins, acrylic resins (e.g., polyacrylonitrile resins copolymerized with acrylic acid esters or methacrylic acid esters, modacrylic resins copolymerized with acrylonitrile and vinyl chloride or vinylidene chloride, etc.), etc., and are fibers composed of known resins.

[0017] Note that the constituent resin of the organic fiber may be composed of either a linear polymer or a branched polymer, and may also be a block copolymer or a random copolymer, and is not particularly limited regardless of the presence or absence of a three-dimensional structure or crystallinity.

[0018] The constituent fibers of the nonwoven fabric may be composed of one type of inorganic component or resin, or may be composed of multiple types of inorganic components or resins. As fibers composed of multiple types of inorganic components or resins, fibers generally called composite fibers, for example, fibers having a fiber cross-section such as a core-sheath type, a sea-island type, a side-by-side type, an orange type, a bimetal type, etc. can be used.

[0019] The constituent fibers of the nonwoven fabric may include fibers with a cross-sectional shape other than substantially circular fibers or elliptical fibers. In addition, as the profiled cross-section fibers, fibers having a fiber cross-section such as a hollow shape, a polygonal shape such as a triangular shape, an alphabetic character type shape such as a Y shape, an irregular shape, a multi-leaf shape, a symbol type shape such as an asterisk shape, or a shape in which a plurality of these shapes are combined may be used.

[0020] In addition, when the constituent fibers of the nonwoven fabric contain organic fibers, a nonwoven fabric having a flexible surface and improved cell mass holding performance can be provided. As a result, it is preferable because it is easy to provide a support substrate for cells in which cells are prevented from unintentionally moving on the main surface. And it is more preferable that the constituent fibers of the nonwoven fabric are only organic fibers so that a support substrate for cells in which cells are more effectively prevented from unintentionally moving on the main surface can be provided.

[0021] Also, it is preferable that the constituent fibers of the nonwoven fabric have hydrophilicity so as to be easily wetted by a liquid containing water such as physiological saline. Due to the good wetting of the nonwoven fabric, the light transmittance of the nonwoven fabric can be increased, and the observability by a microscope can be improved.

[0022] Examples of hydrophilic fibers include, for example, polyacrylonitrile fibers, rayon fibers, nylon fibers, vinylon fibers, and the like. By including these hydrophilic fibers as constituent fibers, a nonwoven fabric that is easily wetted with a liquid containing water can be provided. Alternatively, it may be a nonwoven fabric that is easily wetted with a liquid containing water and is obtained by subjecting the nonwoven fabric to a hydrophilization treatment. The type of hydrophilization treatment can be appropriately selected as long as it does not impair the performance of the cell support substrate. For example, sulfonation treatment, fluorine gas treatment, graft polymerization treatment of vinyl monomers, surfactant treatment, discharge treatment, hydrophilic resin application treatment, surface coating treatment with hydrophilic materials, and the like can be used.

[0023] The constituent fibers of the nonwoven fabric can be obtained by known methods such as, for example, the melt spinning method, dry spinning method, wet spinning method, direct spinning method (for example, meltblowing method, spunbond method, electrospinning method, blow method), a method of extracting fibers with a fine fiber diameter by removing one or more resin components from composite fibers, a method of obtaining fibers by beating and splitting fibers, and the like. The blow method refers to a method in which gas is ejected in parallel to the spinning solution ejected from the liquid ejection part and a shearing force is applied linearly to the spinning solution to fiberize it, as disclosed in JP-A-2009-287138.

[0024] The nonwoven fabric can be prepared, for example, by a dry method in which the above-mentioned fibers are supplied to a carding device or an air-laying device to entangle the fibers, a wet method in which the fibers are dispersed in a dispersion medium and the fibers are entangled in a sheet form by papermaking, a method in which the fibers are spun using the direct spinning method and the spun fibers are collected, and the like.

