Cell support, method for producing the same, method for culturing cells, and cell structure

Abstract

The present invention pertains to a cell support that comprises a base material containing a biocompatible substance or a base material provided with a biocompatible substance on the surface thereof and gelatin particles retained by the biocompatible substance of the base material. The cell support can be produced by a method that comprises allowing the base material, which contains the biocompatible substance, to retain the gelatin particles. A cell structure, in which a reagent or a drug is uniformly incorporated into a larger number of cells, can be obtained by inoculating the cells in the aforesaid cell support and then culturing the cells.

Description

Technical Field

[0001] The present invention relates to a cell support, a method for manufacturing the same, a method for culturing cells, and a cell structure.

Background Art

[0002] Techniques for introducing reagents, drugs, etc. into cultured cells are known. For example, Patent Document 1 describes that a probe solution is added to a culture solution to introduce the probe into cells so that the state of the cells can be detected.

[0003] In addition, a support for culturing cells for transplantation or storing cells (hereinafter, also simply referred to as "cell support") is known. For example, Patent Document 2 describes a biodegradable base material having a biodegradable nonwoven fabric sewn with biodegradable filaments and a biodegradable film-like material superposed on the biodegradable nonwoven fabric. Patent Document 2 describes that when this biodegradable base material is used as a cell support, cells can easily enter inside, do not cause a foreign body reaction in the living body, and can be implanted for a long period of time.

[0004] In addition, Patent Document 3 describes culturing cells by coating a culture dish (UpCell manufactured by CellSeed Co., Ltd.) made of poly N-isopropylacrylamide (PIPAAm) with gelatin or the like. Patent Document 4 describes culturing adherent cancer cells using a cell culture substrate provided with a bioadhesive polymer layer on a substrate made of a material such as silicon, glass, and plastic. Patent Document 5 describes culturing cells with a gelatin nonwoven fabric.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

[0006] As described in Patent Document 1, when attempting to introduce a reagent, drug, etc. into cells, there has been a problem of bias in the uptake of the reagent, drug, etc. into the cells. In particular, when attempting to grow cell colonies in three-dimensional directions, bias in the uptake of reagents, drugs, etc. is likely to occur, resulting in cells that have taken them up and cells that have not, or cells that have taken up a large amount of them and cells that have taken up only a small amount. This bias in cell uptake also occurred when culturing cells using a substrate simply used on the surface in contact with the cells and having a biocompatible substance as described in Patent Documents 2 to 5.

[0007] The present invention has been made based on the above findings, and an object thereof is to provide a cell support, a method for manufacturing the same, a method for culturing cells using the cell support, and a cell structure manufactured using the cell support, which can introduce reagents, drugs, etc. into cells more uniformly. Means for Solving the Problems

[0008] The above problems are solved by a cell support including a substrate containing a biocompatible substance or a substrate having a biocompatible substance applied to its surface, and gelatin particles held on the surface of the substrate on the side in contact with the cells.

[0009] Further, the above problems are solved by a method for culturing cells having a step of preparing the cell support and a step of seeding cells on the cell support.

[0010] Further, the above problem is solved by a cell structure having the cell support and cells held on the cell support.

Advantages of the Invention

[0011] The present invention provides a cell support, a method for manufacturing the same, a method for culturing cells using the cell support, and a cell structure manufactured using the cell support, which can introduce reagents, drugs, etc. into cells more uniformly.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0013] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Note that the present invention is not limited to the following embodiments.

[0014] [Cell Support] A cell support according to an embodiment of the present invention includes a base material containing a biocompatible substance or a base material with a biocompatible substance applied to its surface, and gelatin particles held on the base material.

[0015] Figures 1A and 1B are schematic diagrams showing the configuration of the cell support according to this embodiment. In the example shown in FIG. 1A, the cell support 100 has a base material 112 containing a biocompatible substance and gelatin particles 120 held in the biocompatible substance of the base material 112. In the example shown in FIG. 1B, the cell support 100 has a base material 114 such as glass or plastic, a biocompatible substance 116 applied to the surface of the base material, and gelatin particles 120 held in the biocompatible substance 116. These cell supports 100 can be used as supports for culturing or holding the seeded cells 130 or for storage.

[0016] (Base material) The above base material may be any base material capable of culturing and holding cells, and may be a two-dimensional shaped base material for growing cells into a planar colony, or a three-dimensional shaped base material for growing cells into a three-dimensional colony.

[0017] Examples of the two-dimensional shaped base material include cell culture plates, culture dishes, and Petri dishes. Further, the two-dimensional shaped base material may have a shape such as a bottle shape, a tube shape, a bag shape, a microchannel shape, and a multi-well plate shape.

[0018] Examples of the three-dimensional shaped base material include aggregates of fibers such as non-woven fabrics, woven fabrics, and mesh fabrics, and porous base materials such as membrane filters and mesh sheets.

[0019] The materials of these base materials are not particularly limited, and any material such as metal, resin, glass, and ceramic may be used.

[0020] Examples of the above metal include titanium, nickel, platinum, gold, tungsten, iron, and alloys thereof.

[0021] Examples of the above resin include polyolefins such as polyurethane, polyethylene, and polypropylene, polycarbonate, polylactic acid, polyglycolic acid, poly-ε-caprolactone, and polyvinyl alcohol and their copolymers, polyethylene glycol, polyimide, acrylic resin, polyester, polyvinylidene fluoride, polyvinyl alcohol, polyvinyl chloride, polyvinyl acetate, polyvinyl pyrrolidone, polystyrene and their copolymers, etc. synthetic resins, natural resins such as cellulose, styrene-butadiene copolymers, polyisoprene, isobutylene-isoprene copolymers (butyl rubber), halogenated butyl rubber, butadiene-styrene-acrylonitrile copolymers, silicone polymers, and fluoro silicone polymers, etc. elastomers, and biogenic materials such as polyhydroxybutyric acid, polyhydroxyvaleric acid, proteins, sugars, and glycoproteins (such as fibronectin), etc. are included.

[0022] In addition, the above substrate may be a scaffold material that mimics the extracellular matrix for growing cells into three-dimensional colonies to form spheroids. Examples of the above scaffold material include gel-like substances of biocompatible substances described below, including gelatin, collagen, and hyaluronic acid, etc.

[0023] (Biocompatible substance) The above substrate contains a biocompatible substance on its surface.

[0024] As long as the above substrate contains a biocompatible substance on its surface, its form is not particularly limited. A substrate having the above shape may be formed by a biocompatible substance, or a biocompatible substance may be applied to the surface of a substrate formed into a predetermined shape.

[0025] According to the findings of the present inventors, in a configuration where only gelatin particles are simply imparted to a substrate such as silicon, glass, and plastic, the uptake efficiency of the gelatin particles by cells is not very high. On the other hand, when the substrate contains a biocompatible substance and the biocompatible substance retains the gelatin particles, the uptake efficiency of the gelatin particles by cells is significantly increased (see the comparison between cell supports 3 to 5 and cell supports 1 to 2 in Fig. 4A to Fig. 4 C、 Table 3).

