JP2004271337A - Multi-specimen simultaneous analysis system for cell using surface plasmon resonance phenomenon - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a system, for easily calculating the number of cells contained in a cell group immobilized on a plate. <P>SOLUTION: The method for analyzing the cell includes a step (a) of measuring the intensity values of reflected light being caused by the cell immobilized on the plate by using a surface plasmon resonance imaging method, and a step (b) of calculating parameters relating to the cell from the intensity values of the reflected light. In addition, the system for calculating the parameter of the cell is provided with the plate (a) used for immobilizing the cell, and a surface plasmon resonance imaging apparatus (b) used for observing the plate. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method and a system for accurately measuring a parameter of a cell using a surface plasmon resonance method.
[0002]
[Prior art]
Examining the phenotypic properties of cells based on the interaction between cells and various biomolecules and examining the changes in cells caused by the administration of biomolecules are central issues in cell biology. In general, the effect is investigated by using cells cultured in vitro and adding biomolecules.
[0003]
In recent years, with the advance of combinatorial chemistry, low-molecular-weight drugs having bioactivity and candidate drugs for peptides have been comprehensively synthesized, and an enormous database on genomes and proteins has been constructed. This information is expected to be useful for finding new drugs and understanding complex molecular networks, but the actual functions and actions are often unknown and must be clarified experimentally in the future. Must. However, the conventional method as described above requires a large number of experimental systems, and thus is not suitable for comprehensively analyzing the interaction between each of a huge variety of molecules and cells.
[0004]
The most basic operation in examining the interaction between a biomolecule and a cell is to count the number of interacting cells or measure the number of cells that have proliferated as a result of the interaction. Conventionally, cell counting has been performed directly under a microscope or using a hemocytometer after preparing a uniform cell dispersion. It has also been practiced to determine the DNA content from the amount of radioactive substance taken up and to estimate the number of cells. These are time-consuming and time-consuming methods, such as time-consuming cell counting and the need to label with dyes and radioactive materials. In this sense, it is unsuitable to measure information on the number of many samples at once, and it was not possible to simultaneously detect other information such as morphology while alive. For example, various methods for counting the number of cells interacting with biomolecules make it difficult to measure many types of specimens simultaneously and quickly. A major problem is that it takes time and effort, such as time-consuming cell counting and labeling with a dye and a radioactive substance. The present invention utilizes a microarray substrate on which biomolecules are immobilized, so that cells interacting with the substrate surface can be counted alive or after performing operations such as immobilization, and information on their morphology can be obtained. The present invention provides an apparatus system for obtaining the characteristics, and utilizes a surface plasmon resonance phenomenon for this purpose.
[0005]
Surface plasmon resonance (SPR) is an interaction that occurs between surface plasmons (elastic waves) generated on a metal surface and evanescent waves (light waves) generated by totally reflected electromagnetic waves. Resonance occurs at an incident angle θ of light that provides a condition that the wave number and the wave vector of the plasmon wave and the evanescent wave approximately coincide with each other, and the intensity of the reflected light decreases because the evanescent wave is used for exciting surface plasmons. In order to obtain surface plasmon resonance, a method of arranging a prism made of a medium having a high refractive index as shown in FIG. 1A (Kretschmann arrangement) and injecting laser light and LED light is used. Here, the wave number of the plasmon wave changes due to a change in the dielectric constant of the medium in contact with the metal surface on the opposite side of the prism. That is, when a substance approaches a metal surface, the incident angle of light that gives surface plasmon resonance shifts. By utilizing this fact, it is possible to sense the coating of the metal surface with the substance. This measuring method is excellent in the resolution in the surface vertical direction (on the order of 0.1 nm), and the amount of the substance existing on the surface is determined in ng to pg / cm²It is possible to observe in real time on the order of. In addition, being able to measure in an aqueous medium is also a great advantage in examining the behavior of biomolecules such as proteins. A measuring device utilizing this has been developed as a biomolecular interaction measuring device, and is applied to the analysis of the interaction of proteins and DNA.
[0006]
For the purpose of simultaneously detecting various types of interactions using the above-described surface plasmon resonance analyzer, imaging devices improved from the same have been developed (Non-Patent Documents 1 to 3). In this case, as shown in FIG. 1 (B), uniform parallel light is incident on the sample, and the totally reflected light is captured as a two-dimensional image using a CCD (charge coupled device) camera, and the local dielectric on the sample surface is measured. The change in rate is obtained as image information. However, conventionally, although an attempt was made to measure cells, it was not possible to obtain parameters relating to cells.
[0007]
[Non-patent document 1]
W. Hickel, D.S. Kamp, and W.W. Knoll, Nature, 339, 186, 1989
[0008]
[Non-patent document 2]
W. Hickel and W.C. Knoll, J .; Appl. Phys. , 67, 3572, 1990.
[0009]
[Non-Patent Document 3]
B. P. Nelson, T .; E. FIG. Grimsrud, M .; R. Liles, R.A. M. Goodman, R .; M. Corn, Anal. Chem. 73, 1-7, 2001
[0010]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a method and a system for easily calculating the number included in a cell population fixed on a plate. In particular, it is another object of the present invention to devise an analysis system in which a microarray substrate and a surface plasmon resonance imaging apparatus are combined, and to measure information on many biomolecule-cell interactions at once.
[0011]
[Means for Solving the Problems]
In the present invention, by using a surface plasmon resonance imaging apparatus, multipoint simultaneous counting of cells interacting with the surface of the microarray substrate is performed, and by evaluating their morphology, the behavior of the refractive index and the number of cells can be measured. This was achieved by finding unexpectedly 1: 1 correspondences of individual cell parameters. In the measurement of cells, the versatility and usefulness of this analysis system is improved by devising a device that allows observation while culturing living cells, and setting specifications of an optical system to improve the accuracy of image data. The effect of enhancing the performance was achieved.
[0012]
A microarray in which biomolecules are fixed in the form of microspots is created, and by using this, the interaction between cells and various biomolecules can be performed simultaneously, and the capture, adhesion, and morphology of cells that occur as a result of the interaction The changes can be analyzed at once using a surface plasmon resonance imaging device.
[0013]
The present invention also makes it possible to obtain parameters such as information on the number and morphology of cells interacting with the microarray substrate by applying such a principle of surface plasmon resonance imaging.
[0014]
Accordingly, the present invention provides the following.
[0015]
(1) A method for analyzing cells,
(A) measuring the reflected light intensity due to the cells fixed on the plate using surface plasmon resonance imaging; and
(B) calculating a parameter relating to the cell from the reflected light intensity;
A method comprising:
[0016]
(2) The method according to claim 1, wherein the parameter is a cell number, a cell adhesion area, or a cell size.
[0017]
(3) The method according to claim 1, wherein the step (b) further includes calculating a parameter for the cell by evaluating the reflected light intensity using a known standard curve for the cell.
[0018]
(4) The method according to claim 1, further comprising a step of preparing a standard curve for the cells.
[0019]
(5) The method according to claim 1, wherein the step (b) includes converting the reflected light intensity measured by a surface plasmon resonance imaging device into the number of adherent cells.
[0020]
(6) The method according to claim 1, wherein the step (b) includes obtaining information on cell morphology from reflected light intensity measured by a surface plasmon resonance imaging device.