[0025] And the constituent fibers of the nonwoven fabric may be further integrated. Examples of methods for integrating the constituent fibers include, in addition to the method of adhering the constituent fibers together using a binder, for example, a method of entangling them with a needle or water flow, a method of subjecting them to heat treatment, and the like, and a method of fusing the constituent fibers together by the low melting point component provided in all melt-bonded fibers or partially melt-bonded composite fibers included in the constituent fibers.

[0026] The heat treatment method can be appropriately selected. For example, methods such as heating or heating and pressing with rolls, methods of subjecting to heating machines such as oven dryers, far-infrared heaters, dry heat dryers, and hot air dryers for heating, methods of irradiating infrared rays under no pressure to heat the low melting point components contained in the constituent fibers, etc. can be used.

[0027] The average fiber diameter and fiber length of the fibers constituting the nonwoven fabric are appropriately adjusted so as to realize a cell support substrate in which cells are prevented from moving unintentionally on the main surface.

[0028] The average fiber diameter can be 0.1 to 10 μm, can be 0.3 to 5 μm, and can be 0.5 to 1 μm. Note that the "fiber diameter" referred to in the present invention refers to the length in the direction orthogonal to the direction in which the fiber extends continuously in the fiber shown in the electron micrograph of the main surface of the nonwoven fabric, and the average value of the fiber diameters of 50 fibers to be measured is defined as the "average fiber diameter".

[0029] The constituent fibers of the nonwoven fabric may be short fibers cut to have a specific fiber length. The fiber length of the short fibers can be 5 to 120 mm, can be 10 to 100 mm, and can be 20 to 80 mm. Note that the fiber length of the short fibers refers to the value measured in accordance with JIS L1015(2010), 8.4.1c) direct method (C method).

[0030] Alternatively, the nonwoven fabric may be composed of fibers having a continuous length, such as fibers prepared using, for example, the direct spinning method, which are not cut to have a specific fiber length. Whether the constituent fibers of the nonwoven fabric here have a continuous length can be determined by the following method.

[0031] (Method for determining whether the constituent fibers of the nonwoven fabric have a continuous length) Prepare 20 electron microscope photographs showing the main surface of the nonwoven fabric. At this time, take the electron microscope photographs at a magnification such that one side of the photographed image shown in the electron microscope photograph is about 60 times the average fiber diameter of the nonwoven fabric. The photographing is performed at continuously different locations in the central part of the surface that does not include the cut part of the nonwoven fabric. And when the average value obtained by dividing the total number of fiber ends existing in the 20 prepared electron microscope photographs by 20 (that is, the number of fiber ends existing in one electron microscope photograph) is 0.3 or less, it is determined that the constituent fibers of the nonwoven fabric are fibers having a continuous length. On the other hand, when the average value is greater than 0.3, it is determined that the constituent fibers of the nonwoven fabric are not fibers having a continuous length.

[0032] When the constituent fibers of the nonwoven fabric according to the present invention are fibers having a continuous length, the number of ends of the constituent fibers exposed and present on the surface of the nonwoven fabric is small. Therefore, the main surface of the nonwoven fabric is smooth, and it is considered easy to realize a nonwoven fabric having a main surface with an arithmetic mean roughness Ra defined by the present invention. As a result, since it is easy to realize a support substrate for cells in which cells are prevented from moving unintentionally on the main surface, the constituent fibers of the nonwoven fabric are preferably fibers having a continuous length.

[0033] The nonwoven fabric according to the present invention may have shots. A shot is a non-fibrous structured portion made of the constituent resin of the fibers constituting the nonwoven fabric. As a specific example, when the spinning solution does not fiberize and adheres to the collection surface in the form of droplets when the direct spinning method is used, the adherent becomes a shot.