[0026] In addition, when the above-mentioned substrate is a porous substrate having a three-dimensional shape or the like, the biocompatible substance does not necessarily need to be applied up to the inner surface of the substrate, and it is sufficient that it is applied at least to the outer surface where cells of the substrate are seeded, and it may be applied only to the outer surface where cells of the substrate are seeded.

[0027] The above-mentioned biocompatible substance may be a biogenic polymer or a biodegradable synthetic polymer.

[0028] Examples of the above-mentioned biogenic polymers include biodegradable polyesters such as polyhydroxybutyric acid and polyhydroxyvaleric acid, glycosaminoglycans (such as hyaluronic acid), starch, cellulose or its derivatives (such as carboxymethyl cellulose), alginic acid, chitin, and polysaccharides such as chitosan, as well as poly(amino acids) such as collagen, elastin, gelatin, and laminin, glycoproteins such as fibronectin, and complexes thereof. Among these, collagen, gelatin, fibronectin, laminin, and polysaccharides are preferred.

[0029] Examples of the above-mentioned biodegradable synthetic polymers include polylactic acid, polyglycolic acid, poly-ε-caprolactone, and polyvinyl alcohol and copolymers thereof, polyethylene glycol, polyhydroxybutyric acid, polyhydroxyvaleric acid, and biodegradable polyesters. Among these, polylactic acid, polyglycolic acid, poly-ε-caprolactone, and polyvinyl alcohol and copolymers thereof are preferred.

[0030] According to the findings of the present inventors, among these biocompatible substances, polymer materials with a higher water content are likely to increase and are easy to control the introduction rate of gelatin particles into cells. From the above viewpoints, the biocompatible substances are preferably collagen, gelatin, fibronectin, polyvinyl alcohol and its copolymers, chitin, and chitosan, and more preferably collagen, gelatin, fibronectin, polyvinyl alcohol.

[0031] Among these biocompatible substances, poly(amino acid) is preferable and gelatin is more preferable because it has a high affinity for gelatin particles and is easy to control the introduction rate of gelatin particles into cells.

[0032] The above gelatin may be any known gelatin obtained by denaturing collagen derived from bovine bone, cowhide, pigskin, pig tendon, fish scale, fish meat, etc.

[0033] The above gelatin may be crosslinked. The crosslinking may be crosslinking with a crosslinking agent or self-crosslinking without using a crosslinking agent.

[0034] The above crosslinking agent may be, for example, a compound having a plurality of functional groups that form chemical bonds with hydroxyl groups, carboxyl groups, amino groups, thiol groups, imidazole groups, etc. Examples of such crosslinking agents include glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate (CMC) water-soluble carbodiimide, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether and glycerol polyglycidyl ether compounds having two or more epoxy groups, and propylene oxide. Among these, from the viewpoint of enhancing reactivity, glutaraldehyde and EDC are preferable, and glutaraldehyde is more preferable.

[0035] Examples of the self-crosslinking include crosslinking by application of heat or irradiation with an electron beam or ultraviolet rays.

[0036] In this embodiment, depending on the type of the biocompatible substance, the introduction rate of the gelatin particles into the seeded and grown cells can be controlled. For example, when it is desired to increase the introduction rate of the gelatin particles into the cells, it is preferable to use a biocompatible substance closer to gelatin or to make the degree of crosslinking of gelatin as the biocompatible substance closer to the degree of crosslinking of gelatin in the gelatin particles. Alternatively, when it is desired to suppress the introduction rate of the gelatin particles into the cells to a certain extent, it is preferable to use a biocompatible substance farther from gelatin or to make the degree of crosslinking of gelatin as the biocompatible substance farther from the degree of crosslinking of gelatin in the gelatin particles. Thus, it is desirable to select the type of the biocompatible substance according to the introduction rate of the gelatin particles to be achieved.

[0037] Note that the difference in the degree of crosslinking of the gelatin can be estimated from, for example, the difference in the signal intensity of any energy band in the loss spectrum detected by transmission electron microscope observation (TEM-EELS measurement) combined with electron energy loss spectroscopy, or the peak intensity ratio of COOH to CONH (peak intensity of COOH / peak intensity of CONH) in the spectrum obtained by plotting the wave number on the horizontal axis and the absorbance on the vertical axis and measured with a Fourier transform infrared spectrophotometer (FT-IR).

[0038] By controlling the introduction rate of the gelatin particles, the introduction rate of the reagent or drug carried by the gelatin particles into the cells can also be controlled. Depending on the type of these reagents or drugs, the amount to be introduced into the cells also varies. Therefore, by controlling the introduction rate of the gelatin particles into the cells with the above biocompatible substance and thereby controlling the introduction rate of the reagent or drug into the cells, the amount of the reagent or drug introduced into the cells can be easily controlled.

[0039] (Gelatin particles) The above substrate holds the gelatin particles.

[0040] The above-mentioned gelatin particles are in contact with the biocompatible substance of the above-mentioned substrate and are immobilized on the above-mentioned substrate.

[0041] At this time, the gelatin particles are immobilized and held at a position in contact with the biocompatible substance contained in the substrate. For example, when a biocompatible substance is provided on the surface of the substrate, the gelatin particles are provided and immobilized in the region where the biocompatible substance of the substrate is provided.

[0042] Further, when the above-mentioned substrate is a porous substrate having a three-dimensional shape or the like, the gelatin particles do not need to be provided up to the inner surface of the substrate, and at least, it is sufficient that they are provided with respect to the outer surface on which the cells of the substrate are seeded, and they may be provided only with respect to the outer surface on which the cells of the substrate are seeded.

[0043] The above-mentioned gelatin particles may be nanoparticles made of any known gelatin similar to those described for the biocompatible substance. Gelatin has been used for food and medical purposes for a long time, and it is less likely to harm the human body even when ingested into the body. In addition, since gelatin is dispersed and disappears in the living body, it has the advantage that it does not need to be removed from the living body.

[0044] The weight average molecular weight of the gelatin constituting the above-mentioned gelatin particles is preferably 1000 or more and 100000 or less. The above-mentioned weight average molecular weight can be, for example, a value measured according to the 10th edition of the PAGI method (2006).

[0045] The gelatin constituting the above-mentioned gelatin particles may be crosslinked. The crosslinking may be crosslinking with the above-mentioned crosslinking agent or self-crosslinking without using a crosslinking agent.

[0046] From the viewpoint of facilitating the control of the ease of uptake into cells, the above-mentioned gelatin particles are preferably cationized by introducing a primary amino group, a secondary amino group, a tertiary amino group or a quaternary ammonium group.

