JP4148516B2 - Cell culture kit comprising cell culture substrate and bioactive substance transfer sheet, production method thereof, and cell screening method using the same - Google Patents

Description

  The present invention relates to the function of one or more kinds of biologically active substances having a physiologically active action on cells, or to identify the effect of a combination of two or more kinds of biologically active substances, or screening of physiologically active substances using cells. The present invention relates to a cell culture kit comprising at least a cell culture substrate and a bioactive substance transfer sheet that can be used in the above, a production method thereof, and a cell screening method using the kit.

  In recent years, research on culturing animal and plant cells under various conditions, or research on products by specific cultured cells, has been actively carried out, and in particular, synthesis is impossible artificially or synthesis is extremely difficult. The production of difficult substances using specific cellular activities has been studied in many ways. In addition, research has been conducted to identify substances that affect cell growth and differentiation, and to grow and differentiate desired cells according to the purpose. With rapid progress in cell engineering and medical engineering, The ultra-small biosensors, artificial organs, and neurocomputers that have been used are attracting attention, and active research activities are being conducted to apply them.

  However, as described above, in order to use cells in vitro, it is indispensable to arrange cells as desired and control their growth, differentiation and substance production. The mechanism for controlling substance production has not been fully elucidated, and it is extremely difficult to cultivate cells while controlling cells from this perspective, and this is a major obstacle to the progress of research and development using cells as described above. Yes. In addition, the concept of tailor-made medicine, which takes into account the problems caused by individual differences in drug sensitivity, has been widely recognized in recent years, and the need for it has increased. In many cases, the effect of a substance having a substance is studied only on the function of each substance, and an effective method for easily examining the presence / absence, required amount, and combination effect of a plurality of drugs has not been established.

  As an attempt to control the arrangement of cells, there is an example in which a cell adhesion protein is applied using an ink jet printer to form a pattern, and cells are cultured thereon, as in US Pat. No. 5,108,926. In this method, cells can be cultured on a pattern coated with a cell adhesion protein, but the cells cannot be screened by controlling their growth / differentiation and substance production. In “Proteins / Nucleic Acids / Enzymes”, Volume 45, 727-734 (2000), cell growth factors that affect cell proliferation / differentiation are immobilized on a substrate using photolithography technology. We are studying the effects on differentiation. However, a substrate on which cell growth factors are immobilized is not used as a screening means for cells, and in the photolithography method, these biological materials that are present in a small amount in the living body are wasted, and the processes such as exposure and development must be repeated. The manufacturing process must be complicated.

  In Japanese translation of PCT publication 2000-512009, a method for screening a cell by immobilizing a substance that affects cell adhesion on a substrate has been proposed. Here, the reactive functional group provided on the substrate and the cell adhesive substance are immobilized by a divalent crosslinking reagent. Photolithography is used to bind the reactive functional group and the cell adhesive substance, which not only causes the same problems as described above, but also immobilizes multiple cell adhesive substances when immobilizing them. It is extremely difficult to avoid that a substance to be immobilized and a substance to be newly immobilized are bound with a divalent cross-linking reagent at an undesired position, and it is difficult to place a cell adhesive substance at a desired position. It is extremely difficult. In addition, substances that affect proliferation, differentiation, and substance production are not immobilized, but cells are immobilized in each well with an adhesive substance, and the substances produced by the cells in the process of being cultured in a culture solution are captured. In order to screen for substances that affect at least one of cell adhesion, proliferation, differentiation, survival, maintenance of undifferentiated state, cell death, and substance production as in the present invention. It is not a thing.

  JP 2002-355025 A is characterized in that a plurality of cell screening substances are arranged and fixed in a desired region on the base by a droplet discharge means, and have a plurality of regions having different cell screening functions. A method for forming a cell screening substrate is disclosed. In this invention, since the cell screening substance is all immobilized on the screening substrate in the culture solution, the cell screening substance cannot often be taken into the cells. Therefore, the present invention is effective except when screening cells that affect at least one of proliferation, differentiation, survival, maintenance of undifferentiated state, cell death, and substance production by incorporating a cell screening substance into cells. This is a good proposal.

  Japanese Patent Laid-Open No. 2002-328124 aims at screening high-order combinations of biologically active substances. In the invention, however, the effect on the action of biologically active substances previously applied to a substrate is observed. It was not possible to screen for the effects of biologically active substances acting in an immobilized state on the extracellular matrix in vivo and the effects caused by sequential addition of biologically active substances in the cell culture process.

JP-A-2003-33177 discloses a cell array divided into a plurality of regions in order to easily assay various chemical substances such as drugs and toxicants, and bioactive substances are added to the obtained regions. At the same time, multiple samples are screened. However, in the present invention, since the biologically active substance is applied to the cells in culture using the dispensing means, the risk of contamination in the process. Therefore, it is necessary to construct a device for applying a bioactive substance and a dedicated bioactive substance, and it cannot be used simply.
U.S. Pat.No. 5,108,926 Special Table 2000-512009 JP 2002-355025 JP JP 2002-328124 A JP 2003-33177 A "Protein, Nucleic Acid, Enzyme", 45, 727-734 (2000)

  Accordingly, an object of the present invention is to solve the problems of the above-described conventional examples, and to produce a cell culture kit that can simultaneously observe the effects of a plurality of biologically active substances in an immobilized state and a dissolved state by a simple process, and its production It is to provide a method and a screening method using the method, to further develop research such as cell engineering, and to provide a base technology for producing various devices using cells. Another object of the present invention is to provide a method for screening a substance that affects at least one of all biologically active substances having an action on cells using these cell culture kits. Furthermore, the object of the present invention is to provide a method for screening useful biologically active substances using cells.

The cell culture kit according to the present invention comprises:
A cell culture kit comprising: a substrate having a culture region for culturing cells; and a sheet medium that can be placed on the substrate and has a covering region of the cell region,
Both the culture region and the covering region of the sheet medium with respect to the culture region have one or more types of biologically active substances having physiological activity with respect to cells, and the biologically active substance that one of these areas has is the region. Fixed inside ,
A film in which the sheet medium is stretchable or flexible at least in the covering region
It is a kit for cell culture characterized by having the part which consists of .
A cell culture kit comprising a substrate having a culture region for culturing cells, and a sheet medium that can be placed on the substrate and has a covering region of the cell region,
Both the culture region and the covering region of the sheet medium with respect to the culture region have one or more types of biologically active substances having physiological activity with respect to cells, and the biologically active substance that one of these areas has is the region. Fixed inside,
A support layer for supporting a bioactive substance is partially or entirely formed on the sheet medium.
A cell culture kit.