[0034] Various configurations of the nonwoven fabric, such as basis weight and thickness, can be adjusted as appropriate. The basis weight can be 0.1 to 50 g / m 2 and can be 0.3 to 45 g / m 2 and can be 0.5 to 40 g / m 2It can be. The lower the basis weight of the non-woven fabric, the easier it tends to be to observe cells with a microscope (especially an optical microscope often used in biological observations). This "basis weight" refers to the grammage obtained based on the method defined in JIS P 8124 (Paper and Paperboard - Method for Measuring Grammage).

[0035] The thickness of the non-woven fabric can be 0.3 to 175 μm, can be 1 to 150 μm, and can be 2 to 100 μm. The thinner the non-woven fabric, the easier it tends to be to observe cells with a microscope (especially an optical microscope often used in biological observations). This "thickness" refers to the value measured using an outside micrometer defined in JIS B7502 (measurable thickness: 0 to 25 mm).

[0036] The non-woven fabric according to the present invention has a main surface on one main surface of the non-woven fabric where the arithmetic mean roughness Ra is greater than 0.10 μm and less than 3.02 μm.

[0037] The "arithmetic mean roughness Ra" referred to in the present invention is one of the height-direction parameters defined in 4.2.1 "Arithmetic Mean Height of Profile Curve" of JIS B0601:2013 "Geometrical Product Specifications (GPS) - Surface Texture: Profile Curve Method - Terms, Definitions and Surface Texture Parameters", and refers to the average value (unit: μm) of the height difference from the average surface assuming a flattened surface of the non-woven fabric in the profile curve over the reference length.

[0038] The larger the value of the Ra, the greater the height difference between the portion presenting a fine convex shape and the portion presenting a fine concave shape of the average surface assuming a flattened surface of the non-woven fabric.

[0039] And the arithmetic mean roughness Ra on one main surface of the non-woven fabric can be determined using a non-contact surface roughness measuring instrument (specifically, for example, an optical interference type surface roughness measuring machine (Talysurf CCIMP manufactured by Taylor Hobson)).

[0040] Note that it is disclosed that the cell-retaining substrate according to Patent Document 1 includes an inorganic fiber sheet having a main surface with a maximum cross-sectional height Wt of the undulation curve of less than 35 μm. The "maximum cross-sectional height Wt of the undulation curve" referred to in Patent Document 1 is one of the height-direction parameters of the contour curve defined in 4.1.5 "Arithmetic mean height of the contour curve" of JIS B0601:2013 "Geometrical product specifications (GPS) - Surface texture: Profile method - Terms, definitions and surface texture parameters", and it is disclosed that it means the sum of the maximum value of the peak height Zp and the maximum value of the valley depth Zv of the contour curve in the evaluation length. Note that the sum refers to the maximum difference between the unevenness on the surface of the non-woven fabric whose surface is not flattened.

[0041] And it is disclosed that the "maximum cross-sectional height Wt of the undulation curve" can be determined, for example, using a contact-type or non-contact surface roughness measuring instrument (specifically, for example, a contact-type surface roughness and shape measuring machine (SURFCOM130A, Tokyo Seimitsu)).

[0042] As described above, the arithmetic mean roughness Ra defined in the present invention and the maximum cross-sectional height Wt of the undulation curve disclosed in Patent Document 1 refer to different unevenness. Therefore, as will be clear from the examples described later, just because the maximum cross-sectional height Wt of the undulation curve is less than 35 μm, it is not necessarily the case that the arithmetic mean roughness Ra is greater than 0.10 μm and less than 3.02 μm. That is, just because it is a non-woven fabric having a main surface with a maximum cross-sectional height Wt of the undulation curve of less than 35 μm, it is not necessarily the case that the movement of cells on the main surface is prevented unintentionally.

[0043] Note that Patent Document 1 discloses the finding that, by using a cell-retaining substrate having a maximum cross-sectional height Wt of the undulation curve of less than 35 μm, it is possible to focus uniformly and simultaneously on each cell placed on the cell-retaining substrate. As a result, it is disclosed that it is a cell-retaining substrate from which an easily observable cell image of each cell can be obtained.