[0047] The cationization of gelatin particles can be carried out by a known method of introducing a functional group that cationizes under physiological conditions during production. For example, alkyldiamines such as ethylenediamine and N,N-dimethyl-1,3-diaminopropane, trimethylammonium acetohydrazide, spermine, spermidine, and diethylamide chloride are reacted with a condensing agent containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, cyanuric chloride, N,N'-carbodiimidazole, cyanogen bromide, diepoxy compounds, tosyl chloride, dianhydride compounds such as diethyltriamine-N,N,N',N'',N''-pentanedioic dianhydride, and trityl chloride to introduce the above amino groups into the hydroxyl or carboxyl groups of gelatin.

[0048] The average particle diameter of the above gelatin particles is preferably 100 nm or more and 1000 nm or less. Although the above gelatin particles carry a probe, they do not substantially have a probe in their surface layer. Therefore, even if the average particle diameter is 1000 nm or less, it is easily taken into cells by the activity of the cells themselves. In order to incorporate many gelatin particles into cells in a shorter time, it is more preferable that the average particle diameter of the above gelatin particles is 800 nm or less. On the other hand, gelatin particles with an average particle diameter of 100 nm or more can easily carry a probe in the particles and can increase the capacity of the probe. Also, the larger the average particle diameter of gelatin particles within the range of 1000 nm or less, the more easily they are taken into cells by the activity of the cells themselves. From the above viewpoints, the average particle diameter of gelatin particles is preferably 200 nm or more, and more preferably 300 nm or more.

[0049] The average particle diameter of the gelatin particles can be the apparent particle diameter of the gelatin particles measured by the dynamic light scattering method. Alternatively, the average particle diameter of the gelatin particles can be the value obtained by adding and averaging the major axis and the minor axis. The minor axis and the major axis of the gelatin particles can be the values obtained by analyzing an image obtained by imaging the dried gelatin particles with a scanning electron microscope (SEM) after standing in the atmosphere at 80°C for 24 hours. Since gelatin particles are usually aggregates composed of a plurality of gelatin particles, the major axis, the minor axis, and the particle diameter of the gelatin particles can be the values obtained by adding and averaging the major axis, the minor axis, and the particle diameter of a plurality of gelatin particles (for example, 20 gelatin particles) arbitrarily selected from the above aggregates. When there is a difference between the average particle diameters measured by these methods, the average particle diameter obtained by measurement by the dynamic light scattering method may be adopted.

[0050] The above gelatin particles carry a reagent or a drug.

[0051] That the gelatin particles carry a probe means that the probe is immobilized on the surface of the gelatin particles or incorporated into the inside of the gelatin particles.

[0052] Incidentally, it is preferable that the amount of the probe inside the gelatin particles is larger than the amount of the probe in the surface layer portion. By reducing the amount of the probe in the surface layer portion of the gelatin particles, the amount of the probe exposed on the surface of the gelatin particles can be reduced. Thereby, it is possible to make it difficult for the gelatin particles to be recognized as foreign substances by cells and to make it easy to be taken into cells by activities such as endocytosis. The above surface layer portion means a region up to a depth of 1% with respect to the average particle diameter of the gelatin particles.

[0053] The above-mentioned reagent may be a probe used for applications such as the examination of biological activities, the measurement of substances in vivo, and the quantification of substances in vivo, or a contrast agent for detecting the presence of cells. The detection target of the above-mentioned probe is not particularly limited, and it may be a protein, sugar, nucleic acid such as DNA and mRNA, or a physiological state such as temperature and pH in cells.

[0054] The above-mentioned probe may be, for example, a compound having a site that binds directly or indirectly to the substance to be detected and a site that emits a detectable signal. For example, the above-mentioned probe may be a probe that can specifically bind to the mRNA by a nucleic acid having a sequence complementary to at least a part of the nucleic acid sequence of the mRNA to be detected, or a probe that can specifically bind to the protein to be detected by an antibody. Further, the above-mentioned probe may be a probe that contains a phosphor and emits fluorescence as a signal, or a probe that emits other signals such as chemiluminescence.

[0055] The type of the above-mentioned phosphor is not particularly limited, and it may be a fluorescent dye or semiconductor nanoparticles.

[0056] Examples of the above-mentioned fluorescent dyes include rhodamine-based dye molecules, squarylium-based dye molecules, fluorescein-based dye molecules, coumarin-based dye molecules, acridine-based dye molecules, pyrene-based dye molecules, erythrosine-based dye molecules, eosin-based dye molecules, cyanine-based dye molecules, aromatic ring-based dye molecules, oxazine-based dye molecules, carbopyronine-based dye molecules, and pyromethene-based dye molecules.

[0057] Examples of the semiconductors constituting the above-mentioned semiconductor nanoparticles include II-VI group compound semiconductors, III-V group compound semiconductors, and IV group semiconductors. Specific examples of the semiconductors constituting the above-mentioned semiconductor nanoparticles include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.

[0058] Probes that can specifically bind to the above mRNA may be known probes such as molecular beacons, Taqman probes, cycling probes, and INAF probes. However, since general-purpose fluorescent dyes can be used and detection of various cell types is easy, molecular beacons are preferred.

[0059] A molecular beacon is a nucleic acid derivative having a stem-loop structure, with a fluorescent dye bound to one end of the 5'- and 3'-ends, and a quenching dye bound to the other end. In the state where the molecular beacon forms the above stem-loop structure, since the fluorescent dye and the quenching dye are close to each other, the fluorescence emitted from the fluorescent dye is quenched. However, when it approaches the target sequence, the loop structure is opened and it binds to the mRNA to be detected. As a result, the fluorescent dye and the quenching dye are separated, and fluorescence emission is detected.

[0060] The combination of the above fluorescent dye and quenching dye is not particularly limited, and may be appropriately selected from the above-described fluorescent dyes. The above quenching dye may be a molecule that quenches fluorescence by any of fluorescence resonance energy transfer (FRET), contact quenching, and collisional quenching.

[0061] The probe that can specifically bind to the protein to be detected by the above antibody is preferably a phosphor integrated dot (PID). PID is a nano-sized particle that uses particles made of organic or inorganic substances as a matrix and contains a plurality of phosphors. PID specifically binds directly or indirectly to the protein to be detected by the above antibody to label the protein to be detected. The plurality of phosphors may be present inside the particle or on the surface of the particle. The phosphor integrated particle can emit fluorescence with sufficient intensity to show each molecule of the target substance as a bright spot.

[0062] Examples of the organic substance serving as the matrix material include thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, and furan resin; thermoplastic resins including styrene resin, acrylic resin, acrylonitrile resin, AS resin (acrylonitrile-styrene copolymer), and ASA resin (acrylonitrile-styrene-methyl acrylate copolymer); other resins such as polylactic acid; and polysaccharides. Examples of the inorganic substance serving as the matrix material include silica and glass. It is preferable that the matrix and the fluorescent substance have substituents or sites having opposite charges to each other and an electrostatic interaction acts therebetween.