  This cell culture kit may have two or more combinations of the culture region and the covering region of the culture region.

  Further, in this cell culture kit, a biologically active substance is fixed in the culture area, and the biologically active substance adheres to the covering area with respect to the culture area so as to be releasably attached to the culture liquid. The sheet medium may be a sheet for transferring a biologically active substance to the culture region.

  A method for producing a cell culture kit according to the present invention is a method for producing a cell culture kit having the above-described configuration, wherein at least a liquid discharge means is used for imparting a biologically active substance to the culture region and the covering region. A method for producing a cell culture kit. As the liquid ejecting means, a thermal ink jet method or a piezo ink jet method can be suitably used.

  The cell screening method according to the present invention is a cell screening method using the cell culture kit having the above-described configuration, wherein the sheet medium is placed on the substrate, and the culture region of the substrate corresponds to the culture region. A step of culturing the cell by contacting the cell with a biologically active substance immobilized on one of these regions in a culture solution disposed in the cell region and attaching to the other of these regions. A cell screening method comprising the step of supplying a biologically active substance to a culture solution.

  According to the kit for cell culture of the present invention, the biologically active substance can be surely arranged at a desired position by a simple process, and by using this, the effect of various cell physiologically active substances can be screened.

  Specifically, according to the screening using the cell culture kit according to the present invention, as a result of the screening, for example, necessary for cell proliferation / differentiation, survival and maintenance of undifferentiated state, cell death or substance production. Factors can be identified and a method for efficiently culturing cells can be determined. In addition, not only solid phase but also liquid phase screening substances can be used, so it is possible to screen in a state closer to the living body by combining a biologically active substance in an immobilized state and a biologically active substance in a dissolved state. It is. Furthermore, it becomes possible to examine the difference in effect between a solid phase and a liquid phase or a combination thereof, and to evaluate human sensitivity to, for example, drugs and endocrine disrupting substances called environmental hormones. Furthermore, based on these evaluation results, it becomes possible to determine each person's diagnostic method for various diseases. In addition, it is possible to screen for useful biologically active substances having physiological activity against cells.

  In addition, since the cell culture substrate and the bioactive substance transfer sheet can use the inkjet method as a droplet discharge means, various cell screening substances can be simultaneously applied to the cells by changing the concentration by controlling the number of droplets. It is possible to act on. In addition, since several types of cell screening substances can be mixed and transferred to the cell culture medium with high accuracy, the effects on cell proliferation / differentiation and survival by multi-component biologically active substances, which has been difficult in the past, can be accurately understood. In addition, sequential addition of bioactive substances and changes to different combinations are easy. Therefore, screening for effects caused by these biologically active substances can be simplified and can be an effective means. Further, since it can be easily produced, it can be produced in large quantities, stably stored as a transfer sheet for cell screening, and used as appropriate.

  The cell culture kit of the present invention comprises at least a cell culture substrate having a culture region (compartment) divided into a plurality of compartments, and a sheet medium having a cover region covering each culture region. ing. At least one bioactive substance having a physiological activity on cells is fixed to one of the culture area and the corresponding covering area, and at least one bioactive substance is releasably fixed to the culture medium on the other side. . That is, the bioactive substance may be fixed to the substrate side, or may be fixed to the sheet medium side, and when it is fixed to the substrate side, the bioactive substance on the sheet medium side can be released into the culture medium. When the substrate is fixed to the sheet medium side, the bioactive substance on the substrate side is fixed to the culture solution in a releasable state.

  Hereinafter, the present invention will be described for the case where the biologically active substance on the sheet medium side is releasably fixed to the culture medium and used as a biologically active substance transfer sheet. However, in the configuration described below, the biologically active substance is fixed on the sheet medium side. What can be applied in this case is not limited to the configuration in which the bioactive substance is fixed to the substrate side, but can be applied to the case where the bioactive substance is fixed to the sheet medium side.

  A kit for immobilizing a biologically active substance on the substrate side is a cell culture substrate in which one or more kinds of biologically active substances are arranged and immobilized in a desired region, and one or more kinds of biologically active substances are sheets. It has a bioactive substance transfer sheet arranged in a plurality of areas on the medium.

  In the cell culture substrate, the combination of biologically active substances immobilized in each compartment may differ depending on the compartment. Furthermore, the density of the biologically active substance immobilized on each compartment of the cell culture substrate may vary depending on the compartment. In addition, each section of the cell culture substrate may have a plurality of regions having different cell screening functions by arranging and fixing a plurality of biologically active substances in desired regions. A group of regions formed from two or more regions may be formed in the recess. A group of regions formed from two or more regions may be surrounded by a convex wall-shaped structure.

  In the bioactive substance transfer sheet, it is preferable that a plurality of bioactive substances can be released from the sheet medium. Furthermore, a plurality of regions with different combinations of bioactive materials may be included in the plurality of regions of the bioactive material transfer sheet. Furthermore, in the bioactive substance transfer sheet, the plurality of areas may include a plurality of areas having different densities of bioactive substances. In the bioactive substance transfer sheet, the sheet medium may be a stretchable or flexible film at least in a region holding the bioactive substance. Furthermore, in the bioactive substance transfer sheet, a support layer for supporting the bioactive substance may be partially or entirely formed on the sheet medium. In the bioactive substance transfer sheet, each region may be formed in a recess. In the bioactive substance transfer sheet, each region may be formed in a convex portion formed on the sheet. In the bioactive substance transfer sheet, each region or a group of regions formed from two or more regions may be surrounded by a convex wall-shaped structure.

  In addition, the production of the cell culture substrate and the bioactive substance transfer sheet can be achieved by releasing each bioactive substance by using droplet discharge means, a plurality of sections of the cell culture substrate, a plurality of areas in each section, or / and release. It can be carried out by a method comprising the step of placing in a plurality of regions on a possible bioactive substance transfer sheet medium. Further, as a method for producing the cell culture substrate and the biologically active substance transfer sheet, a thermal ink jet method or a piezo ink jet method can be used as the droplet discharge means. Further, in order to immobilize the biologically active substance on the cell culture substrate, energy for immobilization may be applied from the outside.