[0021]
(7) The method according to claim 1, wherein the surface plasmon resonance imaging includes producing surface plasmon resonance on a metal thin film using a surface light source having a wavelength in a visible to near infrared region.
[0022]
(8) The method according to claim 1, wherein the intensity of the reflected light is obtained by capturing an image of the intensity of the reflected light based on a difference in refractive index between the cell adhered to the thin film surface and the medium as an image with a CCD camera.
[0023]
(9) The method according to claim 1, wherein the plate is a microarray.
[0024]
(10) The method according to claim 1, wherein the plate includes a glass substrate having a metal thin film containing gold, silver or aluminum on one surface.
[0025]
(11) The method according to claim 1, wherein the factor that specifically binds to the cells is immobilized on the plate in a spot shape of 0.01 mm to 10 mm by covalent bond or physical interaction.
[0026]
(12) The method according to claim 1, wherein the plate is a microarray in which biomolecules are immobilized, and the biomolecules recognize a CD antigen, a cell adhesion factor, a cell growth factor receptor, a cytokine receptor, or a sugar chain. .
[0027]
(13) The method according to claim 1, wherein the cells are allogeneic or heterologous.
[0028]
(14) The above-mentioned cells are normal cells of various organs, differentiated cells such as diseased cells, and their precursor cells, embryonic stem cells, fetal stem cells, undifferentiated cells, established cells, or cells with modified genes. The method of claim 1, comprising:
[0029]
(15) A system for calculating a parameter of a cell, the system comprising:
(A) a plate for fixing the cells; and
(B) a surface plasmon resonance imaging device for observing the plate,
A system comprising:
[0030]
(16) The system according to claim 14, wherein the plate is a microarray.
[0031]
BEST MODE FOR CARRYING OUT THE INVENTION
(Summary of the Invention)
The present invention relates to an apparatus for simultaneously and quickly analyzing a large number of specimens for the interaction between biomolecules such as proteins, peptides, nucleic acids, polysaccharides and low molecular weight bioactive substances and various animal cells. About the system. Although the number of samples that can be analyzed is not limited in principle, in practice, several tens to several thousands of samples are simultaneously analyzed. In the analysis by this apparatus system, different types of biomolecules are respectively fixed to spots on a substrate called a microarray, and then the spots are brought into contact with cells, and the resulting biomolecule-cell interaction analysis is performed. I do. The mode of interaction is cell capture based on physicochemical and / or biological interactions, cell adhesion and subsequent spreading via cell adhesion factors, cell proliferation and cell growth based on the action of biomolecules. Many phenomena can be targeted, such as death. More specifically, this device system is characterized by using the principle of surface plasmon resonance imaging in order to analyze cells on a spot, and to simultaneously count cells interacting with a substrate at multiple points, and to provide information on the morphology. By obtaining, it is possible to comprehensively analyze the biomolecule-cell interaction. This apparatus system can be used not only as a high-throughput analysis means in cell biology research and biomaterial development, but also for clinical tests and environmental investigations.
[0032]
(Detailed description)
Hereinafter, the present invention will be described. It is to be understood that throughout this specification, the use of the singular includes the plural concept unless specifically stated otherwise. It is to be understood that the terms used in the present specification are used in a meaning commonly used in the art unless otherwise specified.
[0033]
(Terms and general techniques)
Hereinafter, definitions of terms particularly used in this specification and general techniques will be described.
[0034]
(Substrate / plate / chip / array)
As used herein, "plate" refers to a planar support to which molecules such as antibodies can be immobilized. In the present invention, the plate is preferably made of a glass substrate having a metal thin film containing gold, silver or aluminum on one surface.
[0035]
As used herein, "substrate" refers to the material (preferably solid) from which the chip or array of the invention is constructed. Thus, the substrate is included in the concept of a plate. The material of the substrate may be any solid that has the property of binding to the biomolecule used in the present invention, or that may be derivatized to have such property, either covalently or non-covalently. Materials.
[0036]
For such materials for use as plates and substrates, any material capable of forming a solid surface can be used, for example, glass, silica, silicon, ceramic, silicon dioxide, plastic, metal (including alloys). ), Natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon), including but not limited to: The substrate may be formed from multiple layers of different materials. For example, inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used. Also, polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin Organic materials such as polycarbonate, polyamide, phenolic resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone can be used. In the present invention, a membrane used for blotting, such as a nylon membrane, a nitrocellulose membrane, or a PVDF membrane, can also be used. When analyzing a high-density material, it is preferable to use a material having hardness, such as glass. A preferable material for the substrate varies depending on various parameters such as a measuring instrument, and a person skilled in the art can appropriately select an appropriate material from the various materials described above.
[0037]
As used herein, the term “chip” or “microchip” is used interchangeably, has a variety of functions, and refers to a microminiature integrated circuit that becomes part of a system. Examples of the chip include, but are not limited to, a DNA chip and a protein chip.
[0038]
As used herein, the term “array” refers to a substrate (eg, a chip) having a pattern or a pattern in which one or more (eg, 1000 or more) biomolecules (eg, DNA, proteins such as antibodies) are arranged and arranged. It refers to itself. Among the arrays, those patterned on a small substrate (for example, on a 10 × 10 mm) are referred to as microarrays, but in the present specification, microarrays and arrays are used interchangeably. Therefore, a micropattern that is patterned to be larger than the above-mentioned substrate may be called a microarray. For example, an array is composed of a set of desired antibodies that are themselves immobilized on a solid surface or membrane. The array preferably contains at least 10 identical or different antibodies.², More preferably at least 10³, And more preferably at least 10⁴, Even more preferably at least 10⁵Including These antibodies are preferably placed on a surface of 125 × 80 mm, more preferably 10 × 10 mm. As a format, a size of a microtiter plate such as a 96-well microtiter plate or a 384-well microtiter plate, to a size of a slide glass is contemplated. One or a plurality of antibodies may be immobilized. The number of such types can be any number from one to the number of spots. For example, about 10, about 100, about 500, and about 1000 antibodies can be immobilized.
[0039]
Any number of biomolecules (eg, proteins such as antibodies) can be disposed on a solid surface or membrane, such as a substrate, as described above, but typically, 10 per substrate.⁸Up to 10 biomolecules, in other embodiments up to 10⁷Up to 10 biomolecules⁶Up to 10 biomolecules⁵Up to 10 biomolecules⁴Up to 10 biomolecules³Up to 10 biomolecules, or 10²Up to 10 biomolecules can be placed, but 10⁸Biomolecules exceeding the number of biomolecules may be arranged. In these cases, the size of the substrate is preferably smaller. In particular, the spot size of a biomolecule (eg, a protein such as an antibody) can be as small as the size of a single biomolecule (which can be on the order of 1-2 nm). The minimum substrate area is in some cases determined by the number of biomolecules on the substrate. In the present invention, the factor that specifically binds to a cell is usually immobilized in a spot form of 0.01 mm to 10 mm by covalent bond or physical interaction.
[0040]
On the array, “spots” of biomolecules may be arranged. As used herein, “spot” refers to a certain set of biomolecules. As used herein, “spotting” refers to producing a spot of a certain biomolecule on a certain substrate or plate. Spotting can be performed in any manner, for example, by pipetting or the like, or can be performed by automated equipment, such methods being well known in the art. The biomolecule can be, for example, one that can recognize a CD antigen, a cell adhesion factor, a cell growth factor receptor, a cytokine receptor or a sugar chain.
