WO2023238375A1 - Cell evaluation method and system - Google Patents

Cell evaluation method and system Download PDF

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WO2023238375A1
WO2023238375A1 PCT/JP2022/023435 JP2022023435W WO2023238375A1 WO 2023238375 A1 WO2023238375 A1 WO 2023238375A1 JP 2022023435 W JP2022023435 W JP 2022023435W WO 2023238375 A1 WO2023238375 A1 WO 2023238375A1
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measurement
cell
cells
location
hydrogel layer
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PCT/JP2022/023435
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Japanese (ja)
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鈴代 井上
友海 村井
あや 田中
陸 高橋
倫子 瀬山
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日本電信電話株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

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  • the present invention relates to a cell evaluation method and system.
  • Non-Patent Document 1 In industries related to life science, including medical product development, food safety, cosmetics safety, and plant pesticide development, evaluation of the effects of developed substances on cells (activity evaluation) is important for proceeding with development. It is a good indicator. However, in recent years, regulations and evaluations of animal experiments have been reviewed around the world, and as an alternative to animal experiments, MPS (Microphysiological System) is attracting attention (Non-Patent Document 1).
  • the indicators used to evaluate cell activity include, firstly, the exchange of molecules inside and outside the cell (chemical reactions), and secondly, the deformation of the cytoskeleton itself (physical reactions). Since these are not independent events but events that occur in correlation with each other, it is desirable that the two indicators can be measured simultaneously.
  • Non-patent document 2 Fluorescence labeling observation
  • Non-patent document 3 phase contrast microscopy
  • electrochemistry Non-patent document 1
  • SPR surface plasmon resonance
  • LSPR localized surface plasmon resonance
  • Fluorescent label observation is a technique that visualizes cellular events to be measured by labeling the molecules whose activity is to be measured, such as calcium and amino acids, which are information transmitting substances, in advance with fluorescently labeled probes. Since the labeled molecules are sensitized by fluorescence, it is also possible to observe behavior changes for molecules at low concentrations of several ⁇ M.
  • Phase contrast microscopy is a technique that measures the degree of deformation of cell membrane shapes using a phase contrast microscope that utilizes light diffraction and interference.
  • Cells deform by changing the arrangement of their cytoskeleton when they receive external or internal stimulation, so measuring the shape of cells is one indicator of cell activity.
  • the cytoskeleton exists inside the cell, is composed of protein filaments, and supports the cell (cell membrane) from the inside to maintain the shape of the cell. The cytoskeleton is also involved in cell movement.
  • Phase-contrast microscopes can visualize colorless and transparent specimens by contrasting light and dark, so there is no risk of cell deterioration or death due to staining, which is a problem with bright field observation, and it is possible to observe cell division and the shape of living cells. can be observed.
  • Electrochemical measurement is a technique that measures the amount of secreted charged molecules (calcium, sodium, etc.) produced by cells placed on an electrode, as the amount of charge.
  • SPR measurement is a technique that allows the refractive index at 200 nm near the gold thin film to be measured without a label. By placing cells near the gold thin film, it is possible to observe in real time changes in the cell population present in the measurement region in response to the movement of the cytoskeleton within the cell membrane.
  • the conventional technology has a problem in that measurements for evaluating cell activity cannot be easily carried out at high resolution without stressing the cells to be measured.
  • FIG. 1 is a flowchart for explaining a cell evaluation method according to an embodiment of the present invention.
  • FIG. 2A is a configuration diagram showing the configuration of a cell evaluation system according to an embodiment of the present invention.
  • FIG. 2B is a configuration diagram showing the configuration of the measurement chip 100.
  • FIG. 2C is a configuration diagram showing another configuration of the measurement chip 100.
  • FIG. 2D is a configuration diagram showing another configuration of the measurement chip 100.
  • FIG. 2E is a perspective view showing a partial configuration of the measurement system.
  • FIG. 3A is a photograph showing the observation results of cells at the first location after 3 days of culture using a phase contrast microscope.
  • FIG. 3B is a photograph showing the observation results of cells at the second location after 3 days of culture using a phase contrast microscope.
  • FIG. 3A is a photograph showing the observation results of cells at the first location after 3 days of culture using a phase contrast microscope.
  • FIG. 3B is a photograph showing the observation results of cells at the second location after 3 days