[0044] That is to say, the effects exerted by the arithmetic mean roughness Ra defined in the present invention are different from the effects exerted by the maximum cross-sectional height Wt of the undulation curve defined in the invention of Patent Document 1. Also from this point, even though the nonwoven fabric has a main surface with the maximum cross-sectional height Wt of the undulation curve less than 35 μm, it is not necessarily the case that the unintentional movement of cells on the main surface is prevented.

[0045] As a result of investigations, the applicant of the present application has found that by providing a nonwoven fabric having a main surface with an arithmetic mean roughness Ra greater than 0.10 μm and less than 3.02 μm, a cell-supporting substrate can be realized in which the unintentional movement of cells on the main surface is prevented. Although the reason for this is not fully clear, the following reasons are considered.

[0046] Regarding the main surface of the nonwoven fabric that supports cells in the cell-supporting substrate, when the arithmetic mean roughness Ra thereof is 0.10 μm or less, it is considered that the main surface is too flat and cell movement is likely to occur.

[0047] Also, the cells supported on the main surface of the nonwoven fabric are placed on the portions presenting fine convex shapes formed by the constituent fibers of the nonwoven fabric on the main surface of the nonwoven fabric, and due to the flexibility of the nonwoven fabric, the cells sink into the nonwoven fabric by their own weight, and thus are also in wide-area contact with the portions presenting fine concave shapes formed by the constituent fibers of the nonwoven fabric on the main surface of the nonwoven fabric (in other words, in wide-area contact with the fibers existing in the portions presenting concave shapes). That is to say, it is considered that the area of contact between the cells and the constituent fibers of the nonwoven fabric is large. Therefore, it is considered that cell movement is unlikely to occur.

[0048] However, in the case of cells supported on the main surface of a nonwoven fabric with a large arithmetic mean roughness Ra, since the average value of the height difference between the portion presenting a fine convex shape and the portion presenting a fine concave shape is large, even if the cells sink into the nonwoven fabric due to their own weight because the nonwoven fabric is flexible, it is difficult for the cells to contact the portion presenting a fine concave shape (in other words, the fibers existing in the portion presenting a concave shape). That is, it is considered that the area where the cells and the constituent fibers of the nonwoven fabric are in contact is small. Therefore, it is considered that cell movement is likely to occur. Specifically, when the arithmetic mean roughness Ra is 3.02 μm or more, the contact area between the cells and the constituent fibers of the nonwoven fabric is small, and cell movement is likely to occur.

[0049] Therefore, in order to provide a cell-supporting substrate that more effectively prevents cells from moving unintentionally on the main surface, the arithmetic mean roughness Ra on one main surface of the nonwoven fabric is preferably 0.14 to 2.00 μm, and more preferably 0.21 to 0.95 μm.

[0050] The nonwoven fabric according to the present invention described above can be used as a cell-supporting substrate as it is. However, by reinforcing the nonwoven fabric with a support, even when an external force acts on the nonwoven fabric serving as the cell-supporting substrate, it is possible to prevent the nonwoven fabric from being unintentionally deformed, such as wrinkles occurring on the main surface of the nonwoven fabric. As a result, it is preferable to provide a cell-supporting substrate that also prevents cells from moving unintentionally on the main surface as the nonwoven fabric deforms.

[0051] The type of the support can be appropriately adjusted, and it can be a fabric such as a separately prepared nonwoven fabric or net, a foam, a flat plate, a porous film, or a non-porous film. In particular, since the entire main surface of the nonwoven fabric can be uniformly reinforced, it is preferable to adopt a film as the support.

[0052] The components constituting the support can be appropriately selected, and the inorganic components and resins mentioned above that can constitute the constituent fibers of the nonwoven fabric can be adopted. Further, the support is preferably colorless and transparent with light transmittance so that the observability of cells by an optical microscope is good.