[0063] The average particle diameter of the phosphor-integrated particles is not particularly limited, but in consideration of ease of detection as a bright spot, etc., it is preferably 10 nm or more and 500 nm or less, and more preferably 50 nm or more and 200 nm or less.

[0064] Incidentally, the particle diameter of the phosphor-integrated particles can be measured by measuring the projected area of the phosphor-integrated particles using a scanning electron microscope (SEM) and converting it into an equivalent circle diameter. The average particle diameter and the coefficient of variation of a population composed of a plurality of phosphor-integrated particles are calculated using the particle diameters (equivalent circle diameters) calculated for a sufficient number (for example, 1000) of phosphor-integrated particles.

[0065] The above-mentioned drug may be any drug that can be supported by the gelatin particles. Examples of such drugs include proteins having pharmaceutical activity, plasmids, aptamers, antisense nucleic acids, ribozymes, tRNAs, snRNAs, siRNAs, shRNAs, ncRNAs, nucleic acids used for pharmaceutical purposes including condensed DNA, and antigens used for pharmaceutical purposes.

[0066] Examples of the above-mentioned proteins having pharmaceutical activity include steroids, non-steroidal anti-inflammatory drugs (NSAIDs), vitamin A (retinoids), vitamin D3 and vitamin D3 analogs, antibiotics, antiviral drugs, and antibacterial drugs.

[0067] The above-mentioned agent may be a water-soluble agent or a water-insoluble agent. Examples of water-insoluble agents include immunosuppressants such as cyclosporines including cyclosporine, immunostimulants such as rapamycin, anticancer agents such as paclitaxel, antiviral agents or antibacterial agents, anti-angiogenic agents, analgesics and anti-inflammatory agents, antibiotics, antiepileptic agents, anxiolytics, anti-narcotics, antagonists, neuron blockers, anticholinergic agents, antiarrhythmic agents, antihypertensive agents, hormonal agents, and nutritional agents.

[0068] Among these water-insoluble agents, rapamycin, paclitaxel, docetaxel, and everolimus are preferred. It should be noted that rapamycin, paclitaxel, docetaxel, and everolimus include their analogs and derivatives as long as they have similar drug effects. For example, paclitaxel and docetaxel are in an analog relationship, and rapamycin and everolimus are in a derivative relationship. Among these, paclitaxel is more preferred.

[0069] In addition to being supported on gelatin particles, the above-mentioned reagent or agent may be pre-coated on a substrate. However, when coated on a substrate and taken up by cells, there is likely to be variation in the ease of uptake by cells. Therefore, it is preferable to have a larger amount of loading (by mass) by gelatin particles than the coating amount on the substrate, and it is more preferable not to perform the coating on the substrate.

[0070] (Surface charge) According to the findings of the present inventors, when the total charge of the surface of the cell support having the above-mentioned biocompatible substance and the gelatin particles supported on the biocompatible substance, with which cells come into contact and adhere, is a positive charge, the uptake of gelatin particles is promoted. This is presumably because the surface of the cell is negatively charged, so when the total charge of the surface with which the above cells come into contact and adhere is a positive charge, the cells are likely to approach the substrate and the gelatin particles, and the cells are likely to take up the gelatin particles.

[0071] In addition, according to the findings of the present inventors, when the total charge of the surface contacted by the above cells is a positive charge, the culture efficiency and the observation efficiency are enhanced. It is considered that this is because when the total charge of the adhesion on the surface contacted by the above cells is a positive charge, the adhesion force of the cells to the substrate is increased.

[0072] In addition, it is preferable that Coulomb interaction occurs between the above biocompatible substance and the gelatin particles in a culture environment (a wet environment). Thereby, simply by imparting gelatin particles to the biocompatible substance, it is possible to suppress the detachment of the gelatin particles from the substrate in the culture environment. When the biocompatible substance has a positive charge, it is possible to make the cells approach the biocompatible substance more easily, and to further enhance the ease of uptake of the gelatin particles by the cells and the adhesion strength of the cells to the substrate.

[0073] More specifically, the zeta potential of the surface with which the above cells come into contact and adhere in a solution having a pH of 7.4 is preferably greater than 0 mV and equal to or less than 30 mV. When the above zeta potential is greater than 0 mV, the gelatin particles are easily taken up, and the culture efficiency and the observation efficiency are enhanced. When the above zeta potential is equal to or less than 30 mV, negative effects such as a decrease in the flexibility of the substrate and the gelatin particles can be suppressed. From the above viewpoints, the zeta potential is more preferably 2 mV or more and 30 mV or less, and even more preferably 4 mV or more and 15 mV or less.

[0074] The above zeta potential can be a value measured using a known zeta potential measuring device (for example, a measuring device using the formula described in Hiroyuki Mori, Yoshio Okamoto: Flotation, 27, 1171-124 (1980)).

[0075] To achieve the above state, in the present embodiment, it is preferable that the gelatin particles are cationized. The cationization of the gelatin particles is carried out by crosslinking or PEG-NH 2It can be carried out by a method of reducing the amount of carboxyl groups in the molecule, such as surface modification. In this embodiment, it is preferable that the biocompatible substance is cationized. The cationization of the biocompatible substance can also be carried out by a method of reducing the amount of acidic functional groups such as carboxyl groups, similar to the cationization of gelatin particles. The cationization of gelatin particles and the cationization of the biocompatible substance may be carried out only one of them, or both of them may be carried out. The greater the degree of these cationizations, the easier it is for the gelatin particles to be incorporated, and the higher the culture efficiency and the observation efficiency. On the other hand, in order to suppress a decrease in flexibility and the like due to an excessive degree of these cationizations, the cationization is preferably carried out to such an extent that the zeta potential of the surface with which the above cells come into contact and adhere becomes 30 mV or less.

[0076] [Cell culture method] The cell culture method according to another embodiment of the present invention is a cell culture method using the above-described cell support.

[0077] The cell culture according to this embodiment can be carried out in the same manner as a known cell culture method, except that the above-described cell support is used as the cell support.

[0078] Specifically, the cell culture method according to this embodiment may include a step of preparing the above-described cell support and a step of seeding cells on the cell support.

[0079] For the preparation of the cell support, a cell support that has already been prepared may be used, or a cell support may be prepared.

[0080] The production of the cell support can be carried out including a step of preparing a substrate containing a biocompatible substance and a step of retaining gelatin particles on the substrate.

[0081] The step of preparing a substrate containing a biocompatible substance is the step of preparing a substrate having the above-described shape and material. When the substrate is a molded body formed from a biocompatible substance, it is sufficient to prepare the molded body. Alternatively, a known substrate may be provided with a biocompatible substance and coated or the like. The biocompatible substance can be provided, for example, by applying a solution containing the biocompatible substance to the surface of the substrate and then drying the solution. When the substrate is a porous substrate having a three-dimensional shape or the like, it is not necessary to provide the biocompatible substance up to the inner surface of the substrate, and it is sufficient to provide it at least on the outer surface where cells of the substrate are seeded, or it may be provided only on the outer surface where cells of the substrate are seeded.

