JP2002355025A - Cell screening substrate and method for producing the same, cell screening method and cell screening device each using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell screening substrate formable by a simple process. SOLUTION: This cell screening substrate characterized by having a plurality of areas differing in cell screening function from each other is formed by disposing a plurality of cell screening substances on the respective desired areas on a base by a microdroplet-issuing means to effect immobilizing the substances.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a cell screening substrate which can be used for specifying a substance which contributes to at least one of cell adhesion, proliferation, differentiation, survival, maintenance of an undifferentiated state and cell death. The present invention relates to a method for producing the same, a method for screening cells using the same, and a cell screening apparatus.

[0002]

2. Description of the Related Art In recent years, research on culturing animal and plant cells under various conditions or research on products using specific cultured cells has been actively conducted. In addition, production of substances that are extremely difficult to synthesize using specific cell activities has been studied in various fields.

[0003] 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 the rapid progress of cell engineering and medical engineering, Ultra-small biosensors and artificial organs using cells, as well as neurocomputers, have attracted attention, and active research activities have been conducted to apply them.

[0004] However, as described above, in order to utilize cells in vitro, cells are arranged as desired, and their growth,
It is indispensable to control differentiation and substance production, but cells are arranged and the mechanisms that control their proliferation, differentiation and substance production are not fully understood, so culturing while controlling cells from such a viewpoint This is extremely difficult, and is a major barrier to the progress of research and development using cells as described above.

[0005]

As an attempt to control the arrangement of cells, as described in US Pat. No. 5,108,926, a pattern is formed by applying a cell adhesive protein using an ink jet printer, and the cells are cultured thereon. There is an example. In this method, cells can be cultured on a pattern to which a cell adhesive protein is applied, but cells cannot be screened by controlling proliferation / differentiation or substance production.

[0006] Also, "proteins, nucleic acids and enzymes", 45-5, 7
27-734 (2000), a cell growth factor that affects cell growth / differentiation is immobilized on a substrate by photolithography, and the effect on cell growth / differentiation is examined. However, the substrate on which the cell growth factor is immobilized is not used as a cell screening means, and in photolithography, these biomaterials which are present only in a small amount in a living body are wasted, and processes such as exposure and development must be repeated. The manufacturing process must be complicated.

[0007] In addition, Japanese Patent Publication No. 2000-512009 proposes a method for screening cells by immobilizing a substance that affects cell adhesion on a substrate. Here, the reactive functional group and the cell adhesive substance provided on the substrate are immobilized with a divalent crosslinking reagent. Photolithography is used to bind the reactive functional group to the cell adhesive, which not only poses the same problems as described above, but also when multiple cell adhesives are immobilized, It is extremely difficult to avoid that the immobilized substance and the substance to be newly immobilized are bonded at an undesired position with a divalent crosslinking reagent. Extremely difficult. In addition, without immobilizing substances that affect proliferation and differentiation, and even substance production, cells are immobilized in each well with an adhesive substance, and the substances produced by cells in the process of being cultured in a culture solution are captured. And screening a substance that affects at least one of cell adhesion, proliferation, differentiation, survival, maintenance of an undifferentiated state, cell death and substance production as in the present invention. Not something.

An object of the present invention is to provide a cell screening substrate which can solve the above-mentioned problems of the conventional example and can be formed by simple steps, and a method for producing the same. It is an object of the present invention to provide a base technology for manufacturing various devices.

Further, an object of the present invention is to provide at least one of cell adhesion, proliferation, differentiation, survival, maintenance of an undifferentiated state, cell death and substance production by using cells cultured on these cell screening substrates. And a method of screening for a substance that affects the following.

[0010]

According to the cell screening substrate of the present invention, a plurality of cell screening substances are arranged and immobilized on a desired area on a base by means of microdroplet discharging means.
It has a plurality of regions having different cell screening functions.

[0011] The plurality of regions of the cell screening substrate may include a plurality of regions having different combinations of immobilized cell screening substances. Further, the plurality of regions may include a plurality of regions having different densities of the immobilized cell screening substance.
Further, each region or a group of regions formed from two or more regions may be formed in the concave portion. Further, each region or a region group formed from two or more regions may be surrounded by a convex wall-shaped structure.

On the other hand, according to the method for producing a cell screening substrate of the present invention, a step of arranging each cell screening substance in a desired area on a base by a microdroplet discharging means, and a step of disposing the cell screening substance in each area on the base. It is characterized by including a step of immobilizing.

As the minute droplet discharging means, a discharging means by a thermal ink jet method or a discharging means by a piezo ink jet method can be used. In addition, the immobilization step may further include a step of externally applying energy for immobilization.

The cell screening method according to the present invention comprises:
A cell screening method using the cell screening substrate having the above configuration, comprising a step of culturing cells in a culture solution that comes into contact with a cell screening substance-fixed region of the cell screening substrate. .