  The cell screening method in the case of using a bioactive substance transfer sheet includes culturing cells in a culture solution in contact with the bioactive substance fixing region of the cell culture substrate, and further, living cells on the bioactive substance transfer sheet. A step of bringing the culture solution into contact with the active substance region, allowing the culture solution to freely dissolve in the culture solution, and culturing the cells; The cell screening method may include a step of replenishing a substance necessary for screening of the cells into a culture solution in contact with the region. The cell screening method may have a step of replenishing substances necessary for screening the cells by using a plurality of biologically active substance transfer sheets. In this case, the bioactive substance transfer sheet may be exchanged during culture, and the same bioactive substance may be replenished to the same culture compartment, or the culture conditions may be changed by exchanging with a different sheet and adding a different bioactive substance. Can also be done. This change can be made as many times as necessary. In addition, by using a biologically active substance transfer sheet in which a biologically active substance is disposed on a sustained-release support layer, all or part of the culture is performed, and the sheet is brought into contact with the culture medium for a period of time. You may have the process of culture | cultivating, providing an active substance with respect to a cell gently.

The screening in the cell screening method according to the present invention can be performed based on at least one of the following items, for example.
(1) Evaluate cell shape change in a desired region on the substrate.
(2) Quantification of substances taken into cells in a desired region on the substrate.
(3) The substance synthesized in the cell in a desired region on the substrate is quantified.
(4) The cells are stained for morphology evaluation and substance quantification.
(5) A signal from the reporter gene is detected.
(6) Evaluate by cytoblot assay.
(7) Quantify substances by measuring radiation dose.
(8) The substance is quantified by measuring the amount of fluorescence.
(9) The substance is quantified by measuring the amount of luminescence.
(10) The substance is quantified by measuring the absorbance.

  Hereinafter, the present invention will be described in detail with reference to more preferred embodiments.

  An example of the cell culture kit of the present invention will be described. As shown in FIG. 1 (A), the cell culture substrate 1 has one or more (three types in FIG. 1) biologically active substances 12 desired on a base (substrate) 11 divided into a plurality of sections. Arranged in the compartments, each bioactive substance 12 is immobilized on a base 11. On the other hand, in the bioactive substance transfer sheet 2, two or more kinds of bioactive substances 22 are arranged on the sheet 21 shown in FIG. 1B at desired positions corresponding to the respective sections of the cell culture substrate. In addition, as shown in FIG. 1C, the bioactive substance 22 can be disposed on the convex portion 23 formed on the sheet in advance. For the arrangement, the droplet discharge means 3 can be used. When cells are cultured using the cell culture substrate 1, the biologically active substance transfer sheet is covered with the cell culture substrate 1 before or after the start of the culture, as shown in FIG. 2 (A). The biologically active substance 12 immobilized on the cell culture substrate and the biologically active substance transfer sheet are formed by pressing down the sheet from the back and bringing the sheet into contact with the culture solution 4 to freely dissolve the biologically active substance in the culture solution. By causing the biologically active substance 22 arranged above to act on the cells, it is possible to see higher-order effects due to the combination of the immobilized and dissolved biologically active substances (12, 22). In addition, when a sheet (FIG. 1C) having an arrangement region of the bioactive substance 22 on the convex portion 23 formed on the above-described sheet is used, as shown in FIG. It is possible to bring the culture solution 4 into contact with the region where the biologically active substance is placed and dissolve it by simply covering the cell culture substrate 1 with 2.

  As the cells that can be used in the present invention, any prokaryotic and eukaryotic cells can be used. For example, bacterial cells, yeast cells, neurons, fibroblasts, smooth muscle cells, cardiomyocytes, skeletal muscles Whole cells such as cells, glial cells, embryonic stem cells, hematopoietic stem cells, mast cells, adipocytes, protozoan cells, neural stem cells, and immune cells including T cells and B cells (eg, transformed or non-transformed cells) ); Cell masses can be appropriately selected and used.

  In the present invention, the bioactive substances 12 and 22 are used for adhesion of cells to the base 11, cell proliferation, differentiation, survival and maintenance of undifferentiated state, cell death, substance production, or intracellular signal transduction. Refers to substances that control cultures that affect gene expression, etc., and include substances that bind to cells in addition to extracellular matrix proteins, antibodies that specifically bind to the cell surface, cytokines, or cells Included are chemical substances that are taken into cells and maintain cell growth, differentiation, survival and undifferentiated state, cell death, substance production, inhibition of enzyme activity, or intracellular signaling, gene expression, etc. . These substances can be freely selected to be fixed to the base 11 or arranged on the sheet 21 in consideration of the action on the cells and the localization in the cells.

  For example, examples of the extracellular matrix protein include collagen, fibronectin, laminin and the like. Cytokines include those called cell growth factors and hormones. Cell growth factors include nerve growth factor (NGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), Examples include osteogenic factor (BMP), insulin-like growth factor (IGF), and tumor necrosis factor (TNF). Examples of hormones include peptide insulin and calcitonin, steroidal aldosterone and progesterone, and amino acid derivative epinephrine and thyroxine. Other chemical substances include substances such as allergens that cause allergies, and various chemical substances called so-called endocrine disruptors. In addition, antibodies that can bind to the above-described substances and examine the localization of the substance in cells, and neutralizing antibodies that suppress the function thereof can be used.

  In addition, for the bioactive substances 12 and / or 22, a combination of the bioactive substances 12 and / or 22 arranged by a region on the base 11 and the sheet 21 or a region group formed of two or more regions is used. May be different. This makes it possible to see at least one difference in the effect on the cells by the combination of the bioactive substances 12 and 22.

  Moreover, about the bioactive substance 12 and / or 22, the density of the bioactive substance 12 and 22 arrange | positioned by the area | region group formed from the area | region on the bases 11 and / or 21, or two or more area | regions. May be different. Thereby, the difference in the bioactive effect on the cells due to the change in the density of the bioactive substance 12 and / or the difference in the concentration in the culture medium 22 can be seen in more detail. One of the great advantages of using the droplet discharge means is that the biologically active substance can be easily arranged at an arbitrary ratio in one immobilization region as described above.

  The immobilization of the biologically active substance 12 on the base 11 may use biological affinity through covalent bonds, electrostatic attraction, or the like. When immobilized on the base 11 via a covalent bond, the biologically active substance 12 can be immobilized with a strong force, and the binding force is not easily affected by cells, culture medium, or the like. It is fixed.