  • FIG. 4A is a characteristic diagram showing measurement results by surface plasmon resonance method using a measurement chip loaded with target cells.
  • FIG. 4B is a characteristic diagram showing SPR angle measurement based on the SPR measurement position after T seconds of measurement, using a surface plasmon resonance method using a measurement chip loaded with target cells.
  • FIG. 4C is a characteristic diagram showing the results of observing the passage of time for the P point to which the cells are attached, using a surface plasmon resonance method using a measurement chip loaded with target cells.
  • FIG. 5A is a characteristic diagram showing the SPR angle measured at each measurement point of a cell whose size spans multiple observation points.
  • FIG. 5B is a characteristic diagram showing the results of plotting the SPR angle baseline measured at each measurement point for each observation position of a cell whose size spans a plurality of observation points.
  • FIG. 6A is a characteristic diagram showing the SPR angle measurement results at a certain time in SPR measurement of a reference region to which no cells are attached at a first location where a hydrogel layer is not formed.
  • FIG. 6B is a characteristic diagram showing the SPR angle measurement results after supplying pure water in SPR measurement of the reference region to which no cells are attached at the first location where no hydrogel layer is formed.
  • FIG. 6C is a characteristic diagram showing the SPR angle measurement results after supplying pure water in SPR measurement of the region where cells are attached at the first location where no hydrogel layer is formed.
  • the refractive index (SPR angle) is measured over time at locations where the target cells are located where no hydrogel layer has been formed, and at each location where a hydrogel layer has been formed.
  • the refractive index change (temporal change in SPR angle) in each is acquired.
  • the first measurement result is the refractive index change (temporal change in SPR angle) measured at a location where the hydrogel layer is not formed.
  • the refractive index change measured at the location where the hydrogel layer is formed is the second measurement result.
  • the transparent substrate 101 can be made of, for example, a prism and refractive index glass (BK7 glass, quartz glass), plastic (acrylic), etc. of the SPR measuring device described later.
  • the hydrogel layer 103 is formed to have a thickness sufficiently thick (>1 ⁇ m) for the SPR observation region having sensitivity within 200 nm from the surface of the transparent substrate 101, for example.
  • the cell adhesion layer 104 can be made of a material containing cell adhesion molecules (adhesion factors) such as collagen.
  • the measurement chip 100 can be placed in the flow path 113.
  • the flow path 113 can be formed by bonding the glass substrate 111 to a flow path substrate 112 that includes a groove that becomes the flow path 113, an inlet 114, and an outlet 115.
  • a solution that stimulates the cells to be measured placed (mounted) on the measurement chip 100 is introduced from the inlet 114 and transported through the flow channel 113 to contact (act on) the cells mounted on the measurement chip 100. I can do it.
  • the condensed light irradiated in this manner is reflected on the back surface of the metal layer with which the target solution came into contact, and is photoelectrically converted by the sensor 134 to obtain intensity (light intensity).
  • a change in refractive index (SPR angle change) is determined from the change in light intensity obtained in this manner.
  • the refractive index of the transparent substrate 101 is n
  • the dielectric constant of the metal layer 102 is ⁇ m
  • the dielectric constant of the solution is ⁇ s
  • the angle of incidence of light incident on the interface between the transparent substrate 101 and the metal layer 102 is ⁇
  • n( ⁇ /c) sin ⁇ ( ⁇ /c) [ ⁇ m ⁇ s/( ⁇ m+ ⁇ s)] 1/2 ...(1)''
  • the incident angle and the relationship between the transparent substrate 101 and the metal layer 102 are Resonance of plasmons induced at the interface occurs.
  • This angle ⁇ is the SPR angle.
  • the SPR angle can be determined from the pixel position (pixel value) of the photodiode element where the detected light intensity has decreased, and the refractive index can be obtained as a result.
  • the measuring device 130 performs a first measurement at a first location 202 (second location 203) where a hydrogel layer is not formed in the measurement region 201 and a hydrogel layer is formed in the measurement region 201 by surface plasmon resonance method.
  • a second measurement at a second location 203 is performed.
  • the evaluation of the diffusion rate of substances (molecules) generated from cells in the hydrogel by comparing the first measurement result of the first measurement and the second measurement result of the second measurement by measurement by the measuring device 130 can be performed by, for example, It can be performed using computer equipment. The above-mentioned evaluation can be carried out by using computer equipment and running a predetermined program.
  • the stimulation applying device 140 applies stimulation to cells arranged in the measurement region 201 of the measurement chip 100.