The above-mentioned cell screening method may include a step of replenishing a substance necessary for screening the cells into a culture solution that comes into contact with the immobilization region. Further, an aspect may be employed in which the fixed region is in contact with the flow of the culture solution, for example, the culture is performed by perfusing the culture solution.

The above-described cell screening method may further include a step of observing a change in cell morphology in a desired region, and a method of staining the cell at the time of evaluation may be used. Further, the cell screening method described above,
The method may include a step of quantifying a substance synthesized in cells in a desired region on the substrate, and a step of quantifying a substance taken up in cells in a desired region on the substrate. Can also be included. At least one of radiation measurement, fluorescence measurement, luminescence measurement, and absorbance measurement can be used for quantification of the substance.

That is, the screening in the cell screening method according to the present invention can be performed, for example, based on the following items. 1) Evaluate morphological changes of cells in a desired area on the substrate. 2) Quantification of the substance synthesized in the cell in a desired area on the substrate. 3) Quantification of a substance taken into cells in a desired region on the substrate. 4) Quantify the substance by radiation dose measurement. 5) Quantify the substance by measuring the amount of fluorescence. 6) Quantify the substance by measuring the amount of luminescence. 7) Quantify the substance by measuring the absorbance.

A cell screening apparatus according to the present invention is a cell screening apparatus using the cell screening substrate having the above-described configuration, wherein cells are cultured in a culture solution that comes into contact with a cell screening substance-fixed region of the cell screening substrate. It is characterized by having means.

The apparatus further includes at least one of a means for evaluating a change in cell morphology by the culturing means, a means for quantifying a substance synthesized in the cell, and a means for quantifying a substance taken up in the cell. May be available. Further, it may have a means for producing the cell screening substrate.

According to the screening using the cell screening substrate according to the present invention, as a result of the screening, the cell growth / differentiation, survival and maintenance of the undifferentiated state,
Factors necessary for cell death or substance production can be specified, and a method for efficiently culturing cells can be determined. If the substance to be immobilized is a drug, it is possible to evaluate what combination and amount of the drug in the cultured cells is most effective. For example, if a substrate on which a sustained-release capsule such as acrylamide gel in which a chemical substance called a so-called environmental hormone is encapsulated is used, the environmental hormone gradually flows out of the capsule into the culture solution, and humans against these chemical substances are removed. Can be evaluated. Furthermore, based on these evaluation results, it is possible to determine a method for diagnosing various diseases for each person.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

An example of the cell screening substrate of the present invention will be described. As shown in FIG. 2, in the cell screening substrate 1, two or more (four in FIG. 2) cell screening substances 12 are placed on desired positions (121 to 124) on a base (base) 11 shown in FIG. Each cell screening substance 12 is immobilized on the base 11. By immobilizing two or more cell screening substances 12, it is possible to control at least one of advanced cell adhesion, proliferation, and differentiation.

In the present invention, the cell screening substance 12 influences the adhesion of the cell to the base 11, the proliferation, differentiation, survival and maintenance of the undifferentiated state, cell death, or substance production. A substance for controlling culture, including extracellular matrix proteins, antibodies and cytokines capable of specific binding to the surface of cells, and binding to cells or being taken up by cells to proliferate cells. , Differentiation, survival and maintenance of undifferentiated state, cell death, or a chemical substance that affects substance production.

For example, examples of extracellular matrix proteins include collagen, elastin, fibronectin, laminin and the like. In addition, cytokines include those called cell growth factors and hormones. Cell growth factors include nerve growth factor (NGF) and epidermal growth factor (EG)
F) and fibroblast growth factor (FGF). In addition, examples of hormones include insulin and adrenaline. Other chemical substances include substances such as allergens that cause allergies, and various chemical substances called so-called environmental hormones.

Immobilized cell screening substance 12
Form regions based on differences in chemical and physical properties such as the type and arrangement pattern of the cell screening substance 12 immobilized on the base 11.

As for the cell screening substance 12, the combination of the cell screening substance 12 immobilized may differ depending on the region on the base 11 or the region group formed of two or more regions. As a result, at least one difference among cell adhesion, proliferation, differentiation, survival, maintenance of an undifferentiated state, cell death, and substance production by the combination of the cell screening substances 12 can be seen.

As for the cell screening substance 12, the density of the immobilized cell screening substance 12 may be different depending on the region on the base 11 or the group of regions formed from two or more regions. Thereby, differences in cell adhesion, proliferation, survival of differentiation, maintenance of an undifferentiated state, cell death or substance production due to changes in the density of the cell screening substance 12 can be seen in more detail. One of the great advantages of using the droplet discharging means is that a cell screening substance can be easily arranged at an arbitrary ratio in one immobilization region.

Base 11 for cell screening substance 12
Immobilization on top may be through a covalent bond, through electrostatic attraction, or using biological affinity. When immobilized on the base 11 through a covalent bond, the cell screening substance 12 can be immobilized with a strong force, the binding force is hardly affected by cells, culture media, and the like, and the cell screening substance 12 is stably immobilized on the base 11. It is fixed.