[0082] The step of retaining gelatin particles may be performed by applying a solution containing gelatin particles to a substrate containing a biocompatible substance. At this time, by heating to the temperature at which the gelatin particles solubilize (about 35°C to 45°C) and allowing to stand for about 1 hour, the gelatin particles can be fixed to the substrate and retained on the substrate by the adhesiveness exhibited by the solubilized gelatin particles. Thereafter, the solution may be dried.

[0083] At this time, the gelatin particles are fixed and retained at positions in contact with the biocompatible substance contained in the substrate. For example, when a biocompatible substance is provided on the surface of the substrate, the gelatin particles are provided and fixed in the region where the biocompatible substance is provided on the substrate. When the substrate is a porous substrate having a three-dimensional shape or the like, it is not necessary to provide and fix the gelatin particles up to the inner surface of the substrate, and it is sufficient to provide and fix them at least on the outer surface where cells of the substrate are seeded, or they may be provided and fixed only on the outer surface where cells of the substrate are seeded.

[0084] In addition, the gelatin particles carry a reagent or a drug. The gelatin particles can carry the reagent or the drug by a known method such as mixing the gelatin particles with these reagents or drugs or adding these reagents or drugs to the solution when preparing the gelatin particles.

[0085] At this time too, when the substrate is a porous substrate having a three-dimensional shape or the like, it is not necessary to apply and immobilize the gelatin particles up to the inner surface of the substrate. At least, it is sufficient that the gelatin particles are immobilized and held on the outer surface of the substrate where the cells are seeded.

[0086] Also, at this time, the total charge of the surface where the cells of the cell support come into contact and adhere becomes a positive charge, or a Coulomb interaction occurs between the biocompatible substance and the gelatin particles in the culture environment (humid environment), or the zeta potential in a solution where the pH of the surface where the cells come into contact and adhere is 7.4 is greater than 0 mV and 30 mV or less. The type of the biocompatible substance or the type of the gelatin particles may be selected. For example, a cationized biocompatible substance or cationized gelatin particles may be used to produce the cell support.

[0087] The seeding of the above cells may be performed by a normal method.

[0088] The cells to be seeded may be any cells that are desired to be cultured and stored as necessary. Examples of the above cells include cells derived from biological samples or specimens excised from various organs including bone marrow, heart, lung, liver, kidney, pancreas, spleen, intestinal tract, small intestine, heart valve, skin, blood vessel, cornea, eyeball, dura mater, bone, trachea, and ossicles, commercially available cell lines, and stem cells including skin stem cells, epidermal keratinocyte stem cells, retinal stem cells, retinal epithelial stem cells, cartilage stem cells, hair follicle stem cells, muscle stem cells, bone progenitor cells, adipose progenitor cells, hematopoietic stem cells, neural stem cells, liver stem cells, pancreatic stem cells, ectodermal stem cells, mesodermal stem cells, endodermal stem cells, mesenchymal stem cells, ES cells, and iPS cells, and known cells including cells differentiated from these stem cells. Further, the cells to be seeded may be cells of organisms other than animals, such as plants, fungi, protists, and bacteria.

[0089] At this time, a known medium may be applied to the cell retainer to promote the growth of the cells.

[0090] The seeded cells take in the gelatin particles held by the cell carrier by endocytosis, grow using the cell carrier as a scaffold, and form colonies. Then, the reagent or drug carried by the gelatin particles is gradually released from the gelatin particles taken into the cells. In this way, colonies of cells containing the reagent or drug are formed.

[0091] Conventionally, attempts have been made to introduce the above-mentioned reagent or drug into cells from a solution containing the reagent or drug, or to introduce the above-mentioned reagent or drug into cells by adding a dispersion containing gelatin particles carrying the reagent or drug to a culture medium and having the cells take in the gelatin particles. According to the findings of the present inventors, when the gelatin particles held by the above-mentioned cell carrier are taken into cells, the reagent or drug can be introduced more uniformly into a larger number of cells compared with these methods.

[0092] When the above-mentioned substrate is a porous substrate having a three-dimensional shape or the like, the cells may be seeded in the region of the surface of the cell carrier that holds the gelatin particles. The seeded cells take in the gelatin particles held by the cell carrier by endocytosis. Thereafter, when the cells that have taken in the gelatin particles grow while dividing and enter the inside of the substrate, the reagent or drug carried by the gelatin particles is also passed on to each of the divided cells, and it is considered that colonies of cells containing the reagent or drug are formed.

[0093] [Cell structure] The cell structure according to another embodiment of the present invention is a cell structure including the above-described cell support.

[0094] The above-mentioned cell structure has a substrate containing the above-described biocompatible substance and a plurality of cells directed to the above-mentioned cell support. The gelatin particles held by the substrate and the reagent or drug carried by the gelatin particles are taken into the cells.

[0095] In the above cell culture body, the above gelatin particles, or the above reagent or drug, are uniformly incorporated into more cells. For example,

[0096] The above cell culture body can be used for transplantation into a living body, storage of cells, etc. At this time, since the reagent or drug carried by the gelatin particles is uniformly incorporated into more cells, detection of the state such as cell differentiation and detection of cell life and death can be performed with higher sensitivity.

Example

[0097] Hereinafter, specific examples of the present invention will be described together with comparative examples, but the present invention is not limited thereto.

[0098] 1. Probe The following probes were used.

[0099] GAPDH-MB: A probe in which the 5'-end of a base sequence containing a sequence complementary to the mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is modified with AlexaFlour488 and the 3'-end is modified with IBRQ (lowa black RQ). GAPDH-MB is a molecular beacon in which the 5'-terminal site and the 3'-terminal site are complementary sequences constituting a stem region, and the site between them is a sequence constituting a loop structure.

[0100] It was previously confirmed that the fluorescence intensity from the above molecular beacon emits fluorescence only when reacting with the mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an mRNA constantly expressed in cells, and that the fluorescence intensity increases according to the amount of each mRNA.

[0101] 2. Experiment 1 2-1. Preparation of Gelatin Particles Gelatin (manufactured by Nitta Gelatin Inc., G-2613P) was dissolved in 24 ml of 0.1 M phosphate buffer aqueous solution (pH 5.0) at 37°C. An appropriate amount of ethylenediamine was added to this solution. Further, an aqueous hydrochloric acid solution was added to adjust the pH of the solution to 5.0. Further, an appropriate amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added, and the concentration of gelatin was adjusted to 2% by mass by adding 0.1 M phosphate buffer aqueous solution. This solution was stirred at 37°C for 4 hours to introduce ethylenediamine into the carboxyl groups of gelatin. Thereafter, the reaction product was dialyzed against redistilled water for 3 days to obtain a slurry-like cationized gelatin. Thereafter, acetone as a phase separation inducer was added and mixed at 50°C, and the particles precipitated in the slurry were collected and washed with pure water to obtain cationized gelatin particles. These cationized gelatin particles are designated as cGNS.