[0053] It is preferable that the nonwoven fabric and the support are laminated so that cells can be placed on the main surface having the arithmetic mean roughness Ra according to the present invention. The lamination of the nonwoven fabric and the support as used herein means a mode in which the nonwoven fabric and the support are merely laminated, or a mode in which the nonwoven fabric and the support are laminated and integrated so that they cannot be easily separated by adhesion or the like.

[0054] The cell support substrate formed by laminating and integrating the nonwoven fabric and the support is · A method of collecting fibers spun using the direct spinning method on the main surface of the support to form a nonwoven fabric on the main surface of the support, · A method of melting and fusing a part of the constituent fibers of the nonwoven fabric and / or the constituent components of the support by laminating the nonwoven fabric and the support and subjecting them to a heating or heat and pressure treatment, · A method of adhering the nonwoven fabric and the support with a binder or a hot melt resin such as hot melt powder or hot melt web, can be prepared by

[0055] The prepared cell support substrate can be used by laying it on the bottom surface of the well of a well plate, a petri dish, a slide glass, etc. Alternatively, a pore plate having through holes is prepared, and the periphery of the through holes of the pore plate and the cell support substrate can be joined with an adhesive or the like for use. At this time, it is preferable to employ a non-liquid-permeable cell support substrate such as a cell support substrate formed by laminating a nonwoven fabric and a film so that a liquid such as physiological saline can be held in the portion surrounded by the through holes of the cell support substrate and the pore plate.

[0056] Next, the manufacturing method of the cell support substrate according to the present invention will be exemplified and described. Note that the description will be omitted for the points having the same items and configurations as those already described.

[0057] The manufacturing method of the nonwoven fabric according to the present invention can be appropriately selected. As an example, (Step 1) Prepare a spinning solution obtained by dissolving a resin in a solvent or a spinning solution obtained by dispersing a resin in a dispersion medium. (Step 2) Prepare a film. (Step 3) Collect the fibers obtained by spinning the spinning solution on the main surface of the film to form a fiber web on the main surface of the film. (Step 4) Remove the solvent or dispersion medium contained in the spinning solution from the fiber web to form a nonwoven fabric on the main surface of the film. The manufacturing method of the support substrate for cells comprising these steps can be used.

[0058] First, (Step 1) will be described.

[0059] The type of the solvent or the dispersion medium can be appropriately selected, and examples thereof include water, acetone, methanol, ethanol, propanol, isopropanol, tetrahydrofuran, dimethyl sulfoxide, 1,4-dioxane, pyridine, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, acetonitrile, formic acid, toluene, benzene, cyclohexane, cyclohexanone, carbon tetrachloride, methylene chloride, chloroform, trichloroethane, ethylene carbonate, diethyl carbonate, propylene carbonate, and the like. Note that the solvent or the dispersion medium may be a single type or a mixed solvent or a mixed dispersion medium formed by mixing a plurality of types.

[0060] The mass (solid content mass) of the resin contained in the spinning solution is appropriately adjusted so that the desired nonwoven fabric can be prepared, and it can be 1 to 50% by mass, 5 to 40% by mass, or 10 to 35% by mass.

[0061] Also, the spinning solution may contain a functional material. The mass (solid content mass) of the functional material contained in the spinning solution is appropriately selected, and it can be 0.1 to 30% by mass, 0.5 to 20% by mass, or 1 to 15% by mass.

[0062] The temperature and viscosity of the spinning solution are appropriately selected so that the desired non-woven fabric can be prepared. The temperature of the spinning solution can be 5 to 40°C, can be 10 to 35°C, and can be 15 to 30°C. Also, the viscosity of the spinning solution can be 50 to 8000 mPa·s, can be 100 to 6000 mPa·s, and can be 200 to 5000 mPa·s. Note that this "viscosity" is the value measured at a shear rate of 100 s -1 at a temperature of 25°C using a viscosity measuring device.