Here, as a specific example, when insulin is used as the substance 12 which affects at least one of cell adhesion, proliferation, differentiation, survival, maintenance of an undifferentiated state, cell death and substance production, A method of immobilizing insulin on the base 11 by a covalent bond will be described with reference to FIG. First, 4-Azidobenzoic aci as a linker to insulin
Introduce d N-hydroxysuccinimide ester (see formula below):

[0030]

Embedded image

The thus obtained solution of insulin 12 to which the linker is bound is discharged onto a substrate 11 made of, for example, polyethylene terephthalate (PET) by using a droplet discharge means 13 (ink jet printer). Then, when the substrate is irradiated with light, for example, UV light, from the light source shown as 15 in FIG. 1, the azide group of the linker is cleaved to form an amide bond with a carbon atom of the PET substrate, and as a result, insulin 12 is covalently fixed to the substrate surface as shown in the following formula.

[0032]

Embedded image

Further, in the case of fixing by electrostatic attraction, the fixing can be performed on the base 11 without performing a chemical treatment.
Denaturation of the cell screening substance 12 due to chemical treatment can be suppressed. Furthermore, when immobilizing on the substrate 11 using biological affinity, the treatment required for immobilizing the cell screening substance 12 is relatively easy, and stable immobilization is possible.

The base 11 is composed of the cell screening substance 1
Any material and shape may be used as long as the material 2 can be fixed stably. Specifically, a glass substrate, a plastic plate, a plastic sheet, a polymer film, paper, or the like can be preferably used. Further, the base 11 may be transparent, light-shielding, or colored. Further, in order to immobilize the cell screening substance 12 on the base 11 or to enhance the stability of the cell screening substance 12 on the base 11, a part or the whole of the base 11 is treated with a chemical substance. Alternatively, a process of irradiating radiation may be performed.

In the base 11, individual regions to which the cell screening substance 12 is immobilized, or a group of regions formed from two or more regions may be depressed.
This makes it possible to easily arrange the droplets arranged by the microdroplet discharging means, and furthermore, culture the cells by changing the culture solution for each individual region or group of regions connected by the depression. be able to. A substrate having such a depression can be manufactured by molding a resin material, etching using a photolithography technique, or the like.

The base 11 may have individual regions on which the cell screening substance 12 is immobilized, or a group of regions formed from two or more regions, surrounded by a wall-shaped structure. This makes it possible to easily arrange the droplets arranged by the minute droplet discharging means,
Further, cells can be cultured by changing the culture solution for each region or region group connected by the depression.
With such a substrate, a fine wall-shaped structure can be manufactured by using a photolithography method or the like.

Such a cell screening substrate 1 can be manufactured as follows (see FIG. 1). The base 11 may first perform the above-described processing as needed. Specifically, various chemical and physical treatments such as cleaning of the base 11 to remove undesired substances, irradiation of ultraviolet rays and other radiation, and corona discharge can be performed. Further, 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.

The cell screening substance 12 is placed on such a base 11. For the disposition, the minute droplet discharging means 13 is used. The microdroplet discharging means 13 is capable of discharging droplets having a volume of 100 pl or less per drop, and examples thereof include a micropipette, a microdispenser, and a discharge device using an inkjet method. A discharge device using an inkjet method can be preferably used because a discharge device can be manufactured at low cost and minute droplets can be discharged.
Further, among the ink jet methods, the thermal ink jet method and the piezo ink jet method can be preferably used, and the discharge device by the thermal ink jet method can easily perform fine processing of the discharge port, and the cell screening substance 12
Can be arranged at high density. In addition, the ejection device using the piezo inkjet method uses a displacement of the piezoelectric element to
Since the ejection energy is generated, the cell screening substance 12 can be ejected stably without applying thermal stress to the cell screening substance 12.

The cell screening substance 12 is dissolved in a suitable solvent to make it liquid. As the solvent,
Any substance can be used as long as it can stably dissolve the cell screening substance 12, but water is preferably used. As the water, it is preferable to use various buffers to stably dissolve ion-exchanged water (deionized water) or the cell screening substance 12.

Further, a water-soluble solvent can be used if necessary. The water-soluble solvent may be any 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
Alkyl alcohols having 1 to 4 carbon atoms such as -butyl alcohol; amides such as dimethylformamide and dimethylacetamide; ketone or keto alcohols such as acetone and diacetone alcohol; ethers such as tetrahydrofuran and dioxane; polyethylene glycol and polypropylene Polyalkylene glycols such as glycol; alkylene groups such as ethylene glycol, propylene glycol, butylene glycol, triethylene glycol, 1,2,6-hexanetriol, thiodiglycol, hexylene glycol, and diethylene glycol having 2 to 6
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 monomethyl ether, triethylene glycol Lower alkyl ethers of polyhydric alcohols such as monoethyl ether and triethylene glycol monobutyl ether; and N-methyl-2-pyrrolidone, 2-pyrrolidone, 1,3-dimethyl-2-imidazoline and the like. One or more of these can be appropriately selected and used. Among 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 them, by adding a lower alkyl ether of ethanol, isopropyl alcohol, or polyhydric alcohol, in the case of the thermal jet type, the foaming of the ink on the thin film resistor in the discharge port of the ink jet can be more improved. Since it can be performed stably, it can be suitably used.