  The base 11 and the sheet 21 may be of any material and shape as long as they can stably fix and carry the bioactive substances 12 and 22. Specifically, a glass substrate, a plastic plate, a plastic sheet, a polymer film, paper, or the like can be preferably used. Furthermore, the base 11 and the sheet 21 may be transparent, light-shielding or colored. Further, in order to immobilize the bioactive substance 12 on the base 11, or to improve the stability of the bioactive substances 12 and 22 on the base 11 and the sheet 21, a part or the entire surface of the base 11 is used. May be treated with a chemical substance, or may be irradiated with radiation. In addition, an adhesive or a water repellent may be applied or printed in a region where the bioactive substance is not disposed.

  The sheet 21 can be a stretchable film. For example, synthetic rubber such as silicon rubber, natural rubber, latex, polyolefin film such as polyethylene, polymethylpentene, paraffin film and the like are preferably used. The wall-like structure formed on the cell culture substrate is closely attached so as to surround the region on the sheet 21 where the biologically active substance is disposed, and is pushed down from the back surface until the region contacts the culture solution. The substance can be freely dissolved in the culture solution. In this case, stretchability, elasticity, or flexibility is preferable because it has the advantage that the liquid is hardly leaked because the adhesion is good.

  Further, the bioactive substance 22 on the sheet 21 can be subjected to chemical treatment or electrostatic treatment on the bioactive substance or the sheet so that it can be easily released from the sheet. As a method for transferring the bioactive substance from the sheet 22 to the culture solution, the sheet 22 is brought into close contact with the wall-like structure formed on the cell culture substrate 1, and the culture solution is brought into contact with the region where the bioactive substance is disposed. As described above, the cell culture substrate and the sheet may be shaken, or a rod-shaped protrusion may be pressed from the back of the sheet to bring the region where the bioactive substance is disposed into contact with the culture solution (FIG. 2A). ). Moreover, when releasing, in order to perform releasing efficiently, you may apply a vibration to a culture solution or a sheet | seat. Moreover, the bioactive substance transfer sheet can form a support layer for supporting the bioactive substance on the sheet medium partially or entirely. This makes it possible to stably hold the biologically active substance. The sheet may have a recess for supporting the bioactive substance, and the bioactive substance may be formed in the recess. Alternatively, in the sheet medium, a convex on-wall structure can be formed to surround each region or a region group formed from two or more regions. As a result, the arrangement of the droplets arranged by the droplet discharge means can be facilitated. Further, the sheet may have a convex portion for supporting the bioactive substance, and the bioactive substance may be supported thereon. As a result, the region where the bioactive substance is disposed can be easily brought into contact with the culture solution, and the bioactive substance can be transferred to the culture solution (FIG. 2B).

  As a method for forming concave portions, convex wall-like structures, and convex portions on the bioactive substance transfer sheet, injection molding, casting molding, bonding chips with heat fusion or adhesive, and molding Or a method of press molding with a mold or the like can be used.

  Even if the biologically active substance 12 fixed on the cell screening substrate 1 and the biologically active substance 22 arranged on the biologically active substance transfer sheet 2 are composed of the same biologically active substance, they are different biologically active substances. Or some of the biologically active substances may be the same.

  The cell culture substrate 1 and the bioactive substance transfer sheet 2 as described above can be produced as follows. First, the base 11 and the sheet 21 may be subjected to the above-described processing as necessary. Specifically, various chemical-physical treatments such as cleaning the base 11 or 21 to remove undesired substances, irradiating ultraviolet rays and other radiation, or performing corona discharge can be performed. . For the cell culture substrate, a polymer material, a silane coupling agent, or the like may be applied to a part or the entire surface of the base 11 as necessary.

  Further, the sheet 21 may be subjected to a treatment for easily supporting the biologically active substance. As the treatment, a biologically active substance supporting layer may be formed on the entire surface in advance. For example, gum arabic, agar, gelatin, starch, tragacanth, crystalline cellulose, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, polyvinylpyrrolidone, macrogol, polyvinyl alcohol, polyacrylic acid , Natural methacrylates such as polymethacrylic acid, methacrylic acid copolymer, sodium alginate, sodium fibrin glycolate, sodium starch glycolate, dextrin, gum arabic, carrageenan, agar, gelatin tragacanth, crystalline cellulose and glucose Water-soluble compounds such as sucrose, fructose and xylitol are preferably used. These materials may be applied to a part or the entire surface of the base 11 as necessary.

  By selecting the structure of the support layer, the sustained release property of the bioactive substance attached to the support layer to the culture solution can be obtained. In addition, this sustained release is achieved by adding a substance (for example, a water-soluble styrene-acrylic resin) capable of imparting sustained release to a liquid containing the bioactive substance when the bioactive substance is applied to the sheet. It is also possible to obtain it. The amount of the substance capable of imparting sustained release added to the liquid containing the biologically active substance is 10% by mass or less, preferably 5% by mass or less.

  As a method for forming the carrier layer on the sheet 21, there is a method in which a material as described above is dissolved or dispersed in an appropriate solvent to prepare a coating liquid, and the coating liquid is applied to the sheet to form a film. Can be mentioned. Coating methods include roll coater method, blade coater method, air knife coater method, gate roll coater method, bar coater method, size press method, shim sizer method, spray coating method, gravure coating method, curtain coater method, screen printing method, Examples include a flexographic printing method and an offset printing method. The thickness of the support layer formed as described above may be freely set after drying, but is preferably formed to be about 1 to 100 μm.

  The bioactive substance 12 is disposed on the base 11 and the sheet 21. For the arrangement, a droplet discharge means can be suitably used. The droplet discharge means is a device capable of discharging a droplet having a volume of 100 nl or less, more preferably a droplet of 1 nl or less, and includes a micropipette, a microdispenser, and a discharge device using an inkjet method. It is done. A discharge device using an ink jet method can be preferably used in that the discharge device can be manufactured at low cost and fine droplets can be discharged. Further, among the ink jet methods, the thermal ink jet method and the piezo ink jet method can be suitably used, and the ejection device based on the thermal ink jet method is easy to finely process the ejection port, and the bioactive substance 12 can be arranged at a high density. it can. Further, since the ejection device based on the piezo ink jet method generates ejection energy by displacement of the piezoelectric element, it is possible to stably eject the bioactive substance without applying thermal stress to the bioactive substance.

  When using the droplet discharge means, the bioactive substances 12 and 22 are dissolved in an appropriate solvent for liquefaction. The amount of the biologically active substance to be dissolved is 10% by mass or less, preferably 5% by mass or less. Any solvent can be used as long as it can stably dissolve the biologically active substances 12 and 22, but water is preferably used. The amount of water is 30% by mass or more, preferably 50% by mass or more. As water, it is preferable to use various buffer solutions in order to dissolve ion exchange water (deionized water) and biologically active substances 12 and 22 stably.