[0102] When the apparent average particle diameter of cGNS was determined by dynamic light scattering method at 37°C using DLS-7000 manufactured by Otsuka Electronics Co., Ltd., it was 168.0 nm. Also, when the zeta potential of cGNS was determined by electrophoresis light scattering method using DLS-8000 manufactured by Otsuka Electronics Co., Ltd., it was 8.41 mV.

[0103] 2-2. Loading of Molecular Beacon by Gelatin Particles cGNS and GAPDH-MB were mixed at room temperature for 15 minutes, and then centrifuged and washed with water to obtain gelatin particles carrying the above probe. These gelatin particles are designated as cGNS(GAPDH-MB).

[0104] 2-3. Retention of Gelatin Particles by Substrate As a two-dimensional substrate, a cell culture plate (manufactured by Thermo Fisher, 96well Edge Plate) was prepared. This cell culture plate was coated with gelatin (manufactured by Nitta Gelatin Co., Type B, isoelectric point 5).

[0105] cGNS (GAPDH-MB) was added to this substrate and allowed to stand at 37 °C for 1 hour to immobilize cGNS (GAPDH-MB), thereby obtaining a cell support.

[0106] 2-4. Cell culture on the cell support MC3T3-E1 cells, which are mouse mesenchymal stem cell lines, were seeded on the above cell support and cultured for 1 day.

[0107] 2-5. Observation Figure 2 is a fluorescence image observed 1 day after seeding. As is clear from Figure 2, uniform red fluorescence was observed throughout the plate. This is presumably because GAPDH-MB was uniformly incorporated into all the cells growing on the cell support and reacted with the mRNA of GAPDH in the cells to emit red fluorescence.

[0108] 3. Experiment 2 3-1. Preparation of gelatin particles and loading of molecular beacons onto gelatin particles Similar to Experiment 1, cationized gelatin particles cGNS were prepared. cGNS and GAPDH-MB were mixed at room temperature for 15 minutes, and then centrifuged and washed with water to obtain gelatin particles cGNS (GAPDH-MB) carrying the above probe.

[0109] 3-2. Retention of gelatin particles by the substrate Gelatin hydrogel particles were dispersed in PBS to prepare thermally crosslinked gelatin hydrogel particles (scaffold materials for spheroid formation) swollen with PBS. The particle size of this thermally crosslinked gelatin hydrogel particle was 32 - 53 mm. This thermally crosslinked gelatin hydrogel particle and gelatin particle cGNS (GAPDH-MB) were mixed and allowed to stand at 37 °C for 1 hour to immobilize cGNS (GAPDH-MB) on the thermally crosslinked gelatin hydrogel particle, thereby obtaining a cell support.

[0110] 3-3. Seeding of cells onto the cell support The above cell support and MC3T3-E1 cells, which are mouse mesenchymal stem cell lines, were introduced into a 96-well plate with a U-bottom coated with polyvinyl alcohol, and the cells were cultured for 3 days to form cell aggregates.

[0111] 3-4. Observation After seeding, the cell aggregates were fixed with 4% paraformaldehyde 3 days later, and frozen sections of the central plane of the cell aggregates were prepared. 4’,6-diamidino-2-phenylindole (DAPI) was added to this frozen section to stain the nuclei, and then it was observed with a fluorescence microscope.

[0112] Figure 3 is the fluorescence image observed at this time. As is clear from Figure 3, uniform red fluorescence was observed from the entire plate. This is presumably because GAPDH-MB was uniformly incorporated into the entire cells grown in the cell aggregates and reacted with the mRNA of GAPDH in the cells to emit red fluorescence.

[0113] 4. Experiment 3 4-1. Preparation of gelatin particles Similar to Experiment 1, cationized gelatin particles cGNS were prepared. cGNS and GAPDH-MB were mixed at room temperature for 15 minutes, and then centrifuged and washed with water to obtain gelatin particles cGNS (GAPDH-MB) carrying the above probe.

[0114] 4-2. Retention of gelatin particles by the substrate Gelatin non-woven fabric 1 that was not thermally crosslinked (manufactured by Nippon Woolen Co., Ltd., Genocel (Genocel is a registered trademark of the company)) was prepared. cGNS (GAPDH-MB) was added to this substrate and allowed to stand at 37°C for 1 hour to immobilize cGNS (GAPDH-MB) to obtain a cell support.

[0115] 4-3. Cell culture MC3T3-E1 cells, which are mouse mesenchymal stem cell lines, were seeded on the above cell support and cultured for 1 day. As a comparative example, MC3T3-E1 cells were seeded in a 6-well plate and cultured for 1 day in the presence of OptiMEM culture medium supplemented with cGNS (GAPDH-MB).

[0116] 4-4. Observation One day after seeding, the cultured cells were observed under a fluorescence microscope, and the imaging fluorescence intensity of 6 randomly captured fields was measured. The imaging fluorescence intensity was divided by the number of cells confirmed in the field to calculate the average fluorescence intensity per cell, and the standard deviation for each field was also calculated.

[0117] Table 1 shows the average fluorescence intensity per cell and the standard deviation as described above.

[0118]

Table 1

[0119] As shown in Table 1, when using the cell support, the average fluorescence intensity per cell was higher and the standard deviation was smaller. From this result, it was found that when using a cell support containing a biocompatible material and gelatin particles held on the above substrate, the uptake of molecular beacons (drugs or reagents) by cells was promoted and taken up more uniformly.

[0120] 5. Experiment 4 5-1. Preparation of Gelatin Particles Cationized gelatin particles cGNS were prepared in the same manner as in Experiment 1. At this time, the preparation conditions were changed to adjust 3 types of cGNS (cGNS1 to cGNS3) with different average particle diameters. Each of cGNS1 to cGNS3 was mixed with GAPDH-MB at room temperature for 15 minutes, and then centrifuged and washed with water to obtain gelatin particles cGNS1 (GAPDH-MB) to cGNS3 (GAPDH-MB) carrying the above probe.

[0121] 5-2. Retention of Gelatin Particles by Substrate An uncrosslinked gelatin nonwoven fabric (manufactured by Nippon Woolen Co., Ltd., Genocel) was prepared. Each of cGNS1 (GAPDH-MB) to cGNS3 (GAPDH-MB) was added to this substrate and allowed to stand at 37 °C for 1 hour to immobilize cGNS1 (GAPDH-MB) to cGNS3 (GAPDH-MB), obtaining cell supports 1 to 3.

[0122] 5-3. Cell Culture MC3T3-E1 cells, which are mouse mesenchymal stem cell lines, were seeded on each of cell supports 1 to 3 and cultured for 1 day.

[0123] 5-4. Observation After seeding, the cells cultured after 1 day were observed with a fluorescence microscope, and the imaging fluorescence intensity of 6 randomly captured fields was measured. The imaging fluorescence intensity was divided by the number of cells confirmed in the field to calculate the average fluorescence intensity per cell.