  • the stimulation application device 140 supplies, for example, a predetermined drug to cells arranged in the measurement region 201 of the measurement chip 100.
  • the stimulation application device 140 irradiates, for example, light onto the cells arranged in the measurement region 201 of the measurement chip 100.
  • the stimulation applying device 140 applies, for example, a local voltage to the cells arranged in the measurement region 201 of the measurement chip 100.
  • the stimulation application device 140 irradiates, for example, radio waves to cells arranged in the measurement region 201 of the measurement chip 100.
  • Changes in the SPR signal at the first location 202 reveal (1) the influence of free diffusion (molecular diffusion) due to substances generated from the stimulated cells, and (2) the effect of free diffusion (molecular diffusion) from the stimulated cells at the first location 202.
  • the maximum signal intensity of the generated substance can be determined. This change over time of the SPR signal at the first location 202 is measured as a reference curve.
  • substances generated from the stimulated cells reach the upper surface of the hydrogel layer 103 and then move downward in the hydrogel layer 103. and reaches the SPR measurement area.
  • Example 10 the production of the measurement chip will be explained. First, a glass substrate was prepared, and gold was deposited on the substrate by sputtering or vapor deposition to form a metal layer on the substrate surface. Next, a blue sheet is pasted on the surface of the formed metal layer other than the area where the hydrogel layer is to be formed (the second area) to mask it.
  • the surface of the unmasked metal layer is modified with a compound having a dithiol and an acrylic group, such as bis(2-methacryloyl)oxyethyl disulfide (Bis-thiol).
  • a compound having a dithiol and an acrylic group such as bis(2-methacryloyl)oxyethyl disulfide (Bis-thiol).
  • an acrylamide gel solution is dropped onto the surface of the modified metal layer, sealed with a cover glass, and irradiated with ultraviolet light under a nitrogen purge to cause a polymerization reaction. After sufficient polymerization, remove the cover glass. In this state, a hydrogel layer having the same thickness as the blue sheet is formed on a part of the metal layer (second location).
  • a cross-linker agent such as "Sulfo-SANPAH” is dropped only on the surface of the hydrogel layer to modify the surface of the hydrogel layer with NHS groups.
  • the blue sheet is peeled off to expose the surface of the metal layer in the region (first location) where the hydrogel layer is not formed.
  • an adhesion factor solution including collagen is applied to form a cell adhesion layer on the surface of the metal layer and the upper surface of the hydrogel layer.
  • the measurement chip on which the metal layer, hydrogel layer, and cell adhesion layer were formed was immersed in a cell culture medium containing human vascular endothelial cells HUVEC and cultured for 3 days. Cultivation was performed in Petri dishes. After 3 days of culture, a measurement chip with cells mounted on the metal layer (first location) and hydrogel layer (second location) was obtained by rinsing with phosphate-buffered saline (PBS). Ta.
  • PBS phosphate-buffered saline
  • a reference region in which no target cells are placed is provided at each of the first location and the second location. For example, in each of the first and second locations, a sterilized neodymium magnet may be attached to the front side and the back side of the substrate to provide an area where cells are not mounted.
  • FIG. 3A The state of the cells at the first location after 3 days of culture is shown in FIG. 3A, and the state of the cells at the second location after 3 days of culture is shown in FIG. 3B.
  • FIG. 3B The observation results (photographs) using a phase contrast microscope.
  • FIG. 4A the measurement results by surface plasmon resonance method using a measurement chip loaded with target cells are shown in FIG. 4A.
  • changes in the SPR angle every 10 ⁇ m that is, changes in the refractive index, were measured at regular intervals.
  • PBS was injected onto the measurement chip.
  • Figure 4C shows the results of observing the time course at a certain point (P point) where cells are attached.
  • the observed SPR angle was observed to periodically form large peaks, which is considered to be an event caused by contraction of the cytoskeleton of the stimulated living cells. In this way, the movement of the cytoskeleton over time caused by stimulated cell activity can be measured for each attached cell.
  • both the cell position distribution and the time distribution of the cytoskeletal density for each position can be measured simultaneously.
  • the SPR angle changes over time with sharp peaks at points E and F.
  • hydrogels Since one type or multiple types of hydrogels can be placed simultaneously in the measurement area, it is possible to evaluate the diffusion dynamics of secreted substances when changing the molecular weight filter.
  • SYMBOLS 100...Measurement chip, 101...Transparent substrate, 102...Metal layer, 103...Hydrogel layer, 104...Cell adhesion layer, 105...Cell, 130...Measurement device, 140...Stimulation application device, 201...Measurement area, 202...No. 1 location, 203...2nd location.