The liquid containing at least the cell screening substance 12 according to the present invention may contain a surfactant, an antifoaming agent, a preservative, etc. Agents, 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 cell screening substance 12 such as storage stability. Examples thereof include fatty acid salts, higher alcohol sulfates, and liquid fatty acids. Anionic surfactants such as oil sulfates and alkyl allyl sulfonates; nonionics such as polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters, polyoxyethylene sorbitan alkyl esters, acetylene alcohol and acetylene glycol There are surfactants, and one or more of these can be appropriately selected and used.

After disposing the cell screening substance 12 at a desired position on the base 11 by the microdroplet means 13,
The cell screening substance 12 is immobilized on the base 11. In order to immobilize the cell screening substance 12 on the base 11, a treatment required for immobilization may be performed on the cell screening substance 12 in advance, or a processing necessary for immobilization may be performed on the substrate in advance. As a treatment to the cell screening substance 12, a functional group such as an amino group, a carboxyl group, a disulfide group, an epoxy group, a carbodiimide group, or a maleimide group necessary for covalent bonding may be introduced into the cell screening substance 12, Chargeable substances such as metal and inorganic oxide fine particles, cationic polymers and anionic polymers necessary for binding via attractive force may be bound. In addition, in order to bind using a biological affinity, an avidin molecule or a biotin molecule may be bound, or a substance such as an antigen molecule or an antibody molecule that can bind with a biological affinity may be bound. In addition, the surface of the substrate is coated with a polymer or silane coupling agent,
A functional group such as an amino group, a carboxyl group, a disulfide group, an epoxy group, a carbodiimide group, or a maleimide group required for covalent bonding may be introduced, or a substrate such as gold, silver, platinum, or iron may be charged. A conductive layer or a semiconductor layer such as a metal or an inorganic oxide such as indium tin oxide, titanium oxide, or zinc oxide, or a conductive polymer such as polyacetylene, polypyrrole, polyaniline, or polythiophene may be formed in advance on the substrate surface. . Furthermore, proteins such as biotin molecules and avidin molecules, antibody molecules and antigen molecules, and protein A having antibody binding ability, which have a binding ability with a substance having biological affinity introduced into the cell screening substance 12, are provided on the surface of the base 11. You may. By introducing these substances, the binding force between the surface of the base 11 and the cell screening substance 12 can be strengthened.

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

The cell screening substrate 1 can be manufactured as described above.

Next, a method for culturing cells on the above-described cell screening substrate 1 will be described. By culturing the cells on such a substrate, the cells can adhere, proliferate,
Culture is performed while being affected by differentiation, survival, maintenance of an undifferentiated state, cell death or substance production. The cells are not particularly limited, and any cells can be used. Also, only one cell should be screened at a time.
The type may be two or more. Before culturing the cells, if necessary, the cell screening substrate 1 may be sterilized by irradiating the cell screening substrate 1 with ultraviolet rays or by washing with an alcohol solution. This can prevent culture from being inhibited by undesired microorganisms and the like. The cell screening may be performed by immersing the entire cell screening substrate 1 in a culture solution. However, if at least the region where the cell screening substance is immobilized is immersed in the culture solution, the cell adhesion and proliferation of the cells may be increased. -It is possible to culture while affecting differentiation, survival, maintenance of undifferentiated state, cell death, and substance production.

Further, a desired substance may be added to a culture solution in a desired region during culturing the cells on the cell screening substrate 1 or after culturing the cells for a certain period of time. This allows cells to proliferate and differentiate, survive, maintain undifferentiated state,
Cell death or substance production can be changed, and adhesion to a substrate can be changed. Alternatively, at the time of screening after cell culture, a desired substance such as an indicator may be added to a desired region. Thereby, screening can be easily performed.

Further, during culturing the cells on the cell screening substrate 1 or after culturing the cells for a certain period, the cultured cell group may be removed from the substrate. By doing so, the substrate from which the cultured cells have been removed can be reused, and the removed cultured cell group can be used as an artificially produced biological tissue or a part thereof.
As a specific method, the cultured cell group can be removed by subjecting the screening substrate after culture to trypsin treatment. Thus, the substrate can be used again. This reuse of the substrate is one of the effects obtained by immobilizing the cell screening substance on the substrate and preventing cells from taking up the screening substance into the metabolic system. In addition, when a polymer such as poly (N-isopropylacrylamide) whose water solubility changes depending on the temperature is applied to a substrate in advance, and cell culture is performed thereon,
When the temperature is reduced to about 30 ° C. or lower, the cultured cells can be removed from the substrate by changing the hydration state of the polymer surface. Thereby, the cell group can be used for a living tissue or the like.