[0063] Next, (Step 2) will be described.

[0064] The thickness of the film to be used can be appropriately adjusted and can be 1 to 500 μm, can be 5 to 400 μm, and can be 10 to 300 μm. The film may be a porous film or a non-porous film. However, as described above, it is preferable that the film is a non-liquid-permeable film such as a non-porous film so that a liquid such as physiological saline can be held in the portion surrounded by the through-holes of the cell-supporting base material and the pore plate. Also, the material of the film is not particularly limited, and examples thereof include polyethylene, polystyrene, polypropylene, polycarbonate, polyethylene terephthalate, polyimide, triacetate, polyphenylene sulfide, wholly aromatic polyamide, polyethylene naphthalate, polymethyl methacrylate, fluororesin, and the like. Among these, polyethylene terephthalate is highly transparent, so it is easy to observe cells with an optical microscope. Also, it has excellent chemical resistance, and furthermore, it is tough even when thin, so it is suitable.

[0065] Note that by laminating and integrating the non-woven fabric with the film, a cell-supporting base material can be provided in which the non-woven fabric is more effectively reinforced by the film. As a result, even when an external force acts on the non-woven fabric of the cell-supporting base material, it is possible to prevent the non-woven fabric from being unintentionally deformed, such as wrinkles occurring on the main surface of the non-woven fabric. This makes it easy to uniformly focus on each cell held, and when observing the cells, a cell-supporting base material that can obtain a clear cell image can be provided, which is preferable.

[0066] Then, step (3) will be described.

[0067] The method of spinning the spinning solution is appropriately selected so that the desired nonwoven fabric can be prepared, and the electrospinning method can be adopted. When the electrospinning method is adopted, a voltage is applied to the spinning solution, and a voltage opposite to the voltage is applied to a counter electrode such as a metal plate provided at a distance from the discharge portion of the spinning solution, or the counter electrode is grounded, so that the spinning solution flies toward the counter electrode and is thinned. Then, the thinned spinning solution is collected on the main surface of the film to form a fiber web on the collector. The spinning amount is appropriately adjusted so that a nonwoven fabric with the desired basis weight and thickness can be obtained.

[0068] By directly spinning fibers onto the non-porous film using the electrospinning method, a cell support substrate comprising a nonwoven fabric having an arithmetic mean roughness Ra defined in the present invention can be realized.

[0069] Furthermore, step (4) will be described.

[0070] The method of removing the solvent or dispersion medium contained in the fiber web to form a nonwoven fabric can be appropriately selected. As an example, a method of subjecting the fiber web formed on the main surface of the film together with the film to a heat treatment can be adopted. The type of heating device can be appropriately selected. For example, a method using a device that heats or heats and presses with a roll, an oven dryer, an infrared heater, a dry heat dryer, a hot air dryer, a device that can irradiate infrared rays and heat, etc. can be adopted.

[0071] The heating temperature is appropriately selected, but it is adjusted so that the remaining solvent or dispersion medium can be volatilized and removed, and the constituent components such as the constituent fibers are not decomposed or denatured unintentionally. For example, when the remaining solvent is N,N-dimethylformamide (boiling point: 153°C), the heating temperature can be set to 170°C.

[0072] The prepared cell-supporting substrate may be used as it is, or it may be subjected to various processing steps such as being fed into a pressing device such as a calendar to smooth the surface or adjust the porosity, being subjected to a hydrophilic treatment, or being punched into a shape according to the usage mode.

Examples

[0073] Examples of the present invention are described below, but the present invention is not limited to the following examples.

[0074] (Comparative Example 1) A polyester resin film with a thickness of 250 μm (non-porous film, manufactured by Toray Industries, Inc., Lumirror (registered trademark) S10) was prepared. And this was used as the cell-supporting substrate.