  Moreover, a water-soluble solvent can be used as needed. As the water-soluble solvent, it is added in an amount of 50% by mass or less, preferably 30% by mass or less per type. Any water-soluble solvent may be used as long as it is soluble in water. For example, methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, etc. 1-4 alkyl alcohols; amides such as dimethylformamide and dimethylacetamide; ketones or ketoalcohols such as acetone and diacetone alcohol; ethers such as tetrahydrofuran and dioxane; polyalkylene glycols such as polyethylene glycol and polypropylene glycol ; Alkylene such as ethylene glycol, propylene glycol, butylene glycol, triethylene glycol, 1,2,6-hexanetriol, thiodiglycol, hexylene glycol, diethylene glycol, etc. Alkylene glycols containing 2 to 6 carbon atoms; glycerin; ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monobutyl ether, triethylene glycol Examples include lower alkyl ethers of polyhydric alcohols such as monomethyl ether, triethylene glycol monoethyl ether, and triethylene glycol monobutyl ether; N-methyl-2-pyrrolidone, 2-pyrrolidone, 21-dimethyl-2-imidazoline, and the like. One or more of these can be appropriately selected and used. Of these many water-soluble organic solvents, polyhydric alcohols such as diethylene glycol and lower alkyl ethers such as triethylene glycol monomethyl ether are preferred.

  Among these, by adding a lower alkyl ether of ethanol or isopropyl alcohol, or a polyhydric alcohol, in the case of the thermal jet type, foaming of the bioactive substance imparting ink on the thin film resistor in the discharge port of the ink jet Can be suitably used because it can be carried out more stably.

  In addition to the above components, the liquid containing at least the biologically active substances 12 and 22 according to the present invention can contain at least one hydrophilic resin. Examples of hydrophilic resins include natural polymers such as lignin sulfonate and shellac, polyacrylates, styrene-acrylic acid copolymer salts, styrene-acrylic acid-ethyl acrylate copolymer salts, styrene- Acrylic acid-acrylic acid alkyl ester copolymer salt, styrene-maleic acid copolymer salt, styrene-maleic acid-alkyl acrylate copolymer salt, styrene-maleic acid half ester copolymer salt, styrene-methacrylic acid Copolymer salt, vinyl naphthalene-acrylic acid copolymer salt vinyl naphthalene-maleic acid copolymer salt, β-naphthalene sulfonic acid formalin condensation salt, anionic polymer such as polyphosphate, polyvinyl alcohol, methylolated melamine polyvinyl Pyrrolidone, methylcellulose, hydroxymethylcellulose , And the like cellulose derivatives such as carboxymethyl cellulose. In the present invention, one of these resins can be selected, or two or more of these resins can be mixed and used. As others, many can be listed, for example, natural resins such as albumin, gelatin, casein, starch, cationized starch, gum arabic, and sodium alginate. The amount of the hydrophilic resin is 10% by mass or less, preferably 5% by mass or less. Of course, the present invention is not limited to these.

  Also, when the bioactive substance 12 is released from the bioactive substance transfer sheet by imparting sustained release properties, a hydrophilic polymer compound can be added. For example, sustained release of polyvinyl alcohol, polyacrylate, polymethacrylate, methacrylic acid copolymer, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, polyvinylpyrrolidone, etc. It is suitably used for application. The hydrophilic resin is added in the range of 1 to 10% by mass, more preferably in the range of 2 to 5%. Of course, the present invention is not limited to these.

  In addition to the above components, the liquid containing at least the biologically active substances 12 and 22 according to the present invention includes a surfactant, an antifoaming agent, a preservative, in order to obtain a solution having desired physical properties as required. Inorganic salts, organic salts, and the like can be added.

  For example, any surfactant can be used as long as it does not adversely affect the bioactive substances 12 and 22 such as storage stability. For example, fatty acid salts, higher alcohol sulfate salts, liquid fatty oils Anionic surfactants such as sulfate ester salts and alkylallyl sulfonates, nonionic interfaces such as polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters, polyoxyethylene sorbitan alkyl esters, acetylene alcohol and acetylene glycol There is an active agent, and one or more of these can be appropriately selected and used.

  With respect to the cell culture substrate, the biologically active substance 12 is fixed on the base 11 after the biologically active substance 12 is arranged at a desired position on the base 11 by the droplet discharge means. In order to immobilize the bioactive substance 12 on the base 11, a process necessary for immobilization may be performed on the bioactive substance 12 in advance, or a process necessary for immobilization may be performed on the substrate in advance. As the treatment to the biologically active substance 12, functional groups such as amino group, carboxyl group, disulfide group, epoxy group, carbodiimide group, maleimide group and the like necessary for covalent bond may be introduced into the biologically active substance 12, Metals necessary for bonding through attractive force and chargeable substances such as inorganic oxide fine particles, cationic polymers, and anionic polymers may be bonded. Further, in order to bind using biological affinity, an avidin molecule or biotin molecule may be bound, or a substance that can be bound by biological affinity such as an antigen molecule or an antibody molecule may be bound. Alternatively, the substrate surface may be coated with a polymer or a silane coupling agent, and functional groups such as amino groups, carboxyl groups, disulfide groups, epoxy groups, carbodiimide groups, and maleimide groups necessary for covalent bonding may be introduced. In order to charge the substrate surface, metals such as gold, silver, platinum and iron, inorganic oxides such as indium tin oxide, titanium oxide and zinc oxide, and conductive polymers such as polyacetylene, polypyrrole, polyaniline and polythiophene A conductor layer or a semiconductor layer may be formed on the substrate surface in advance. Furthermore, a protein such as a biotin molecule or avidin molecule having a binding force with a substance having biological affinity introduced into the bioactive substance 12, an antibody molecule or an antigen molecule, or a protein A having an antibody binding ability is provided on the surface of the base 11. May be. By introducing these substances, the bonding force between the surface of the base 11 and the bioactive substance 12 can be strengthened.

  At the time of immobilization, energy may be applied from the outside by irradiation with light such as light or heating. By adding these energies from the outside, the binding between the surface of the base 11 and the bioactive substance 12 can be promoted.

  As described above, the cell culture substrate 1 and the bioactive substance transfer sheet 2 can be produced.