Next, a method for culturing cells on the above-described cell screening substrate 1 and screening the cells and the substance immobilized on the substrate will be described. As the screening means, the method for observing the morphological change of the cells cultured on the cell screening substrate 1 described above can be used. The microscope may be any optical microscope, such as a scanning electron microscope, a transmission electron microscope, a scanning probe microscope, and a fluorescence microscope, as long as the cell morphology can be observed. The cell screening substrate on which the cells have been cultured is placed at the observation position of the microscope, and the morphology of the cells is observed under a microscope. Screening is possible only by observing the cell morphology with a microscope, and evaluation can be performed by a simple method. In the evaluation, the cells may be stained. By performing cell staining, evaluation by observation with a microscope can be easily performed when cells proliferate at high density or when cells fuse with each other due to differentiation to become multinucleated cells.

In addition to the morphological observation, a process in which cells adhere to a substrate or proliferate or differentiate, and as a result, substances produced by cells or substances taken into cells are quantified.
It may be used as a screening means. Here, when the quantification target cannot be directly evaluated, a substitute substance may be quantified. Specifically, using a genetic engineering technique, quantification can be performed in the vicinity of the gene of the desired quantification target protein. A desired protein may be quantified by incorporating a protein gene and quantifying the quantifiable protein. By evaluating these substances, it is possible to investigate in detail what kind of change has occurred in the cell due to the substance immobilized on the substrate, and this will lead to elucidation of the information transmission mechanism in the cell. When the evaluation is performed using a substance taken up into a cell, a substance that can be taken up may be provided with an index that can be evaluated in advance, and quantification can be performed relatively easily.

For quantification of these substances, a method of measuring the amount of radiation emitted from a radioactive compound, a method of measuring the amount of fluorescence emitted from a substance labeled with a fluorescent substance, and the method of measuring the amount of luminescence emitted from a luminescent substance There are methods for measuring the amount and methods for measuring the absorbance of the dye.

In the method of measuring the radiation dose emitted from a radioactive compound, a compound which is replaced by a radioisotope of an element such as hydrogen, carbon, nitrogen, phosphorus and sulfur which is contained in a living body in a large amount is used. The method of measuring the amount of radiation emitted from is very sensitive, and since these substances have the same chemical properties as ordinary compounds, they do not affect the metabolic activity of cells and are similar to those in vivo. The phenomenon is observable.

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

Further, in the method of measuring the amount of light emitted from a luminescent substance, the amount of light can be measured with high sensitivity.
Very small changes can be captured. If a gene that is expressed by adhesion, growth, differentiation, or substance production by a screening substance is specified, a firefly luciferase gene or the like is introduced near the gene, and firefly luciferase produced together with the gene expression is introduced. The amount is measured by the amount of luminescence generated by the addition of ATP and luciferin. This makes it possible to evaluate the effect of the screening substance on the basis of the amount of luminescence.

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

An apparatus for culturing cells on the above-described cell screening substrate and screening the cells and the substance immobilized on the substrate will be described. Two or more types of cell screening substances are arranged and fixed in a desired region on a substrate by a microdroplet discharging means, and
The cell screening substrate having a plurality of regions having different cell screening functions is characterized by having a means for culturing cells in a culture solution that comes into contact with the cell screening substance-fixed region of the cell screening substrate. Or at least one of a means for evaluating a change in cell morphology by the culturing means, a means for quantifying a substance synthesized in a cell, and a means for quantifying a substance taken up in a cell. FIG. 6 shows an example of the apparatus according to the present invention. The substrate (600) is loaded from the substrate loading chamber (601) of the apparatus, and the screening substance is applied to the substrate using the fine droplet discharging means in the screening substance application chamber (602). In 603), the screening substance is immobilized by light or heat irradiation, and a cell screening substrate is manufactured. Next, the substrate is carried to the culture room (604), and after culturing the cells on the substrate by the method described above, the detection room (605)
Then, cell morphological changes, cell adhesion, proliferation / differentiation, survival, maintenance of undifferentiated state, cell death, and substance production are observed, or cells are screened by the above-described quantification means.

Here, 601 to 603 may be, for example, devices as shown in FIG. In FIG. 7, reference numeral 710 denotes a minute droplet discharging device. The substrate 600 is a stocker 71
It is set to 1 and is sent to the belt conveyor 713 by the conveyor 712 and sent out to the tray 715.
714 is an auxiliary roller for feeding. Tray 71
The substrate 600 sent to 5 is firmly sucked and fixed on the tray by suction by the pump 716. Tray 715
The upper substrate 600 is sent to a region where the first processing step is performed. Reference numeral 704 denotes a UV / O ₃ lamp for performing a pre-treatment of the substrate. When the substrate is unloaded from the region of the first step by the feed motor 717, the cell screening substance is applied by the microdroplet discharging means 710. The substrate to which the screening substance has been applied is immediately transported to a region where the solidification treatment in the third step is performed, and the substance is fixed on the substrate. Here, reference numeral 705 denotes a UV irradiation lamp. The substrate that has gone through the above three processing steps is a belt conveyor 72
The sheet is conveyed to the next step 604 via the feed roller 721 and the feed roller 721.