[0041]
As used herein, the term "address" refers to a unique location on a substrate that may be distinguishable from other unique locations. The address is suitable for association with the spot with that address, and may take any shape, such that the entity at every each address can be distinguished (eg, optical) from the entity at another address. The address defining shape may be, for example, a circle, an ellipse, a square, a rectangle, or an irregular shape. Therefore, “address” indicates an abstract concept, and “spot” may be used to indicate a specific concept. However, when there is no need to distinguish between the two, in the present specification, “address” is used as “address”. “Spot” may be used interchangeably.
[0042]
The size defining each address is, inter alia, the size of the substrate, the number of addresses on a particular substrate, the amount of analyte and / or reagents available, the size of the microparticles and any array in which the array is used. Depends on the degree of resolution required for the method. The size can be, for example, in the range of 1-2 nm to several cm, but any size consistent with the application of the array is possible.
[0043]
The spatial arrangement and shape defining the address are designed to suit the particular application in which the microarray is used. The addresses can be closely spaced, widely distributed, or sub-grouped into a desired pattern appropriate for a particular type of analyte.
[0044]
Microarrays are described in Shujunsha, edited by Cell Engineering, “DNA Microarrays and the Latest PCR Method”, F. See Templin, et al. , Protein microarray technology, Drug Discovery Today, 7 (15), 815-822 (2002).
[0045]
Since the data obtained from microarrays is enormous, data analysis software for managing the correspondence between clones and spots and performing data analysis is important. As such software, software attached to various detection systems can be used (Ermolaeva O et al. (1998) Nat. Genet. 20: 19-23). The format of the database includes, for example, a format called GATC (Genetic analysis technology consortium) proposed by Affymetrix.
[0046]
For microfabrication, see, for example, Campbell, S.M. A. (1996). The Science and Engineering of Microelectronic Fabrication, Oxford University Press; V. (1996). Microarray Fabrication: a Practical Guide to Semiconductor Processing, Semiconductor Services; Madou, M .; J. (1997). Fundamentals of Microfabrication, CRC15 Press; Rai-Choudhury, P .; (1997). Handbook of Microlithography, Micromachining, & Microfabrication: Microlithography, and the like, the relevant portions of which are incorporated herein by reference.
[0047]
Various methods such as a microcontact printing method and a photolithography method can be used for producing a microarray, but a method using a micropatterned surface of an alkanethiol monomolecular film is preferable. In this case, first, a monomolecular film of alkanethiol having a hydrophobic functional group such as a methyl group or a fluoromethyl group is formed on a glass substrate having a gold thin film deposited on one surface. A photomask on which a number of light-transmitting spots each having a diameter of several μm to about 1 mm is arranged on the monomolecular film is irradiated with ultraviolet rays. Thereby, the alkanethiol in the irradiated part can be decomposed and removed in the form of a spot. Next, a monomolecular film of alkanethiol having a reactive functional group such as a carboxyl group, an amino group, an aldehyde group, an imide group, an acid chloride, an epoxy group, and an isocyanate group is formed in the exposed gold spot. Using the reactive functional group introduced into the spot, a test substance such as a protein, DNA, polysaccharide, peptide, or synthetic drug is chemically immobilized. For example, in the case of a carboxyl group-containing spot, immobilization can be performed by converting a carboxyl group into an active ester using N-hydroxysuccinimide and then dropping a small amount of a biomolecule-containing solution onto each spot. . The hydrophobic monomolecular film formed around the spot is effective for suppressing the diffusion of the solution. Blocking is performed with an inactive protein such as bovine serum albumin or a hydrophilic polymer such as polyethylene glycol to suppress non-specific interaction between the background area around the spot and the cells.
[0048]
As can be seen from the progress of analysis technology using DNA microarrays, protein chips, etc., microarrays can perform high-throughput analysis on many samples on a single substrate, making it extremely effective Means. The present invention applies such a microarray concept to quickly measure various types of biomolecule-cell interactions. In this case, integration of microarrays is important in order to simultaneously analyze an extremely large number of specimens and to minimize the amount of biomolecules and cells required for the analysis. On the other hand, when acquiring information on cells on the microarray, only data with a large error can be obtained unless measurement is performed on a population consisting of a certain number of cells or more. From such a viewpoint, the size of each spot constituting the microarray is desirably a size that allows at least several tens to several thousands of cells to interact with each other.For example, in the case of a circular spot, Its diameter is about several μm to about 1 mm.
[0049]
Various methods such as a microcontact printing method and a photolithography method can be used for producing a microarray, but a method using a micropatterned surface of an alkanethiol monomolecular film is preferable.
[0050]
As used herein, the term “biomolecule” refers to a molecule associated with a living organism. As used herein, the term “organism” refers to a biological organism, and includes, but is not limited to, animals, plants, fungi, viruses, and the like. A biomolecule includes, but is not limited to, a molecule extracted from a living body, and is included in the definition of a biomolecule as long as it is a molecule that can affect a living body. Such biomolecules include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, including DNA such as cDNA, genomic DNA, RNA such as mRNA), polysaccharides. , Oligosaccharides, lipids, small molecules (eg, hormones, ligands, signal transducers, small organic molecules, combinatorial library compounds, etc.), and composite molecules thereof, but are not limited thereto.
[0051]
(cell)
As used herein, a "cell" is defined in the broadest sense as used in the art, and is a whole unit of a tissue of a multicellular organism or a single-celled organism, a membrane structure that isolates the outside world. An organism that has a self-renewal ability inside and has genetic information and its expression mechanism. The cells used herein may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells). The source of cells can be, for example, a single cell culture or such as cells from embryos, blood, or body tissues of a normally grown transgenic animal, or cells from a normally grown cell line. Examples include, but are not limited to, cell mixtures, or bacterial cells.
[0052]
As used herein, the term “stem cell” refers to a cell that has self-renewal ability and has pluripotency (ie, pluripotency) (“pluripotency”). Stem cells can usually regenerate tissue when it is damaged. As used herein, a stem cell can be, but is not limited to, an embryonic stem (ES) cell or a tissue stem cell (also referred to as a tissue stem cell, a tissue-specific stem cell, or a somatic stem cell). In addition, artificially created cells (eg, the fused cells, reprogrammed cells, etc. described herein) can also be stem cells, as long as they have the above-mentioned ability. Embryonic stem cells refer to pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981, and have been applied to the production of knockout mice since 1989. Human embryonic stem cells were established in 1998 and are being used in regenerative medicine. Tissue stem cells, unlike embryonic stem cells, are cells in which the direction of differentiation is limited, are present at specific positions in tissues, and have an undifferentiated intracellular structure. Thus, tissue stem cells have a low level of pluripotency. Tissue stem cells have a high nucleus / cytoplasm ratio and poor intracellular organelles. Tissue stem cells generally have pluripotency, have a slow cell cycle, and maintain their proliferative potential for more than an individual's life.