  The prepared cell screening substrate 1 and bioactive substance transfer sheet 2 can be stored in a container for a long time. If the container is sealed together with a desiccant or an oxygen scavenger, it can be stored more preferably and stably. It can also be simply laminated with a film or the like.

  Next, a method for culturing cells using the cell culture kit comprising the cell culture substrate 1 and the bioactive substance transfer sheet 2 will be described. By culturing the cells using the cell culture kit, the cells are cultured while being affected variously by the biologically active substance. Before culturing the cells, the cell culture substrate and the sheet may be sterilized by irradiating them with radiation or ultraviolet rays or washing with an alcohol solution as necessary. Thereby, it is possible to prevent the culture from being adversely affected by unwanted microorganisms.

  Further, when cells are cultured using the cell culture kit comprising the cell culture substrate 1 and the bioactive substance transfer sheet 2, the bioactive substance transfer sheet may be immersed in the culture solution throughout the culture. However, it may be removed along the way. Further, a desired substance may be added to a culture solution in a desired region after cell culture for a certain period. This makes it possible to control the concentration of the biologically active substance with respect to the cells, add a substance to be acted on, change the influence, and change the adhesion on the substrate. As a method of adding a desired substance to the culture solution, a plurality of sheets may be brought into contact with the culture solution at different times. Alternatively, the culture solution may be exchanged during the culture, and a different bioactive transfer sheet may be used. As a result, different combinations of biologically active substances can be allowed to act at any time in the culture, and the culture conditions can be easily changed. Alternatively, a desired substance such as an indicator may be added to a desired region during screening after cell culture. Thereby, screening can be easily performed.

  Further, the cultured cell group may be removed from the substrate when cells are cultured using the cell culture kit including the cell culture substrate 1 and the biologically active substance transfer sheet 2 or after the cell culture for a certain period. . As a result, the substrate from which the cultured cells have been removed can be used again, and the removed cultured cell group can be used as a biological tissue prepared artificially or a part thereof. As a specific method, the cultured cell group can be removed by trypsinizing the cultured screening substrate. As a result, the substrate can be used again. This reuse of the substrate is one of the effects obtained by immobilizing the bioactive substance on the substrate so that the cells cannot take the screening substance into the metabolic system. In addition, when a temperature-responsive polymer such as poly (N-isopropylacrylamide) is applied on a substrate in advance and cell culture is performed on the substrate, if the temperature is reduced to about 30 ° C. or less, the cultured cell group is polymer surface. It can be removed from the substrate by changing the hydration state. Thereby, a cell group can be utilized for a biological tissue etc.

  Next, cells are cultured with the cell culture kit comprising the cell culture substrate 1 and the biologically active substance transfer sheet 2 described above, and the substances immobilized on the cells and the substrate and the substances dissolved in the culture solution from the sheets are obtained. The screening method is described. As the screening means, the above-described method for observing the morphological change of the cells cultured on the cell culture substrate 1 can be used. The microscope may be any optical microscope, scanning electron microscope, transmission electron microscope, scanning probe microscope, fluorescent microscope, or the like as long as the cell morphology can be observed. A cell culture substrate on which cells are cultured is placed at the observation position of the microscope, and the morphology of the cells is observed by microscopic observation. Screening is possible only by observing cell morphology with a microscope, and evaluation can be performed by a simple method. Moreover, you may dye | stain a cell in the case of evaluation. By performing cell staining, evaluation by observation with a microscope can be easily performed when cells proliferate at a high density or when cells are fused by differentiation to become multinucleated cells.

  In addition to morphological observation, the process of cell adhesion or proliferation and differentiation, and as a result, the substance produced by the cell or the substance taken into the cell can be quantified and used as a screening tool. Good. Here, if the quantification target cannot be directly evaluated, the alternative substance may be quantified. Specifically, it can be quantified in the vicinity of the gene of the desired protein to be quantified using genetic engineering techniques. A desired protein may be quantified by incorporating a protein gene and quantifying the quantifiable protein. By evaluating these substances, it is possible to examine in detail what kind of changes are occurring in the cells due to the substances immobilized on the substrate, which leads to the elucidation of the information transmission mechanism in the cells. In the case where the evaluation is performed with a substance taken up into cells, it is possible to provide an index that can be evaluated for a substance that will be taken up in advance, and it is possible to quantify relatively easily.

  These substances are quantified by measuring the amount of radiation emitted from radioactive compounds, measuring the amount of fluorescence emitted from substances labeled with fluorescent substances, and measuring the amount of luminescence emitted from luminescent substances. And a method for measuring the absorbance of the dye.

  In the method of measuring the amount of radiation emitted from radioactive compounds, compounds substituted with radioactive isotopes of elements such as hydrogen, carbon, nitrogen, phosphorus, sulfur, etc. The method of measuring the radiation dose is very sensitive, and the chemical properties of these substances are not different from those of ordinary compounds, so it does not affect the metabolic activity of cells and the same phenomenon as in vivo is observed. Is possible.

  In addition, labeling with a fluorescent substance is relatively easy, and since it is a low molecular compound, it has little influence on the metabolic activity of cells. In addition, when quantifying substances produced in cells by a quantification method using an antigen-antibody reaction, various antibodies labeled with fluorescent substances are commercially available and have high measurement sensitivity, so evaluation by fluorescence measurement is effective. is there.

  Furthermore, in the method of measuring the amount of luminescence emitted from a luminescent substance, the amount of luminescence can be measured with high sensitivity, so that even a slight change can be captured. If a gene that is expressed as a result of adhesion, proliferation, differentiation, or substance production by the screening substance is specified, a luciferase gene or the like is introduced near the gene, and the amount of luciferase produced along with gene expression is determined. Measured by the amount of luminescence produced by the addition of ATP and luciferin. Thus, the influence of the screening substance can be evaluated by the amount of luminescence.

  In the method of measuring the absorbance of the dye, the absorbance can be amplified by the dye by using an enzyme reaction or the like together, and a trace amount of substance can be quantified.

The present invention will be described in more detail with reference to the following examples. These examples are specific examples shown for a deeper understanding of the present invention, and the present invention is not limited to these specific examples. It is not limited to examples. Unless otherwise indicated, “%” is based on mass.
Example 1
The following method was used to act on cells in combination with biologically active substances. Basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), and bone morphogenetic protein-2 (BMP-2) were used as bioactive substances. First, in order to immobilize the biologically active substance on the substrate, the substrate was treated on a 96-well cell culture plate made of polystyrene by the following method.