However, the cell screening apparatus according to the present invention is not particularly limited, even in other embodiments, as long as the above object is achieved.

[0060]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which are specific examples for a deeper understanding of the present invention. However, the present invention is not limited to these specific examples. Example 1 (Cell screening method for evaluating cell growth factor) In order to immobilize a cell screening substance on a substrate, a functional group was introduced by the following method. 50 mmol of dicyclohexylcarbodiimide (DCC) in tetrahydrofuran (T
HF) The solution was added dropwise to a solution of 50 mmol of N-hydroxysuccinimide and 45 mmol of 4-azidobenzenecarboxylic acid in THF at 4 ° C.
With stirring for 24 hours. After drying the reaction product under reduced pressure, it was recrystallized and purified in an isopropanol / diisopropanol solution. Subsequently, the reaction product was dissolved in dimethylformamide, dissolved in an isotonic phosphate buffer (pH 7.0), and the cell screening substance was added dropwise little by little. The mixture was stirred at 4 ° C for 48 hours to introduce an azido group into the cell screening substance.

Here, insulin, basic fibroblast growth factor (basic-FG)
F), an azide group was introduced into each using epidermal growth factor (EGF) and transforming growth factor β (TGF-β). After washing the ink cartridge with purified water, a 50% methanol solution is used to dilute a phosphate isotonic buffer (pH 7.0) containing each cell screening substance into which a functional group has been introduced, to a concentration of 50 μg / ml. Was charged.

Subsequently, as shown in FIG. 1, each cell screening substance 12 was discharged onto a polyethylene terephthalate (PET) film base 11 by an ink jet printer. Each cell screening substance 12
As shown in FIG. 2, the insulin-immobilized region 121, the basic fibroblast growth factor-immobilized region 122, and the epithelial cell growth factor-immobilized region 12 do not overlap each other.
3. Discharged to the transforming growth factor fixing region 124, respectively. After the droplets were dried, the surface of the base 11 was irradiated with ultraviolet rays using an ultraviolet lamp to immobilize the cell screening substance 12. To remove unreacted cell screening substance 12, a phosphate isotonic buffer (pH =
7.0), the base 11 was washed. Thus, the cell screening substrate 1 was produced.

The cells were screened on the cell screening substrate 1.

A screening substrate previously sterilized with a germicidal lamp was placed in a glass Petri dish, and DMEM (Dulbecco's Modified Eagle's minimum esse) containing 10 μg / ml Transferrin was added.
ntial medium) medium was used as the culture solution. Vascular endothelial cells were cultured on the screening substrate, and cultured at 37 ° C. for 24 hours in humid air containing 5% CO ₂ . Also, in the culture solution,
³ H- thymidine was added, in order to assess the degree of proliferation was determined by radiation dose amount of ³ H of ³ H- thymidine incorporated into cells by proliferation after the culture.

When the substrate after culturing was observed under a microscope, it was observed that the cells were growing in the region where insulin, basic-FGF and EGF were immobilized. However, no cell proliferation was observed in the TGF-β immobilized region.

When the growth density of the cells was determined from the ³ H radiation dose, the insulin-immobilized area was 10,000 cells / mm ² , b
The asic-FGF immobilized region, in 6000 / mm ^2, EGF immobilization region was 8,000 / mm ^2. In the TGF-β immobilized region, the number was 100 cells / mm ² . From the above results, it was found that insulin, basic-FGF and EGF had a growth effect on vascular epithelial cells, and TGF-β had no growth effect.

Example 2 (Cell screening method for evaluating growth / differentiation factors of cells) Azide was added to insulin-like growth factor-1 (IGF-1), basic FGF122, EGF and TGF-β by the immobilized functional group introduction method. A group was introduced. Each cell screening substance was discharged onto a PET film using an inkjet printer in the same manner as in Example 1, and immobilized on a substrate. At this time, the arrangement pattern is as shown in FIG. As shown in FIG. 3, a single immobilization region 12a in which four cell screening substances are individually immobilized on a substrate, and a two-substance immobilization in which two cell screening substances are immobilized in close proximity. There is an area 12b. In this way, on the same substrate,
The ability to easily form a plurality of regions in which the relationship of positions at which more than one kind of cell screening substances are immobilized is changed is one of the great advantages of using a droplet discharging means for applying a cell screening substance to a substrate. One.