[0053]
As used herein, “somatic cells” are cells other than germ cells such as eggs and sperm, and refer to all cells that do not directly deliver their DNA to the next generation. Somatic cells usually have limited or lost pluripotency. The somatic cells used herein may be naturally occurring or genetically modified.
[0054]
As used herein, the term “isolated” refers to a substance that naturally accompanies at least a reduced amount in a normal environment, and preferably is substantially free from the substance. Thus, an isolated cell refers to a cell that is substantially free from other attendant materials (eg, other cells, proteins, nucleic acids, etc.) in the natural environment. When referring to nucleic acids or polypeptides, "isolated" refers to, for example, substantially free of cellular material or culture media when produced by recombinant DNA technology, or precursor when chemically synthesized. Refers to nucleic acids or polypeptides substantially free of somatic or other chemicals. An isolated nucleic acid preferably includes sequences that naturally flank the nucleic acid in the organism from which the nucleic acid is derived (ie, sequences located at the 5 ′ and 3 ′ ends of the nucleic acid). Absent. In the present invention, the cells are preferably isolated at the time of measurement.
[0055]
As used herein, an “established” or “established” cell is one in which a particular property (eg, pluripotency) is maintained and the cell continues to proliferate stably under culture conditions. State. Therefore, the established stem cells maintain pluripotency. In the present invention, the use of the established cells is preferable because the properties are constant, the coefficient for calculating the linearity with the reflected light intensity is easy to specify, and the coefficient hardly fluctuates. It is preferable to use established stem cells as a measurement target because a step of newly collecting stem cells from a host can be avoided.
[0056]
Cells used in the present invention may be cells from any organism (eg, unicellular organisms of any type (eg, bacteria, yeast) or multicellular organisms (eg, animals (eg, vertebrates, invertebrates), plants ( For example, monocotyledonous plants, dicotyledonous plants, etc.) may be used. For example, cells from vertebrates (e.g., black lampreys, lampreys, cartilaginous fish, teleost fish, amphibians, reptiles, birds, mammals, etc.) are used, and more specifically, mammals (e.g., monopores, Marsupials, oligodonts, winged wings, winged fins, carnivores, insectivores, longnoses, equinoids, artiodactyls, tubulars, scales, sponges, whales, primates , Rodents, lagomorphs, etc.). In one embodiment, cells from primates (eg, chimpanzees, Japanese macaques, humans), particularly cells from humans, are used, but are not limited thereto.
[0057]
As used herein, “cell parameters” refers to various elements that specify the shape, properties, etc. of a cell. Such parameters include, for example, cell size, number, density, shape (spherical, fibrous, etc.), internal state (differentiation state, etc.), cell surface state (marker expression, etc.), cell adhesion area And the like, but not limited thereto. Important parameters of the present invention include, for example, the size and number of cells. In the method of the present invention, it has been unexpectedly found that the size of such cells and the number in a population can be calculated in an accurate manner from the reflected light intensity, and therefore, the cells can be visually observed or the like. No counting step is required.
[0058]
Among the parameters of the present invention, secondary information such as the shape can also be estimated from the reflected light intensity in the method of the present invention. Examples of such a method include, but are not limited to, a method in which extension and flattening accompanying cell adhesion are read from an increase in reflected light intensity. As an exemplary method, for example, cells are initially adhered onto a spot having a biomolecule promoting cell adhesion such as fibronectin or collagen fixed on the surface, and the SPR reflected light intensity is measured over time while culturing in the presence of a medium. Methods include, but are not limited to. As the adhesion of the cells progresses, the cells spread, flatten, or extend projections, which increase the contact area of the individual cells with the substrate surface, and are detected as an increase in SPR reflected light intensity. This makes it possible to perform a detailed real-time analysis of the cell adhesion process and to examine in detail the effect of matrix molecules on cell adhesion.
[0059]
In the present specification, the “standard curve” refers to a curve that linearly represents the relationship between the intensity of reflected light and the cell parameter (preferably the number of cells). Such a standard curve is created by measuring a plurality of reflected light intensities, calculating corresponding cell parameters (eg, counting the number), and correlating them (eg, plotting them on a graph). be able to. The creation of such a curve may be performed manually, or may be performed by a computer or the like. Alternatively, in the method of the present invention, if such information is known, a standard curve may simply be provided. By providing such a standard curve, the reflected light intensity can be converted into information on cell number or shape.
[0060]
(Surface plasmon resonance)
As described above, the surface plasmon resonance (SPR) method used in the present specification is a measurement method commonly used in the art. SPR utilizes the interaction of light-surface plasmon waves generated under the condition of total reflection of light in a metal thin film provided on a light-transmitting medium, and the principle of measurement is as follows.
[0061]
Surface plasmon waves are electromagnetic waves generated at the interface between a metal and an insulator. By satisfying the resonance condition determined by the refractive index (or dielectric constant) near the interface between the metal and the insulator and the thickness thereof, such a surface plasmon wave can be generated optically. First, in a light-transmitting medium having a metal thin film, light polarized so that total reflection occurs on the light-transmitting medium surface is incident. At this time, an evanescent wave (light wave) having a wave number with the incident angle of light as a variable is generated at the interface between the metal thin film and the light transmitting medium. On the other hand, a surface plasmon wave is generated on a metal thin film by a tunnel effect of light. When the wave numbers of the evanescent wave and the surface plasmon wave generated on both surfaces of the metal thin film match, surface plasmon resonance occurs, and a part of the energy of the incident light is used as the excitation energy of the surface plasmon wave.
[0062]
According to the law of conservation of energy, the light intensity reflected on the metal thin film surface is the difference between the incident light intensity and the light intensity lost by the excitation of the surface plasmon wave. Therefore, by measuring the incident angle dependence of the reflected light intensity, surface plasmon resonance can be observed. This resonance condition is defined by the wavelength of the incident light, the incident angle, the complex permittivity of the light transmitting medium and the metal thin film, the complex permittivity of the sensor-sensitive film provided on the metal thin film, and the like. If the complex dielectric constant changes due to the cell attachment reaction on the membrane, the resonance condition changes, and under a constant wavelength condition, the incident angle of light at which surface plasmon resonance occurs changes. By obtaining this, the state of the cell can be obtained.
[0063]
Since a surface plasmon wave occurs in a region of about several hundred nm on a metal thin film, a biochemical reaction between a substrate and a cell that causes a change in dielectric constant needs to occur in this region. Therefore, an extremely thin film is sufficient as the sensitive film to which the biological material is fixed. Further, since surface plasmon resonance can measure only the vicinity of the metal thin film, it is possible to measure even a colored sample or a suspended sample without being affected. A protein antigen detection sensor and the like have been put to practical use by utilizing surface plasmon resonance (for example, BIAcore by BIAcore). A person skilled in the art can also perform surface plasmon resonance measurement using such a commercially available device, and may appropriately manufacture a desired product.
[0064]
As described above, surface plasmons can be said to be compression waves of electrons existing on a metal surface, and this speed is limited by contact of the surface with another substance. Then, an evanescent wave is generated on the metal surface by the light incident through the prism, and the entering light angle that resonates with the surface plasmon wave is obtained, whereby the speed of the plasmon wave can be estimated. It has been said that it is possible to know the amount of substances existing on the surface from the change in the speed of plasmon waves.