[0075] (Comparative Example 2) Polyacrylonitrile (manufactured by Sigma-Aldrich, molecular weight 150,000) was dissolved in N,N-dimethylformamide (boiling point: 153 °C) to prepare a spinning solution (solid content concentration: 12% by mass). Next, the polyester resin film prepared in Comparative Example 1 was prepared and subjected to a gold vapor deposition treatment for 30 seconds for imparting conductivity. Then, the polyester resin film (thickness: 250 μm) on which gold was vapor-deposited was wound around the surface of an earthed stainless steel drum so that the gold vapor-deposited surface was exposed. And by spinning the spinning solution by the electrospinning method under the following conditions, it was collected on one main surface of the polyester resin film on which gold was vapor-deposited to form a fiber web on one main surface of the polyester resin film. (Electrospinning conditions) · Shape of the spinning solution discharge part in the metal nozzle (spinning solution discharge part): circular shape with an inner diameter of 0.44 mm · Distance between the tip of the metal nozzle and the earthed stainless steel drum: 120 mm · Voltage applied to the spinning solution: 12 kV · Spinning solution discharged from the metal nozzle: 0.5 g / hour · Spinning environment: temperature 25 °C, humidity 20% RH Finally, a polyester resin film vapor-deposited with gold and having a fiber web on one main surface was subjected to a dryer device (heating temperature: 170 °C, heating time: 30 minutes) to remove N, N-dimethylformamide contained in the fiber web, and a laminate in which a nonwoven fabric was laminated and integrated on the main surface of the polyester resin film vapor-deposited with gold was prepared. Furthermore, the prepared laminate was subjected to a pressure press machine (pressure: 8 MPa) to press between both main surfaces thereof, thereby preparing a support substrate for cells. The constituent fibers of the nonwoven fabric included in the support substrate for cells were polyacrylonitrile fibers having a continuous length.

[0076] (Example 1) The laminate prepared in Comparative Example 2 was used as a support substrate for cells without being subjected to a pressure press machine. The constituent fibers of the nonwoven fabric included in the support substrate for cells were polyacrylonitrile fibers having a continuous length.

[0077] (Example 2) A laminate in which a nonwoven fabric was laminated and integrated on the main surface of a polyester resin film vapor-deposited with gold was prepared in the same manner as in Comparative Example 2 except that the spinning amount was changed. Then, the prepared laminate was used as a support substrate for cells without being subjected to a pressure press machine. The constituent fibers of the nonwoven fabric included in the support substrate for cells were polyacrylonitrile fibers having a continuous length.

[0078] (Example 3) A laminate in which a nonwoven fabric was laminated and integrated on the main surface of a polyester resin film vapor-deposited with gold was prepared in the same manner as in Comparative Example 2 except that a spinning solution in which the solid content concentration of polyacrylonitrile was adjusted to 8% by mass was used. Then, the prepared laminate was used as a support substrate for cells without being subjected to a pressure press machine. The constituent fibers of the nonwoven fabric included in the support substrate for cells were polyacrylonitrile fibers having a continuous length.

[0079] (Comparative Example 3) On one main surface of a polyester resin film (manufactured by Toray Industries, Inc., Lumirror (registered trademark) S10, thickness: 50 μm), a polyester staple fiber nonwoven fabric with an average fiber length of 64 mm was simply overlaid, and then subjected to heat and pressure treatment to form a cell support substrate in which the film and the nonwoven fabric were laminated and integrated.

[0080] The physical properties of the cell support substrates prepared in the examples and comparative examples, and the performance exhibited by these cell support substrates were evaluated as follows and summarized in Table 1. For configurations that were not present, a "-" mark was described in the table.