The chicken skeletal muscle cells were cultured on this substrate. Using a DMEM medium supplemented with 10 μg / ml fibronectin as a culture solution, culture was performed under the same conditions as in Example 1. Amersham Cell proliferat
Evaluation was performed using an ion kit. This is 5-bromo-2'-deoxyu
ridine (BrdU) fluorescein isothiocyanate (FI
TC) The amount of DNA synthesis is evaluated by a fluorescent antibody method using a labeled antibody. In addition, the cells after culture were stained to evaluate the proliferation density. In this procedure, cells after culturing were treated with methanol for 30 minutes, dried, then Hoechst33258 was diluted 10,000 times, reacted for 5 minutes, and stained for nuclei. Excess stain was washed with an isotonic phosphate buffer. This substrate was placed on a slide glass, glycerin was dropped, and a cover glass was placed thereon. Observe the substrate prepared in this way with a fluorescence microscope,
The number of stained nuclei was evaluated. In addition, the number of nuclei containing BrdU-labeled DNA was quantified by a fluorometric method. As a result, with IGF-1 and EGF, it was observed that the amount of fluorescence increased and the nuclei of the cells were dense. This indicates that cell proliferation and differentiation are promoted. Also, basi
With cFGF, the amount of fluorescence was increased, but the cell nuclei were dispersed. This indicated that proliferation was activated but differentiation was not promoted. For TGF-β, no significant increase in the amount of fluorescence was observed. Therefore, it is considered that the proliferation was not promoted. In the region where the two cell screening substances were combined, the increase in the amount of fluorescence was not observed in the region where IGF-1 and EGF, which were active for proliferation and differentiation, were combined, and proliferation was suppressed. Was. As described above, it was found that when used alone, an inhibitory effect is exhibited when an active cell screening substance is also combined.

Example 3 (Allergen Screening Method) In this example, a substance that could become an allergen was immobilized on a substrate, and an inflammation-inducing substance produced from cells when cells collected from a human were cultured on the substrate. By quantifying a certain histamine, it is evaluated whether the person has an allergic predisposition.

In this embodiment, cedar pollen as shown in FIG.
Substrates were prepared to evaluate whether they were allergic to milk, house dust, and wheat. A two-substance immobilized region 12b in which each allergen is immobilized close to each other in order to be able to diagnose whether allergens develop due to a synergistic effect of the region 12a in which each allergen is immobilized alone and a plurality of allergens. , Three substance immobilization area 12
c, a four-substance immobilized region 12d was provided.

The cedar pollen, house dust, and wheat were sufficiently ground using a homogenizer in advance. Then, after the aqueous solution containing each allergen was centrifuged, the dissolved components excluding the sediment were fixed.
Subsequently, each allergen solution was diluted with a 50% methanol solution to a concentration of 50 μg / ml, and the concentration of P was determined by the method described in Example 1.
It was immobilized on an ET film using an inkjet printer. The immobilization pattern was in accordance with FIG. The non-immobilized region was covered with bovine serum albumin so as to prevent non-specific adsorption.

The cells are obtained by collecting blood from the person to be evaluated,
Blood components were separated by density gradient centrifugation, and allergic cells were removed.

The culture was diluted with D containing 10% fetal bovine serum (FBS).
The cells were cultured on a substrate with an anti-histamine antibody (rabbit) added as a MEM medium.

The substrate after the culture was taken out and washed with a phosphate isotonic buffer. After treatment with methanol for 30 minutes and drying, the amount of histamine was quantified from horseradish peroxidase-labeled anti-rabbit Ig antibody by a change in the absorbance of o-phenylenediamine by the enzyme antibody method. As a result, a large amount of histamine was detected in the region where the substance extracted from house dust was immobilized, and this person is considered to be allergic to house dust. Although cedar pollen, milk, and wheat are considered negative, histamine was detected in large amounts in areas where milk and wheat were fixed in close proximity. It turned out that it could develop.

As described above, by using the present substrate, it is possible to diagnose what allergy is present. In particular, allergic reactions to a plurality of allergens can be diagnosed as in the present embodiment. Example 4 (Proliferation / differentiation screening substrate based on density of immobilized substance) The effect of EGF and TGF-β on cell proliferation of NRK cells, which are fibroblasts, was examined. According to Example 1, an azide group was previously introduced into each of the two cell growth factors EGF and TGF-β. As the substrate, a PET film was used, and each cell growth factor was discharged onto the substrate according to FIG. 5 using an inkjet printer so as to have a combination having different densities depending on the position of the substrate. The ejection amount was controlled by setting the dot density of drawing data transmitted from a computer connected to a printer in each area. The numbers in FIG. 5 indicate the relative dot densities of the respective growth factors in the row direction or the column direction. In the area where the two numbers intersect, each growth factor is fixed at the dot density set by these numbers. It indicates that

NRK cells were cultured on the substrate thus prepared. The culture solution is supplemented with BrdU and 0.5 wt% FBS
The cells were cultured at 37 ° C. for 48 hours in a DMEM medium containing 5% CO ₂ in humid air at a concentration of 5%.

After culture, Amersham Cell proliferation
Evaluation was performed using the kit. The evaluation results are as shown in Table 1.

[0078]

【table 1】

[0079] NRK cells are considered to be promoted in growth by the sole action of EGF and TGF-β, but it is considered that the combination of the two exerts a growth inhibitory effect. Thus, from the effect of EGF and TGF-β on the cell proliferation of NRK cells, it was found that it is better to culture in a culture medium in which EGF and TGF-β do not coexist.