  A solution of activated dextran tosylated with Tresylchloride (1.5 mg / ml) was added to the culture substrate coated with Poly-L-lysine to bind the substrate to the activated dextran. Next, prepare each carbonate buffer containing bFGF and IGF-I, add them to the substrate, add them in different amounts, and leave them at 4 ° C for 12 hours. Thus, a fixed region and a non-immobilized region were prepared to prepare a cell culture substrate.

  Next, a solution containing 5% glycerin and BMP-2 at 20 μg / ml was prepared, and the ink cartridge was washed with 70% ethanol and then filled with this solution. Next, BMP-2 is placed on the projections of the polystyrene sheet having projections at positions corresponding to the holes of the 96-well plate previously sterilized with a germicidal lamp at a stepwise density change using a Canon inkjet printer PIXUS950i. A bioactive substance transfer sheet was prepared.

  Next, DMEM containing 2% of FBS was injected as a culture solution into the 96-well cell culture substrate.

Next, the biologically active substance transfer sheet was placed so as to correspond to each hole of the 96-well cell culture substrate, and the wall and sheet of each region of the cell culture substrate were brought into close contact with each other. Thereby, the arrangement | positioning area | region of the bioactive substance formed on protrusion was contacted, the vibration was given in order to accelerate | stimulate dissolution of a bioactive substance, and BMP-2 was dissolved in the culture solution. Next, the number of mouse skeletal muscle cell lines C2C12 suspended in DMEM (Dulbecco's Modified Eagle's minimum essential medium) medium containing 2% fetal bovine serum (FBS) in each hole of the cell culture substrate is 500 cells / hole. In addition, the cells were cultured at 37 ° C. for 96 hours in humid air containing 5% CO ₂ .

  When the cell culture substrate after culture is observed with an optical microscope, it is observed that cells proliferate in the bFGF presence region, muscle differentiation is promoted in the IGF-I presence region, and bone differentiation is promoted in a concentration dependent manner in the BMP-2 presence region. It was done. Next, the cultured cells were treated with 10% formalin for 15 minutes and methanol for 15 minutes, reacted with a fluorescent dye for 30 minutes, and DNA was fluorescently stained. The amount of fluorescence of DNA at 700 nm was measured and used as an index of proliferation. In addition, the enzyme activity values of creatine kinase (CK) and alkaline phosphatase (ALP) in a solution obtained by freezing and pulverizing the cell groups in each compartment were measured, and the specific activities determined by the ratio to the total protein amount in the solution were respectively determined. It was used as an index of differentiation and bone differentiation. As a result, a combined analysis of the effects of the immobilized bFGF, the immobilized IGF-I, and the dissolved BMP-2 on the cells was made.

Example 2
As bioactive substances, epidermal growth factor (EGF) and nerve cell growth factor (NGF) were used. First, using the same method as in Example 1, a region where EGF is immobilized and a non-immobilized region are prepared on a 96-well cell culture plate made of polystyrene by changing the density stepwise, and cell screening is performed. A substrate was prepared. Next, hydroxypropylcellulose was applied to the stretchable polyolefin sheet as a support layer using a bar coater, and heated at 60 ° C. for 10 minutes to form a support layer. Next, using a Canon inkjet printer PIXUS 950i, EGF and NGF were arranged on the sheet to produce a bioactive substance transfer sheet having a plurality of bioactive substance areas. Cells were screened using these cell culture substrates and bioactive substance transfer sheets.

Cover the substrate for cell culture previously sterilized with a germicidal lamp, cover the biologically active substance transfer sheet, press the protrusions from the back of the sheet, push each area containing the biologically active substance down to the culture surface, and touch for 15 minutes while gently shaking After the biologically active substance is dissolved in the culture solution, 500 cells / compartment of the neuronal cell line PC12 suspended in RPMI (Roswell Park Memorial Institute) 1640 medium supplemented with 2% FBS is added and contains 5% CO ₂ 37 The cells were cultured at 37 ° C. for 48 hours in humid air at 0 ° C. Next, the culture solution is removed, and the cells are fixed and stained with hematoxylin and eosin staining in order to evaluate the degree of cell proliferation and differentiation, and the thus prepared substrate is observed with an optical microscope. Nuclear and cell morphology was evaluated. As a result, it was confirmed that the immobilized EGF had a different action on PC12 cells than the dissolved EGF. As a result, the effect on the cells by the combination of the growth factor in the dissolved state and the growth factor in the immobilized state could be easily examined.

Example 3
From the hybridoma obtained by immunizing rats with mouse bFGF as an antigen, taking out mouse spleen cells two months later and fusing them with myeloma cells, a number of monoclonal antibody-producing hybridomas were obtained, from which 96 types of monoclonal antibodies were purified. did. Next, mouse bFGF was immobilized at 200 pg / mm ² on a polystyrene 96-well plate as a culture substrate. Next, hydroxypropylcellulose was applied to the entire surface of the parafilm using a bar coater, and the obtained monoclonal antibody was placed using a Canon inkjet printer PIXUS 950i at a position corresponding to each hole of the 96-well plate. A sheet was produced. 75 μl of DMEM culture solution was added onto the culture substrate, and the sheet was further covered and adhered, the substrate was brought into contact with the antibody placement region of the culture solution and the screening sheet, and the antibody was transferred into the culture solution.

Next, 75 μl of DMEM containing 6 × 10 ⁴ cells / ml of myocyte C2C12 cells was added to each well and cultured at 37 ° C. in 5% CO ₂ humidified air. After 48 hours, 0.002% 5-bromo-2'-deoxyuridine (BrdU) was added to the culture solution, and after 3 hours, the culture solution was removed, and the cells were treated with methanol for 30 minutes. Next, in order to evaluate the state of proliferation, staining was performed with an FITC-labeled anti-BrdU antibody, Hoechst33258 was further diluted 10,000 times, reacted for 5 minutes, and the nucleus was stained. Excess staining solution was washed with phosphate isotonic buffer. The substrate thus prepared was observed with a fluorescence microscope, and the number of stained nuclei was evaluated. In addition, the number of nuclei containing DNA labeled with BrdU was quantified by fluorescence quantification.

  As a result, in the three compartments, there is a compartment where BrdU fluorescence is lower than other compartments, and the monoclonal antibodies A, B, and C corresponding to the compartments are neutralizing antibodies that efficiently suppress the growth promoting effect of bFGF. I was able to confirm.