[0065]
Binarization is also an important consideration. When obtaining information about cells adhered to the substrate surface, binarization of an image is also an important consideration. The signal based on one cell bound to the substrate reflects the information in the vertical direction of the cell, that is, the refractive index of the substance constituting the cell, its distribution, and the total information of the thickness. When trying to obtain the number or morphology of cells from digitized SPR image data, it is necessary that the information of each cell is made uniform horizontally with respect to the substrate. Therefore, the intensity of the incident light at which the surface cotton plasmon resonance occurs due to the binding of cells shifts greatly, that is, the intensity of the reflected light greatly changes when measured using an optical system fixed near the SPR angle. The wavelength of the light source, the refractive index of the prism, the thickness of the gold thin film, etc. must be optimized.
[0066]
In practice, light is incident at an angle slightly smaller than the surface plasmon resonance angle, and the intensity of the reflected light at that time is determined, whereby a change in the surface plasmon resonance angle due to the attachment of a substance can be known. The change in the surface plasmon resonance angle is the intensity of reflected light at the incident angle, and varies depending on the surface occupancy, refractive index, shape, and layer structure of the attached matter. It is also affected by the linearity (reflected light intensity-electric signal) of the detector and the shape of the reflected light-angle spectrum (which greatly varies depending on the wavelength of the light source used, the thickness of the metal film, etc.).
[0067]
It is understood that the relationship between the number of cells adhered to a certain area and the average reflected light intensity depends on the above various parameters. Considering the case of measuring the intensity of reflected light at a certain incident angle, it is necessary to consider the following conditions to define the relationship between the cells adhered to the substrate and the intensity of reflected light.
(1) Whether the area occupied by one cell can be regarded as constant regardless of the cell. (2) Whether the roughness of the cell adhesion surface can be neglected.
(3) Whether the shape of the reflected light spectrum is not significantly changed.
(4) As a comprehensive result of the microstructure of cells (cell membrane, cytoplasm, nucleus, and organelles) and their refractive indices, one cell adhesion is large and constant enough to enable two gradations. Whether to give an angle change.
(5) Whether the resolution of the two-dimensional plane is high enough to give a line between the cell number and the reflected light intensity.
And so on.
[0068]
When considering these conditions, the relationship between the cells adhered to the substrate and the reflected light intensity is strongly influenced by the characteristics of the device and plate (eg, microarray), up to a positive correlation. Even if it is known, for example, it is unknown whether there is linearity or not, but rather it is expected that there is an indefinite relationship.
[0069]
In the present invention, without being bound by theory, it was found that, considering the various conditions described above, the relationship between the cells adhered to the substrate and the intensity of the reflected light was unexpectedly linear. Therefore, in the method of the present invention, cell information can be accurately measured in a 1: 1 relationship only by measuring the intensity of reflected light.
[0070]
Surface plasmon resonance imaging can use a surface light source having a wavelength in the range from visible light to near infrared (light having a wavelength of 520 nm to 3390 nm). Preferably, it may be advantageous to utilize light with a wavelength between 800 nm and 1200 nm. By using such a wavelength, surface plasmon resonance can be advantageously created on the metal thin film.
[0071]
In the present specification, the "reflected light intensity" means that the intensity of the reflected light intensity based on the difference in the refractive index between the cell adhered to the thin film surface and the medium can be captured as an image with a CCD camera. Not limited to that.
[0072]
As the surface plasmon resonance imaging apparatus, an apparatus having a configuration as shown in FIG. 2 is used. It has a horizontal sample stage so that it can be observed even while culturing living cells, and uses white light with little damage to cells as a light source. In order to enhance the accuracy of image data, it is effective to use a CCD camera that can detect light in the near infrared region. For the measurement, a microarray and a prism are arranged in Kretschmann, and uniform parallel light is incident from the back surface of the microarray at an acute angle of about 0.3 to 0.5 degrees from the plasmon resonance angle. In this case, various light sources such as a laser light source, an LED light source, an LSED light source, a halogen lamp, a mercury arc lamp, and an LD light source can be used. However, when using highly coherent light such as laser light, each part of the optical path is used. Care must be taken to avoid diffraction at To obtain a two-dimensional image, a CCD camera is used. In this case, an optical system and a detection system that can detect only near-infrared light having a wavelength of about 900 nm using an interference filter or the like are used. Improves the sensitivity of surface plasmon resonance images.
[0073]
In order to examine the interaction between a biomolecule and a cell, first, a target cell is suspended in an appropriate medium such as a cell culture medium and a phosphate buffer, and the suspension is dropped on a microarray. After the biomolecule-cell interaction, non-interacting cells are removed by washing. The microarray thus obtained is mounted on a surface plasmon resonance imaging apparatus, and a two-dimensional image corresponding to a change in the dielectric constant of the array surface is obtained. Interaction between the biomolecule and the cell occurs, and in the region where the cell adheres, as shown in FIG. 3, it is observed as a region with high reflected light intensity, and conversely, the region where no cell is present is dark. As a result, the presence / absence of cell attachment is obtained as two-graded image data. This is explained by the fact that the surface plasmon resonance spectrum shifts greatly to the high angle side due to cell adhesion as shown in FIG. In other words, when the measurement is performed with the light incident angle fixed near the surface plasmon resonance angle, the surface plasmon resonance gives a low reflected light intensity in the absence of cells, but the reflection at the same angle is caused by the adhesion of the cells. The light intensity increases significantly. As a result, a two-dimensional image such as a binarized image is obtained corresponding to a portion where the cell exists and a portion where the cell does not exist. The two-dimensional image obtained in this manner is read into a computer as digitized information, and the average lightness per unit area in each spot is determined using appropriate software, thereby obtaining an index of the number of cell adhesion. It is possible to obtain.
[0074]
(Description of a preferred embodiment)
Hereinafter, preferred embodiments of the present invention will be described.
[0075]
In one aspect, the present invention provides a method for analyzing a cell. This method comprises the steps of (a) measuring the intensity of reflected light caused by cells fixed on a plate using surface plasmon resonance imaging; and (b) calculating a parameter relating to the cells from the reflected light intensity . Surface plasmon resonance imaging can be manipulated using techniques well known in the art. The fixation of the cells to the plate may be performed directly, or may be performed by previously binding a binding agent (eg, an antibody, a ligand, a small molecule, a sugar chain, etc.) to the plate. The measurement of the reflected light intensity can also be performed using a method well known in the art. Such techniques are described herein above and are described, for example, in "Surface Analytical Technologies for Probing Biomaterial Processes", J. Am. Davies, ed. CRC Press, Boca Raton, FL, 1996; "Biomolecules Sensors", E.C. Gizelli, C .; R. Low eds. B., Taylor & Francis, New York, NY, 2002; P. Nelson, A .; G. FIG. Frutos, J .; M. Brockman, R.A. M. Corn. , Near-infrared surface plasmon resonance measurements of ultrathin films, 1. Angle shift and SPR imaging experiments, Anal. Chem. , 71, 3928-3934 (1999).
[0076]
Here, preferably, the cell parameter calculated in the present invention may be the number of cells, the cell adhesion area, or the cell size. In the present invention, since the reflected light intensity and the cell number are unexpectedly correlated with linearity, a special effect that the cell number can be calculated by simply measuring the reflected light intensity is achieved. Was. This has not been possible in the past, and heretofore, a step of counting the number of cells has been indispensable. Therefore, the present invention has an effect that such complicated steps can be omitted.