[0080]

The cell screening substrate of the present invention can reliably immobilize a cell screening substance at a desired position in a simple step, and can maintain cell adhesion, proliferation, differentiation, survival, undifferentiated state, and cell death. And a substance that affects at least one of the substance production can be screened.

According to the cell screening method of the present invention,
It is possible to examine the effect of various substances on cells on a single substrate, and to examine the difference in the effect of various substances on various cells.

[Brief description of the drawings]

FIG. 1 is an example of a method for manufacturing a cell screening substrate of Example 1.

FIG. 2 is an example of a method for arranging cell screening substances on the cell screening substrate of Example 1.

FIG. 3 is an example of a method for arranging cell screening substances on the cell screening substrate of Example 2.

FIG. 4 is an example of a method for arranging allergens on an allergen screening substrate of Example 3.

FIG. 5 is an example of a method for arranging cell screening substances on the screening substrate of Example 4.

FIG. 6 is an example of a cell screening device according to the present invention.

FIG. 7 is an example of a cell screening substrate manufacturing means of the cell screening apparatus according to the present invention.

DESCRIPTION OF SYMBOLS 1 Cell screening substrate 11 Base substrate 12 Cell screening substance 12a Single immobilization area 12b Material immobilization area 12c Material immobilization area 12d Material immobilization area 13 Microdroplet ejection means 15 Ultraviolet irradiation lamp 121 Insulin immobilization Region 122 Basic fibroblast growth factor immobilized region 123 Epithelial cell growth factor immobilized region 124 Transforming growth factor β immobilized region

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Kohei Watanabe 4-14-3 Motomachi Jindaiji, Chofu City, Tokyo F-term (reference) 2G045 BB20 BB48 CB01 DA13 FB03 FB08 FB12 GC10 GC15 2G054 AA08 AB04 CE02 EA03 EA04 GA02 4B029 AA07 BB11 CC01 FA04 FA09 4B063 QA01 QA05 QA18 QA19 QQ08 QR48 QR66 QR77 QR82 QX02 QX07

Claims (20)

[Claims] 1. A cell screening substrate having a plurality of regions having different cell screening functions by arranging and immobilizing a plurality of cell screening substances in desired regions on a base by means of microdroplet discharge means. . 2. The cell screening substrate according to claim 1, wherein the plurality of regions include a plurality of regions having different combinations of immobilized cell screening substances. 3. The cell screening substrate according to claim 1, wherein the plurality of regions include a plurality of regions having different densities of immobilized cell screening substances. 4. The method according to claim 1, wherein each region or a group of regions formed from two or more regions is formed in the recess.
4. The cell screening substrate according to any one of items 1 to 3. 5. The cell screening substrate according to claim 1, wherein each region or a group of regions formed from two or more regions is surrounded by a convex wall-shaped structure. 6. arranging each cell screening substance in a desired area on a base by a microdroplet discharging means;
A method for producing a cell screening substrate, comprising a step of immobilizing a cell screening substance in each region on the base. 7. The method for producing a cell screening substrate according to claim 6, wherein an ejection means by a thermal inkjet method is used as the minute droplet ejection means. 8. The method for producing a cell screening substrate according to claim 6, wherein an ejection means by a piezo inkjet method is used as the minute droplet ejection means. 9. The method for producing a cell screening substrate according to claim 6, further comprising a step of externally applying energy for fixing to the fixing step. 10. A cell screening method using the cell screening substrate according to any one of claims 1 to 5, wherein the cells are cultured in a culture solution in contact with a cell screening substance fixing region of the cell screening substrate. A cell screening method, comprising the step of: 11. The cell screening method according to claim 10, further comprising a step of replenishing a substance necessary for screening the cells into a culture solution in contact with the fixed region. 12. The cell screening method according to claim 10, wherein the fixed region is in contact with the flow of the culture solution. 13. The cell screening method according to claim 10, further comprising a step of observing a morphological change of the cell. 14. The method according to claim 13, wherein the cells are stained during the evaluation.
3. The cell screening method according to item 1. 15. The cell screening method according to claim 10, further comprising a step of quantifying the substance synthesized in the cells. 16. The cell screening method according to claim 10, further comprising a step of quantifying the substance taken up into the cells. 17. The cell screening method according to claim 15, further comprising a step of quantifying the substance by at least one of radiation dose measurement, fluorescence dose measurement, luminescence measurement, and absorbance measurement. 18. A cell screening apparatus using the cell screening substrate according to claim 1, wherein cells are cultured in a culture solution that comes into contact with a cell screening substance-fixed region of the cell screening substrate. A cell screening device comprising a means. 19. The method according to claim 19, further comprising at least one of a means for evaluating a change in cell morphology by said culturing means, a means for quantifying a substance synthesized in the cell, and a means for quantifying a substance taken up in the cell. Item 19. The cell screening device according to Item 18. 20. The cell screening device according to claim 18, further comprising means for manufacturing the cell screening substrate.