[0124] Table 2 shows the average fluorescence intensity per cell number described above.

[0125]

Table 2

[0126] As shown in Table 2, it was found that the larger the average particle diameter of the gelatin particles, the easier it is for the gelatin particles and the molecular beacons (drugs or reagents) supported on the gelatin particles to be taken into the cells by the cells' own activities.

[0127] 6. Experiment 5 6-1. Preparation of Gelatin Particles Similar to Experiment 1, cationized gelatin particles cGNS were prepared. cGNS and GAPDH-MB were mixed at room temperature for 15 minutes, and then centrifuged and washed with water to obtain gelatin particles cGNS (GAPDH-MB) carrying the above probe.

[0128] 6-2. Retention of Gelatin Particles by Substrate As three-dimensional shaped substrates, uncrosslinked gelatin nonwoven fabric 1 (manufactured by Nippon Woolen Co., Ltd., Genocel), crosslinked gelatin nonwoven fabric 2 (manufactured by Nippon Woolen Co., Ltd., Genocel, heat treatment time 4 - 24 hours), and nonwoven fabric made of polypropylene (manufactured by Toray Industries, Inc., polypropylene long fiber nonwoven fabric) were prepared. Regarding the polypropylene nonwoven fabric, substrates with gelatin coating and substrates without gelatin coating were prepared.

[0129] cGNS (GAPDH-MB) was added to these substrates and allowed to stand at 37°C for 1 hour to immobilize cGNS (GAPDH-MB), thereby obtaining cell supports.

[0130] 6-3. Seeding of Cells onto Cell Supports MC3T3-E1 cells, which are mouse mesenchymal stem cell lines, were seeded onto the above cell supports and cultured for 1 day.

[0131] 6-4. Observation After seeding, PBS containing CYTO13 was added to the cell supports 1 day later to stain the nuclei, and the mixture was allowed to stand at 37°C for 1 hour. Then, each cell support was imaged in a glass bottom dish and observed with a confocal laser microscope.

[0132] Figure 4A is a fluorescence image observed from the cell support using gelatin nonwoven fabric 1. Figure 4B is a fluorescence image observed from the cell support using gelatin nonwoven fabric 2. Figure 4C is a fluorescence image observed from the cell support using the polypropylene nonwoven fabric coated with gelatin. 。

[0133] As is clear from FIGS. 4A to 4C, uniform red fluorescence was observed throughout the plate. This is presumably because GAPDH-MB was uniformly incorporated into all of the cells grown into cell aggregates, reacted with the mRNA of GAPDH in the cells, and emitted red fluorescence. Also, the fluorescence intensity differed depending on the type of substrate. From this result, it was found that the introduction rate of GAPDH-MB differed depending on the state of the substrate, and it was found that the introduction rate of GAPDH-MB could be changed by changing the state of the substrate.

[0134] On the other hand 、 raw When cells were cultured with cGNS (GAPDH-MB) immobilized on a substrate without applying a biocompatible substance, no red fluorescence was observed. From this result, it was found that the presence of a biocompatible substance in the substrate promoted the uptake of cGNS (GAPDH-MB).

[0135] 7. Experiment 6 7-1. Preparation of gelatin particles In the same manner as in Experiment 1, cationized gelatin particles cGNS were prepared. cGNS and GAPDH-MB were mixed at room temperature for 15 minutes, and then centrifuged and washed with water to obtain gelatin particles cGNS (GAPDH-MB) carrying the above probe.

[0136] 7-2. Retention of gelatin particles by the substrate As substrates, a well plate made of polypropylene (PP), a non-woven fabric made of polypropylene (PP) (manufactured by Toray Industries, Inc., polypropylene long fiber non-woven fabric), a well plate coated with polyvinyl alcohol (PVA), a well plate coated with gelatin, and a gelatin non-woven fabric (manufactured by Nippon Woolen Co., Ltd., Genocel) were prepared. Each of cGNS (GAPDH-MB) was added to these substrates and allowed to stand at 37° C. for 1 hour to fix cGNS1 (GAPDH-MB), which were used as cell supports 1 to 5, respectively.

[0137] 7-3. Cell culture MC3T3-E1 cells, which are mouse mesenchymal stem cell lines, were seeded on each of cell supports 1 to 5, and the cells were cultured for 1 day.

[0138] 7-4. Observation After seeding, the cells cultured 1 day later were observed with a fluorescence microscope, and the imaging fluorescence intensity of 6 randomly captured fields was measured. The imaging fluorescence intensity was divided by the number of cells confirmed in the field to calculate the average fluorescence intensity per cell.

[0139] Table 3 shows the average fluorescence intensity per cell number described above.

[0140]

Table 3

[0141] As shown in Table 3, when the substrate contained a biocompatible substance, the average fluorescence intensity per cell was high. From this result, it was found that the incorporation of cGNS (GAPDH-MB) was promoted by the substrate having a biocompatible substance.

[0142] 8-1. Experiment 7 In the same manner as in Experiment 1, cationized gelatin particles cGNS were prepared. cGNS and GAPDH-MB were mixed at room temperature for 15 minutes, and then centrifuged and washed with water to obtain gelatin particles cGNS (GAPDH-MB) carrying the above probe.

[0143] 8-2. Retention of Gelatin Particles by Substrate As substrates, an unthermally crosslinked gelatin nonwoven fabric (manufactured by Nippon Woolen Co., Ltd., Genocel), a thermally crosslinked gelatin nonwoven fabric 2 (manufactured by Nippon Woolen Co., Ltd., Genocel, heat treatment time: 4 hours), and a thermally crosslinked gelatin nonwoven fabric 3 (manufactured by Nippon Woolen Co., Ltd., Genocel, heat treatment time: 24 hours) were prepared. Also, a cationized gelatin nonwoven fabric 4 obtained by introducing amino groups into an unthermally crosslinked gelatin nonwoven fabric (manufactured by Nippon Woolen Co., Ltd., Genocel), and an anionic gelatin nonwoven fabric 5 obtained by introducing succinic anhydride into the amino groups of gelatin in an unthermally crosslinked gelatin nonwoven fabric (manufactured by Nippon Woolen Co., Ltd., Genocel) were prepared. Each of cGNS (GAPDH-MB) was added to these substrates, and they were allowed to stand at 37°C for 1 hour to immobilize cGNS1 (GAPDH-MB), which were used as cell supports 5 to cell support 9, respectively. Note that cell support 5 is the same as cell support 5 in Experiment 6.

[0144] The zeta potential of cell supports 5 to 7 in a solution with a pH of 7.4 was measured using a nanoparticle analyzer nanoPartica SZ-100V2 (product name) manufactured by Horiba, Ltd.

[0145] 8-3. Cell culture MC3T3-E1 cells, which are mouse mesenchymal stem cell lines, were seeded on each of cell supports 1 to 5, and the cells were cultured for 1 day.