[0077]
Such linearity can be represented by the following relationship.
D (D = cell density = cell number / unit area) = kR (R = SPR reflected light intensity)
Here, the coefficient k (cm²/ Cell) varies depending on the type and state of the cell, and may be, for example, 0.1 to 0.2 for lymphocytes, 0.3 to 0.4 for nerve cells, and stromal. For cells, it can be 2-4. This coefficient is a constant value when the prepared cells have a certain property or a similar preparation method. Therefore, it is possible to calculate the density and the number of cells simply by calculating the reflected light intensity. In addition, the coefficients are merely examples, and these values may fluctuate significantly depending on not only the state of the cell but also the specifications of the apparatus, measurement conditions, and the like. Such a variation is within the range understood by those skilled in the art, and the coefficient can be appropriately determined in consideration of these variations.
[0078]
By analyzing the coefficient k, the shape of the cell can be identified. For example, when a standard curve for a cell having a known shape (for example, size) is known, the size of the unknown cell is identified by analyzing the standard curve of the unknown cell and identifying the coefficient k. It is also possible. For example, for the first cell,
D = k₁R
And the size is s₁If it is known that the second cell
D = k₂R
If the relationship becomes clear, its size s₂Is
s₂= (K₂× s₁) / K₁
Can be obtained by
[0079]
In a preferred embodiment, the step (b) in the method of the present invention includes providing a known standard curve for the cell and calculating a parameter for the cell by comparing with the reflected light intensity. By using a known standard curve, cell parameters can be immediately determined. A typical example in which such a known standard curve can be used is when the cells used are normal. Alternatively, step (b) may include generating a standard curve for the cells. Such an approach may be advantageous if a standard curve is not available, or if a standard curve needs to be generated again. More preferably, it is advantageous to check the state of the cells daily by creating a standard curve, and more accurate results can be obtained. It may not be necessary to generate a standard curve for relative values between different cells.
[0080]
In a preferred embodiment, the step (b) in the method of the present invention includes converting the reflected light intensity measured by a surface plasmon resonance imaging device into the number of adherent cells. Such a conversion can be automatically performed from the reflected light intensity once the standard curve is obtained.
[0081]
In a preferred embodiment, step (b) in the method of the present invention involves obtaining information on cell morphology from reflected light intensity measured with a surface plasmon resonance imaging device. Such a conversion can be automatically performed from the reflected light intensity once the standard curve is obtained. As such a method, a method for determining the size can be applied. This is because a form change in a certain prediction range can be inferred from the size.
[0082]
In a preferred embodiment, surface plasmon resonance imaging involves creating surface plasmon resonance on a metal thin film using a surface light source having a wavelength in the visible to near infrared region. Here, preferably, the wavelength in the visible to near-infrared region has a wavelength of 520 nm to 3390 nm, and more preferably a wavelength of 800 nm to 1200 nm. Such wavelengths are advantageous because the reflected light intensity has a remarkable dependence on the incident angle, and a large change in reflected light intensity due to the attachment of a substance to the surface of a metal (such as gold) makes it possible to perform highly sensitive measurement. Because it becomes.
[0083]
In another preferred embodiment, the reflected light intensity may be obtained by capturing an image of the intensity of the reflected light intensity based on the difference in the refractive index between the cell adhered to the thin film surface and the medium as an image with a CCD camera. By capturing such an image, processing as a computer image can be performed, and automation processing is achieved. Capturing such reflected light intensity as an image of a CCD camera is well known in the art and is described, for example, in W.W. Knoll, M .; Liley, D .; Pisevivic, J. et al. Spinke and M.S. J. Tarlov, Supramolecular architecturals for the functionalization of solid surfaces, Adv. Biophys. , 34, 231-251 (1997); P. Nelson, A .; G. FIG. Frutos, J .; M. Brockman. , R .; M. Corn, Near-infrared surface plasmon resonance Measurements of ultrathin films, 1. Angle shift and SPR imaging experiments, Anal Chem. , 71, 3928-3934 (1999).
[0084]
In a preferred embodiment, the plate used in the present invention is a microarray. Methods for making and using such microarrays are known, as described herein above, and can be readily implemented by those skilled in the art.
[0085]
In a preferred embodiment, the plate used in the present invention may include a glass substrate having a metal thin film containing gold, silver or aluminum on one side. Methods for using and manufacturing such glass substrates are well known in the art and have been described herein above.
Preferably, it may be advantageous that the factor which specifically binds to the cells is immobilized on a plate in a spot form of 0.01 mm to 10 mm by covalent bond or physical interaction. This is because the arrangement is fixed in this manner, and the subsequent information processing becomes easy.
[0086]
Preferably, the plate is a microarray in which biomolecules are immobilized, wherein the biomolecules recognize a CD antigen, a cell adhesion factor, a cell growth factor receptor, a cytokine receptor or a sugar chain. Such recognizing biomolecules include, but are not limited to, for example, antibodies, ligands, receptors, specific nucleic acid sequences, and the like. Preferably, it is an antibody, and more preferably, it is a monoclonal antibody.
[0087]
The cells measured herein are allogeneic or heterologous. Preferably they are the same. That is, the measurement of the number and the state of the cells can be more accurate because the uniform cell population is the measurement target. However, the present invention can be used for various purposes, even if different types are mixed.
[0088]
In a preferred embodiment, the cells to be measured by the present invention include normal cells of various organs, differentiated cells such as diseased cells and their precursor cells, embryonic stem cells, fetal stem cells, undifferentiated cells, and established cells. Includes cells or genetically modified cells. Such cells can be appropriately selected or prepared by those skilled in the art depending on the purpose.
[0089]
The invention also provides, in another aspect, a system for calculating a parameter of a cell. The system comprises: (a) a plate for fixing the cells; and (b) a surface plasmon resonance imaging device for observing the plate. Those skilled in the art can appropriately select and use a combination of a plate and an apparatus for specular plasmon resonance imaging described above in this specification.
[0090]
Preferably, the plate used in the system of the present invention may be a microarray. The method for manufacturing and using the microarray is as described above.
[0091]
The references cited herein, such as scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each were specifically described.
[0092]
The present invention has been described above by showing preferred embodiments for easy understanding. Hereinafter, the present invention will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only, and are not provided for limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but only by the claims.
[0093]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples. Reagents such as antibodies used below were obtained as follows unless otherwise noted. The following reagents were obtained from an agent (Wakken Pharmaceutical Co., Ltd., Kyoto, Japan): alkanethiol (Sigma Chemical Co., St. Louis, MO, USA), antibody (Fujisawa Pharmaceutical, Osaka, Japan) Beckman Coulter Co., Ltd., Tokyo, Japan; Nichirei Co., Ltd., Tokyo, Japan) and other reagents: Wako Pure Chemical (Osaka, Japan).