[0081] (Method for measuring the cell migration distance in the cell support substrate) (Step 1) Prepare a 24-well plate, and fix and lay a cell support substrate cut out to the same size as the well bottom surface of the well plate on the well bottom surface of the well plate using an adhesive. At this time, when the cell support substrate includes a nonwoven fabric, the cell support substrate is laid on the well bottom surface so that the nonwoven fabric is exposed. (Step 2) Place the 24-well plate with the cell support substrate laid on the well bottom surface on the shaking part of a shaker (OS-762RC manufactured by Optim). (Step 3) Inject 200 μL of physiological saline into the well of the 24-well plate on which the cell support substrate is laid. (Step 4) Place one of the following four types of cell aggregates A1 to B2 on the cell support substrate laid on the well bottom surface (on the main surface of the nonwoven fabric when the cell support substrate includes a nonwoven fabric). At this time, place it randomly at a location other than the center so that the cell aggregate does not exist on the center of the cell support substrate. Cell aggregate A1: A spherical cell aggregate of HEK293, a human embryonic kidney cell line, average diameter: 300 μm Cell aggregate A2: A spherical cell aggregate of HEK293, a human embryonic kidney cell line, average diameter: 560 μm Cell aggregate B1: A spherical cell aggregate of HepG2, a human liver cancer-derived cell line, average diameter: 550 μm Cell mass B2: A spherical cell mass of HepG2, a human liver cancer-derived cell line, average diameter: 900 μm (Step 5) Photograph A of each cell-supporting substrate on which the cell mass is stationary, prepared through Step 4, is taken using a digital microscope. (Step 6) Intending to reproduce the act of transporting while placing the cell mass on the main surface of the cell-supporting substrate, the shaker is operated under the conditions of a rotation speed of 100 rpm for 3 seconds to shake the 24-well plate on which the cell mass is stationary. Then, after stopping the shaker, after visually confirming that the movement of the cell mass due to the shaking has stopped, photograph B of the cell-supporting substrate on which the cell mass is placed is taken using a digital microscope. (Step 7) Photograph A and photograph B are overlaid, and a line segment connecting the position A of the cell mass before operating the shaker and the position B of the same cell mass after operating the shaker to form the shortest distance is drawn. Then, the length of the drawn line segment is measured. (Step 8) The length of the obtained line segment is taken as the distance (cell movement distance, unit: mm) that each cell has moved. The above measurement is performed for the four types of cell masses A1 to B2 described above. Note that the smaller the cell-supporting substrate with a cell movement distance (unit: mm), the more it means that the cell-supporting substrate is one in which cells are prevented from unintentionally moving on the main surface.

[0082] [Table 1]

[0083] From the results of comparing the comparative examples and the examples, it was found that the cell movement distance on the cell-supporting substrate prepared in the examples was shorter than half of the cell movement distance on the cell-supporting substrate prepared in the comparative examples for any of the four types of cell masses A1 to B2. Therefore, it was found that the cell-supporting substrate according to the present invention prevents cells such as cell masses from unintentionally moving on the main surface.

[0084] For this reason, it was considered that the cell-supporting substrate according to the present invention includes a nonwoven fabric having an arithmetic mean roughness Ra on one main surface that is greater than 0.10 μm and less than 3.02 μm.

Industrial Applicability

[0085] The cell-supporting substrate according to the present invention can be used for transporting or observing cells such as cell aggregates. In particular, it can be used for transporting or observing cell aggregates having a shape close to a sphere. Further, cells (particularly, cell aggregates) statically placed on the cell-supporting substrate according to the present invention can be used for observing the cells using an optical microscope, an electron microscope, etc. for pathological diagnosis such as cytodiagnosis or for confirming the shape of the cells, together with the cell-supporting substrate.

Claims

1. A support substrate for cells, comprising a non-woven fabric, wherein an arithmetic mean roughness Ra on one main surface of the non-woven fabric is greater than 0.10 μm and less than 3.02 μm, The support substrate for cells.

2. The support substrate for cells according to claim 1, further comprising a support on the main surface of the non-woven fabric opposite to the one main surface. The support substrate for cells according to claim 1.

3. The support substrate for cells according to claim 2, wherein the non-woven fabric and the support are integrally laminated.

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