[0146] 8-4. Observation One day after seeding, the cultured cells were observed with a fluorescence microscope, and the imaging fluorescence intensity of 6 randomly captured fields was measured. The imaging fluorescence intensity was divided by the number of cells confirmed in the field to calculate the average fluorescence intensity per cell.

[0147] Table 4 shows the zeta potential of each cell support and the average fluorescence intensity per cell number described above.

[0148]

Table 4

[0149] As shown in Table 4, it was found that the incorporation of gelatin particles was promoted by anionizing the biocompatible substance contained in the substrate and adjusting the zeta potential of the cell support.

[0150] 9. Experiment 8 9-1. Preparation of gelatin particles In the same manner as in Experiment 1, cationized gelatin particles cGNS were prepared. cGNS and GAPDH-MB were mixed at room temperature for 15 minutes, and then centrifuged and washed with water to obtain gelatin particles cGNS(GAPDH-MB) carrying the above probe.

[0151] 9-2. Preparation of substrates An uncrosslinked gelatin nonwoven fabric (Genocel, manufactured by Nippon Woolen Co., Ltd.) was prepared. cGNS(GAPDH-MB) was added to this substrate and allowed to stand at 37°C for 1 hour to immobilize cGNS(GAPDH-MB), obtaining a cell support.

[0152] As a comparative example, a drug (paclitaxel) dissolved in a solvent (anhydrous ethanol) was applied to a substrate (gelatin nonwoven fabric (Genocel, manufactured by Nippon Woolen Co., Ltd.)), dried, and a substrate coated with the drug was prepared.

[0153] 9-3. Cell culture SK-BR-3 cells, which are a human breast cancer cell line, were seeded on each of the above-prepared cell support and the substrate coated with the drug, and the cells were cultured for 24 to 48 hours.

[0154] 9-4. Observation Cell aggregates were fixed with 4% paraformaldehyde, and frozen sections of the central plane of the cell aggregates were prepared. 4’,6-diamidino-2-phenylindole (DAPI) was added to this frozen section to stain the nuclei, and then observed with a fluorescence microscope, and the imaging fluorescence intensity of 6 randomly captured fields was measured. The imaging fluorescence intensity was divided by the number of cells confirmed in the field to calculate the average fluorescence intensity per cell, and the standard deviation for each field was also calculated.

[0155] Table 5 shows the average fluorescence intensity and standard deviation per the above cell count.

[0156]

Table 5

[0157] As shown in Table 5, when using the cell support, the standard deviation was smaller. In contrast, when only coating the drug on the substrate, it can be seen that the drug was sufficiently taken up by the cells from the average fluorescence intensity per cell. However, on the other hand, it was found that the standard deviation was large and there was variation in the uptake of the drug by the cells. From this result, it was found that when using a cell support containing a substrate containing a biocompatible substance and gelatin particles held on the substrate, the drug or reagent was taken up more uniformly by the cells.

[0158] This application claims priority based on International Application No. PCT / JP2020 / 035197 filed on September 17, 2020, and the contents described in the claims, specification, and drawings of that application are incorporated herein by reference.

Industrial Applicability

[0159] According to the present invention, a cell structure in which a reagent or drug is taken up more uniformly by a larger number of cells can be obtained. This cell structure can be suitably used for transplantation and cell storage.

Explanation of Signs

[0160] 100 Cell support 112 Substrate containing a biocompatible substance 114 Substrate such as glass or plastic 116 Biocompatible substance 120 Gelatin particles 130 Cell

Claims

1. A substrate containing a biocompatible substance, or a substrate with a biocompatible substance applied to its surface, and gelatin particles carrying a reagent or drug, held by the biocompatible substance of the substrate, wherein a Coulomb interaction occurs between the biocompatible substance and the gelatin particles in a culture environment, and the total charge of the surface to which cells attach is a positive charge, a cell support.

2. The cell support according to claim 1, wherein the substrate is a molded body of a biocompatible substance.

3. The cell support according to claim 1 or 2, wherein the substrate has a molded body and a biocompatible substance applied to the surface of the molded body.

4. The cell support according to any one of claims 1 to 3, wherein the biocompatible substance is a biopolymer derived from a living body or a biodegradable synthetic polymer.

5. The cell support according to claim 4, wherein the biocompatible substance is at least one biocompatible substance selected from the group consisting of collagen, gelatin, fibronectin, polyvinyl alcohol, and copolymers thereof.

6. The cell support according to any one of claims 1 to 5, wherein the zeta potential of the surface to which cells attach in a solution having a pH of 7.4 is greater than 0 mV and not more than 30 mV.

7. The cell support according to any one of claims 1 to 6, wherein the zeta potential of the surface to which cells attach in a solution having a pH of 7.4 is 4 mV or more and 15 mV or less.

8. The cell support according to any one of claims 1 to 7, wherein the biocompatible substance is cationized.

9. The cell support according to any one of claims 1 to 8, wherein the gelatin particles are cationized.

10. The cell support according to any one of claims 1 to 9, wherein the biocompatible substance is a material selected according to the introduction rate of the gelatin particles to be achieved for the cells supported by the cell support.

11. The cell support according to any one of claims 1 to 10, wherein the substrate has a two-dimensional shape.

12. The cell support according to any one of claims 1 to 11, wherein the substrate has a three-dimensional shape.

13. The cell support according to any one of claims 1 to 12, wherein the reagent or drug is a molecular beacon.

14. A step of preparing a substrate containing a biocompatible substance, A step of applying gelatin particles carrying a reagent or a drug to the substrate to hold the gelatin particles on the substrate; having; By selecting the substrate or the gelatin particles, a Coulomb interaction is generated between the biocompatible substance and the gelatin particles in a culture environment, and the total charge of the surface to which cells adhere is made positive; A method for manufacturing a cell support.

15. The method for manufacturing a cell support according to claim 14, wherein the zeta potential of the surface to which cells adhere is made greater than 0 mV and 30 mV or less by selecting the substrate or the gelatin particles.

16. The method for manufacturing a cell support according to claim 14 or 15, wherein the zeta potential of the surface to which cells adhere is made 4 mV or more and 15 mV or less by selecting the substrate or the gelatin particles.

17. The method for manufacturing a cell support according to any one of claims 14 to 16, wherein the substrate contains the cationized biocompatible substance.

18. The method for manufacturing a cell support according to any one of claims 14 to 17, wherein the gelatin particles are cationized gelatin particles.

19. The method for manufacturing a cell support according to any one of claims 14 to 18, wherein the biocompatible substance is a material selected according to the introduction rate of the gelatin particles to be achieved for the cells supported by the cell support.

20. A step of preparing a cell support according to any one of claims 1 to 13; A step of seeding cells on the cell support; A method for culturing cells having.

21. A cell support according to any one of claims 1 to 13; Cells held on the cell support; A cell structure having.

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