[0094]
(Example 1)
A monomolecular film of alkanethiol (n-hexadecylmercaptan) having a methyl group at a terminal was formed on a glass substrate having a gold thin film deposited on one surface. A photomask in which spots having a diameter of 1 mm were arranged in a 5 × 5 matrix was superimposed on the monomolecular film and irradiated with ultraviolet rays. Thereby, the alkanethiol in the irradiated part was decomposed and removed in the form of spot. Next, a monomolecular film of alkanethiol (11-mercaptoundecanoic acid) having a carboxyl group was formed in the exposed gold spot. After converting the carboxyl on the spot surface to an active ester using N-hydroxysuccinimide, about 100 nl of a 0.1 mg / ml anti-CD90 monoclonal antibody solution was dropped on each spot to immobilize the antibody. Blocking with bovine serum albumin was performed to suppress cell adhesion to the background area around the spot.
[0095]
PC-12 cells derived from rat adrenocortical pheochromocytoma were suspended in a serum-containing medium, and 0.2 ml of the suspension was dropped on an antibody-immobilized substrate. This is done at 37 ° C and 5% CO₂The cells were allowed to adhere by allowing to stand under the atmosphere described in (1). After 30 minutes, the antibody array was washed with a phosphate buffer to remove non-adherent cells. Thereafter, the cells adhered to the spot were fixed using a glutaraldehyde solution.
[0096]
To measure the number of cells adhered to each spot, an SPR image of the substrate was obtained, and the reflected light intensity of each spot was measured. During the observation, the setting was made such that the intensity of the reflected light in the area other than the antibody spot (background area) was 100,000.
[0097]
FIG. 5 shows an SPR image of the substrate to which PC-12 cells are adhered. The area where the cells adhered was observed as a bright area in the SPR image. The average value was 162,490 as a result of measuring the reflected light intensity of the rectangular area at the center of each spot. The standard deviation was determined to be 11,511 as an index of non-uniformity of cell adhesion.
[0098]
(Example 2)
The cell density different from Example 1 and the combination of the immobilized molecule were tested for the density of adherent cells and their heterogeneity. That is, mouse skull-derived stromal cells PA6 were used as cells, and fibronectin was used as an immobilized molecule. Preparation of the substrate and cell adhesion were performed in the same manner as in Example 1, except that the molar composition of the alkanethiol monomolecular film in the spot was 11-mercaptoundecanoic acid: n-11-mercaptoundecanol = 1: 9. . The substrate-cell contact time for adhering the cells was 2.5 hours.
[0099]
FIG. 6 shows an SPR image of the substrate to which PA6 cells are adhered. Also in this case, the area where the cells adhered was observed as a bright area in the SPR image. As a result of measuring the reflected light intensity of the rectangular area at the center of each spot, the average value was 214.388. In addition, the result of calculating the standard deviation was 23.590, which was a large value as compared with the result of the adhesion test of PC-12 cells. As shown in this example, it is possible to easily evaluate the variation in cell adhesion to each spot by using the SPR imaging apparatus.
[0100]
(Example 3)
As in Example 1, the adhesion of PC-12 cells to the anti-CD90 antibody-immobilized array was evaluated using an SPR imaging device. At this time, the composition of the alkanethiol monomolecular film in the spot was changed from 11-mercaptoundecanoic acid: n-11-mercaptoundecanol = 0: 10 to 10: 0, and the concentration of the antibody solution used for immobilization was also changed. It varied between 0.01 μg / ml and 5 mg / ml.
[0101]
The density of the adhered cells varied depending on the carboxyl group density in the spot and the antibody concentration in the immobilization reaction (FIG. 7). Even when the carboxyl group content was 0%, cell adhesion was observed when a high-concentration antibody solution was used, indicating the presence of nonspecifically adsorbed antibodies. As shown by these results, it is possible to quickly examine the substrate preparation conditions by performing cell adhesion evaluation by SPR imaging.
[0102]
(Example 4)
An antibody array was prepared in the same manner as in Example 1. At this time, antibodies include CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD14, CD16, CD19, CD20, CD21, CD33, CD34, CD38, CD41, CD45, CD56, CD71, HLA. Monoclonal antibodies against a total of 22 leukocyte surface antigens of -DR were used, and IgG1, IgG2a, and IgG2b were used as controls.
[0103]
The cells used were CCRF-CEM (T-cell acute lymphoma) and Ramos (B-cell Burkitt's lymphoma), which are cell lines for hematopoietic organs (both were purchased from Human Science Foundation). 5 × 10 cells⁶The cells were dispersed at a concentration of cells / ml in a phosphate buffer solution containing 0.53 mM EDTA and 1 mg / ml human IgG, and subjected to an adhesion test. 400 μl of the cell suspension is dropped on the substrate surface, and CO 2₂The cells were allowed to adhere by standing in an incubator. After 30 minutes, the floating cells were washed away by gently washing the substrate with a phosphate buffer solution, and the cells adhered to the substrate were fixed using a 2% glutaraldehyde solution.
[0104]
In the surface plasmon observation, the reflection intensity of the background was set to 100,000, and the reflection intensity was measured for an area of 10 × 10 pixels in each spot. FIGS. 8 and 9 show the SPR image of the antibody array to which the cells were adhered and the measured values of the reflected light intensity.
[0105]
In CCRF-CEM, many cells adhered to each spot of CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD38, CD45, and CD71, and slight adhesion was observed to spots of CD21. On the other hand, in Ramos, many cells adhered to spots of CD10, CD19, CD20, CD38, CD45, and CD71, and slight adhesion was observed to spots of CD21 and HLA-DR. In the Ramos CD7 spot, the SPR reflected light intensity showed a relatively high value even though almost no cell adhesion was observed. In addition, slight adhesion of cells was observed in the Ramos HLA-DR spot, but the SPR reflection intensity was relatively low. As can be seen from these results, when the cell density is low, the measurement error by SPR imaging seems to be large, and attention is required.
[0106]
The adhesion profile of the two cell lines to each antibody spot was nearly identical to the profile of surface markers known to be expressed on T-cell acute lymphoma as well as B-cell Burkitt's lymphoma.
The above results indicate that by using the method of the present invention in which a microarray and a surface plasmon resonance imaging device are combined, it is possible to rapidly examine the interaction between various types of antibodies and cells. Further, as can be seen from this example, application to clinical tests such as cell immunodiagnosis is also possible.
[0107]
(Example 5)
FIG. 10 shows the relationship between the number of adherent cells of PC-12, PA6, and CCRF-CEM cells and the intensity of SPR reflected light. In each case, a correlation was observed between the cell number and the reflection intensity, and a linear relationship was observed between the correlation coefficients. However, the slope of the straight line varied greatly from cell to cell. As shown in FIG. 11, the slope of the straight line was large in the remarkably expanded PA6 cells, and the slope of the straight line became smaller as the spread of the cells became lighter, such as PC-12 and CCRF-CEM.
[0108]
(Example 6: Demonstration in embryonic stem (ES) cells)
(Preparation of embryonic stem (ES) cells)
Cultured on a gelatin-coated cell culture dish using a GMEM medium containing 1% FBS, 10% Knockout Serum Replacement, LIF (Leukemia Inhibitory Factor), blasticidin, 2-mercaptoethanol, non-essential amino acids, pyruvate. The mouse ES cells (EB5) thus obtained are detached from the culture dish using 0.25% trypsin / EDTA. This was added to a serum-free medium at 5 × 10⁶The cells were suspended so as to be cells / mL, and 0.2 mL thereof was dropped on the antibody-immobilized substrate to prepare an array.