Example 4
In order to examine the concentration dependency of the bFGF function inhibitory effect of the three neutralizing antibodies obtained in Example 3, a cell culture substrate was prepared by immobilizing bFGF on polystyrene 96-well plates at different concentrations. Next, hydroxypropylcellulose is coated on the front surface of the parafilm using a bar coater, and bFGF neutralizing antibodies A, B, and C are placed in positions corresponding to the holes of the cell culture substrate using an inkjet printer. A sheet printed on was obtained. Next, 75 μl of the culture solution was added to the screening substrate, and the screening sheet was placed over, the region where the neutralizing antibody was placed and the culture solution were brought into contact with stirring, and the neutralizing antibody was transferred into the culture solution. Next, 75 μl of DMEM containing 6 × 10 ⁴ myocyte C2C12 cells / ml was added to each hole of the cell culture substrate and cultured at 37 ° C. in 5% CO ₂ humidified air.
After 48 hours, the culture solution was removed, and the cells were treated with methanol for 30 minutes, dried and fixed. Next, in order to evaluate the state of proliferation, Hoechst33258 was diluted 10,000 times, reacted for 5 minutes, and the nucleus was stained. Excess staining solution was washed with phosphate isotonic buffer. The substrate thus prepared was observed with a fluorescence microscope, and the number of stained nuclei was evaluated. As a result, it was found that in the compartment where the neutralizing antibody for bFGF was transferred, the number of cells was smaller as the concentration of the neutralizing antibody was higher, and the growth promoting effect of bFGF was suppressed. Moreover, it was found from the comparison of the number of cells that the inhibitory effect was strong in the order of B, C, and A.

(A)-(C) is a figure for demonstrating an example of the manufacturing method of the kit for cell cultures using the liquid discharge means. (A) And (B) is a figure for demonstrating an example of the cell culture process using the kit for cell cultures.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Cell culture substrate 2 Bioactive substance transfer sheet 3 Discharge means 4 Culture solution 11 Base 12 Bioactive substance 21 Sheet 22 Bioactive substance 23 Convex part

Claims (27)

A cell culture kit comprising: a substrate having a culture region for culturing cells; and a sheet medium that can be placed on the substrate and has a covering region of the cell region,
Both the culture region and the covering region of the sheet medium with respect to the culture region have one or more types of biologically active substances having physiological activity with respect to cells, and the biologically active substance that one of these areas has is the region. Fixed inside ,
A film in which the sheet medium is stretchable or flexible at least in the covering region
A kit for cell culture comprising a part consisting of A cell culture kit comprising: a substrate having a culture region for culturing cells; and a sheet medium that can be placed on the substrate and has a covering region of the cell region,
Both the culture region and the covering region of the sheet medium with respect to the culture region have one or more types of biologically active substances having physiological activity with respect to cells, and the biologically active substance that one of these areas has is the region. Fixed inside,
A support layer for supporting a bioactive substance is partially or entirely formed on the sheet medium.
A cell culture kit characterized by comprising: The carrier layer can release the biologically active substance or allows the biologically active substance imparted with sustained release to migrate.
The cell culture kit according to claim 2, which can be separated. The cell culture kit according to any one of claims 1 to 3, comprising two or more combinations of the culture region and the sheet and medium-side cover region with respect to the culture region. A bioactive substance is fixed to the culture area, and the bioactive substance is releasably attached to the culture area by contact with the culture liquid in a covering area with respect to the culture area, and the sheet medium is a bioactive substance. The cell culture kit according to any one of claims 1 to 3, wherein the kit is a sheet for transfer to the culture region. The kit for cell culture according to claim 5 which has two or more of combinations of said culture field and a covering field of said culture field. The cell culture kit according to any one of claims 4 to 6 , comprising two or more culture regions in which the types of the biologically active substances or combinations of two or more kinds of biologically active substances are different. The cell culture kit according to any one of claims 4 to 7 , comprising two or more culture regions having different densities of the biologically active substance. The kit for cell culture according to any one of claims 4 to 8 , which has two or more covering regions in which the kind of the biologically active substance or the combination of two or more kinds of biologically active substances are different. The cell culture kit according to any one of claims 4 to 9 , comprising two or more covering regions having different densities of the biologically active substance. The cell culture kit according to any one of claims 1 to 10 , wherein the culture region is formed in a recess provided in the substrate. The cell culture kit according to any one of claims 1 to 11 , wherein the culture region is surrounded by a convex wall-shaped structure. The cell culture kit according to any one of claims 1 to 12 , wherein the covering region is formed in a recess provided in the sheet medium. The kit for cell culture according to any one of claims 1 to 12 , wherein the covering region is formed in a convex portion formed on the sheet medium. The cell culture kit according to any one of claims 1 to 14 , wherein the covering region is surrounded by a convex wall-shaped structure formed on the sheet medium. A method for producing the cell culture kit according to any one of claims 1 to 15 ,
A method for producing a cell culture kit, wherein at least a liquid discharge means is used for applying a biologically active substance to the culture region and the covering region. The method for producing a cell culture kit according to claim 16 , wherein the liquid discharge means is discharge means by a thermal ink jet method. The method for producing a cell culture kit according to claim 16 , wherein the liquid discharge means is discharge means by a piezo ink jet method. The method for producing a cell culture kit according to any one of claims 16 to 18 , further comprising a step of immobilizing the biologically active substance by applying energy for immobilization from the outside. A cell screening method using the cell culture kit according to any one of claims 1 to 15 , wherein the sheet medium is placed on the substrate, and a culture region of the substrate corresponds to the culture region. A step of culturing the cell by contacting the cell with a biologically active substance immobilized in one of these regions in a culture solution that is covered with the covering region and placed in the cell region, and attached to the other of these regions A cell screening method comprising a step of supplying a biologically active substance to a culture solution. The cell screening method according to claim 20 , further comprising a step of supplementing the culture medium with a substance necessary for screening the cells. The cell screening method according to claim 20 or 21 , further comprising a step of replacing the sheet medium with the same or different sheet medium. The cell screening method according to any one of claims 20 to 22 , further comprising a step of observing the morphological change of the cells. The cell screening method according to claim 23 , wherein the cells are stained at the time of evaluation. The cell screening method according to any one of claims 20 to 24 , comprising a step of quantifying a substance synthesized in the cells. The cell screening method according to any one of claims 20 to 25 , comprising a step of quantifying the substance taken up into the cells. 27. The cell screening method according to claim 25 or 26 , comprising a step of quantifying the substance by at least one of the radiation dose measurement, fluorescence measurement, luminescence measurement, and absorbance measurement.

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