[0109]
(result)
Using this array, it was confirmed whether or not the same measurement can be performed. As a result, it was found that the measurement could be performed while maintaining the linearity unexpectedly, as in the case of the leukemia cells.
[0110]
(Example 6: Demonstration in mouse insulinoma cells)
Mouse insulinoma (MIN6) (pancreatic beta cell-like insulin producing cell line)
MIN6 cells cultured in a DMEM medium containing 10% fetal bovine serum and glucose, 2-mercaptoethanol, sodium carbonate, streptomycin sulfate, and penicillin were detached from the culture equipment using 0.1% trypsin / EDTA. This was added to a serum-free medium at 5 × 10⁶The cells were suspended so as to be cells / mL, and 0.2 mL thereof was dropped on the antibody-immobilized substrate to prepare an array.
[0111]
(result)
Using this array, it was confirmed whether or not the same measurement can be performed. As a result, it was found that the measurement could be performed while maintaining the linearity unexpectedly, as in the case of the leukemia cells.
[0112]
As described above, it is possible to evaluate the degree of cell extension by measuring the SPR reflection intensity.
[0113]
As described above, the present invention has been exemplified by the preferred embodiments of the present invention, but it is understood that the scope of the present invention should be construed only by the appended claims. Patents, patent applications, and references cited herein are to be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.
[0114]
【The invention's effect】
As described above in detail, according to the present invention, it is possible to rapidly and comprehensively analyze the interaction between various types of biomolecules and cells. Thus, for example, by examining the type and frequency of a marker protein expressed on the cell surface, it is possible to know the phenotype, differentiation stage, and disease state of the cell, which is useful not only in cell biology but also in clinical tests. It becomes an analysis system. Further, the present method can be applied not only to the interaction with biomolecules but also to the case of artificial surfaces and environmental pollutants.
[0115]
In the field of clinical testing, immunoassay using flow cytometry has become an important tool for diagnosis along with other pathological tests, as represented by immunocytotyping of leukocytes . However, in this case as well, it is largely based on the empirical judgment of the person performing the test, and it is necessary to determine the optimal analysis conditions for each sample. In addition, difficulties in comparison between cells, such as those involving significant changes in cell morphology, the need to use labeled antibodies, difficulty in quantitative comparison of fluorescence intensity, and accurate diagnosis. In many cases, there are many types of antigens to be used as indices, which are disadvantageous from an economical point of view. The method of the present invention is an effective means for easily obtaining a quantitative analysis result, for example, in classifying leukemia.
[Brief description of the drawings]
FIG. 1A shows an arrangement of a prism and a metal thin film for obtaining surface plasmon resonance. (B) Injection of uniform parallel light for performing surface plasmon resonance imaging. 1. Medium, 2. 2. metal thin film; Prism, 4. Incident light; 5. 5. Angle of incidence, 6. Evanescent wave potential, 7. surface plasmon waves; 8. reflected wave; 10. Uniform parallel light; cell.
FIG. 2 is a schematic diagram of an exemplary surface plasmon resonance imaging device used in the present invention. 1. Light source, 2. Lens, 3. Pinhole, 4. 4. Deflection plate; Prism, 6; Microarray, 7. Flow cell, 8. 8. rotating stage; Interference filter, 10. 10. CCD camera, Personal computer.
FIG. 3 is an example of a surface plasmon resonance image when cells adhere to spots on a microarray.
FIG. 4 shows an example of a shift of a surface plasmon resonance spectrum observed when a cell adheres to a substrate surface. (A) In the absence of cells, (b) In the presence of cells.
FIG. 5 shows an example of a surface plasmon resonance image of a microarray when an anti-CD90 monoclonal antibody is immobilized on all spots under the same conditions and allowed to interact with PC-12 cells.
FIG. 6 shows an example of a surface plasmon resonance image of a microarray when fibronectin is immobilized on all spots under the same conditions and interacts with PA6 cells.
FIG. 7 shows an example of a surface plasmon resonance image of a microarray when an anti-CD90 monoclonal antibody is immobilized under different conditions and allowed to interact with PC-12 cells.
FIG. 8 shows examples of surface plasmon resonance images when a microarray on which antibodies against various CD antigens are immobilized interacts with two types of leukemia model cells. (A) arrangement of antibodies, (b) acute lymphocytic leukemia cells (CCRF-CEM cells), (c) Burkitt lymphoma cells (Ramos cells).
FIG. 9 shows an example of a histogram of reflected light intensity obtained from a surface plasmon resonance image when a microarray on which antibodies against various CD antigens are immobilized and two types of leukemia model cells interact. In each column, the bar on the left is acute lymphocytic leukemia cells, and the bar on the right is Burkitt's lymphoma cells.
FIG. 10 illustrates the relationship between the number of adherent cells of PC-12 cells, PA6 cells, and CCRF-CEM cells and the intensity of SPR reflected light, and an optical micrograph of a representative spot to which those cells adhere.

Claims (16)

A method for analyzing cells, comprising:
(A) measuring the reflected light intensity due to the cells fixed on the plate using surface plasmon resonance imaging; and (b) calculating a parameter related to the cells from the reflected light intensity;
A method comprising: The method according to claim 1, wherein the parameter is a cell number, a cell adhesion area, or a cell size. The method according to claim 1, wherein the step (b) further comprises calculating a parameter for the cell by evaluating the reflected light intensity using a known standard curve for the cell. 2. The method of claim 1, further comprising generating a standard curve for said cells. The method according to claim 1, wherein the step (b) includes converting the reflected light intensity measured by a surface plasmon resonance imaging device into the number of adherent cells. The method according to claim 1, wherein the step (b) includes obtaining information on cell morphology from reflected light intensity measured by a surface plasmon resonance imaging device. The method of claim 1, wherein the surface plasmon resonance imaging includes creating a surface plasmon resonance on a thin metal film using a surface light source having a wavelength in the visible to near infrared region. 2. The method according to claim 1, wherein the reflected light intensity is obtained by capturing an intensity of the reflected light intensity based on a difference in refractive index between a cell adhered to the thin film surface and the medium as an image with a CCD camera. 3. The method of claim 1, wherein the plate is a microarray. The method according to claim 1, wherein the plate includes a glass substrate having a metal thin film containing gold, silver or aluminum on one side. 2. The method according to claim 1, wherein the factors that specifically bind to the cells are immobilized on the plate by covalent bonding or physical interaction in a spot shape of 0.01 mm to 10 mm. The method according to claim 1, wherein the plate is a microarray in which biomolecules are arranged and immobilized, and the biomolecules recognize a CD antigen, a cell adhesion factor, a cell growth factor receptor, a cytokine receptor, or a sugar chain. 2. The method of claim 1, wherein said cells are allogeneic or xenogenic. The cells include normal cells of various organs, differentiated cells such as diseased cells and their precursor cells, embryonic stem cells, fetal-derived stem cells, undifferentiated cells, established cells, or cells with modified genes, The method of claim 1. A system for calculating a parameter of a cell, the system comprising:
(A) a plate for fixing the cells; and (b) a surface plasmon resonance imaging apparatus for observing the plate.
A system comprising: 15. The system of claim 14, wherein said plate is a microarray.

Images