Abstract

In Step S101, cells to be measured are arranged in a measurement area. In Step S102, a first measurement result is obtained by the surface plasmon resonance method at a location in the measurement area where a hydrogel layer is not formed, and a second measurement result is obtained by the surface plasmon resonance method at a location in the measurement area where the hydrogel layer is formed (measurement step). In Step S103, a change in the cellular membrane of the cells is evaluated on the basis of the first measurement result (first evaluation step). In Step S104, the diffusion rate of a substance generated from the cells in the hydrogel is evaluated by comparing the first measurement result with the second measurement result (second evaluation step).

Description

細胞評価方法およびシステムCell evaluation method and system
　本発明は、細胞評価方法およびシステムに関する。 The present invention relates to a cell evaluation method and system.
　医療品開発をはじめとして、食の安全性、化粧品の安全性、植物の農薬開発等のライフサイエンスに関わる業界において、開発物質が細胞に及ぼす影響評価(活性評価)は、開発を進める上で重要な指標となる。しかし、近年、動物実験に対する規制や評価の見直しが世界中で高まってきているため、動物実験の代替え実験法として、生体内を模倣した環境を人工的な流路チップなどで再現するＭＰＳ（Microphysiological System）が注目を集めている（非特許文献１）。 In industries related to life science, including medical product development, food safety, cosmetics safety, and plant pesticide development, evaluation of the effects of developed substances on cells (activity evaluation) is important for proceeding with development. It is a good indicator. However, in recent years, regulations and evaluations of animal experiments have been reviewed around the world, and as an alternative to animal experiments, MPS (Microphysiological System) is attracting attention (Non-Patent Document 1).
　細胞の活性を評価する際に指標となるものとして、第１に、細胞内外の分子のやり取り(化学的な反応) と、第２に、細胞骨格自体の変形(物理的反応)とがある。これらは、独立的な事象ではなく、互いに相関して発生する事象であることから、２つの指標が同時に測定できることが望ましい。 The indicators used to evaluate cell activity include, firstly, the exchange of molecules inside and outside the cell (chemical reactions), and secondly, the deformation of the cytoskeleton itself (physical reactions). Since these are not independent events but events that occur in correlation with each other, it is desirable that the two indicators can be measured simultaneously.
　現在、ＭＰＳで細胞の活性測定を行う検出システムとして、蛍光標識観察（非特許文献２） 、位相差顕微鏡観察（非特許文献３） 、電気化学（非特許文献１） 、表面プラズモン共鳴（ＳＰＲ）測定もしくは局在表面プラズモン共鳴（ＬＳＰＲ）（非特許文献４）が存在する。 Currently, detection systems for measuring cell activity using MPS include fluorescence labeling observation (Non-patent document 2), phase contrast microscopy (Non-patent document 3), electrochemistry (Non-patent document 1), and surface plasmon resonance (SPR). measurement or localized surface plasmon resonance (LSPR) (Non-Patent Document 4).
　蛍光標識観察は、活性の測定対象とする分子、例えば情報伝達物質であるカルシウムやアミノ酸を、予め蛍光標識プローブによって標識することで、測定対象となる細胞イベントを可視化する技術である。標識された分子は蛍光により増感されるため、数μＭレベルの低濃度の分子に対する挙動推移の観察も可能となる。 Fluorescent label observation is a technique that visualizes cellular events to be measured by labeling the molecules whose activity is to be measured, such as calcium and amino acids, which are information transmitting substances, in advance with fluorescently labeled probes. Since the labeled molecules are sensitized by fluorescence, it is also possible to observe behavior changes for molecules at low concentrations of several μM.
　位相差顕微鏡観察は、光の回折と干渉を利用した位相差顕微鏡を用いて細胞膜形状の変形度合いを測定する技術である。細胞は、外部からもしくは内部からの刺激を受けると細胞骨格の配置を変化させて変形することから、細胞の形状を測定することが細胞活性の１つの指標となる。なお、細胞骨格は、細胞の内部に存在し、タンパク質フィラメントから構成され、細胞（細胞膜）を内部から支えて細胞の形を維持している。また、細胞骨格は、細胞運動にも関わっている。 Phase contrast microscopy is a technique that measures the degree of deformation of cell membrane shapes using a phase contrast microscope that utilizes light diffraction and interference. Cells deform by changing the arrangement of their cytoskeleton when they receive external or internal stimulation, so measuring the shape of cells is one indicator of cell activity. The cytoskeleton exists inside the cell, is composed of protein filaments, and supports the cell (cell membrane) from the inside to maintain the shape of the cell. The cytoskeleton is also involved in cell movement.
　位相差顕微鏡は、明暗のコントラストにより無色透明な標本を可視化することができるため、明視野観察で問題となる染色による細胞の変質・死滅のリスクがなく、細胞分裂や生きたままの細胞の形状を観察することができる。 Phase-contrast microscopes can visualize colorless and transparent specimens by contrasting light and dark, so there is no risk of cell deterioration or death due to staining, which is a problem with bright field observation, and it is possible to observe cell division and the shape of living cells. can be observed.
　電気化学測定は、電極上に配置した細胞が産生する電荷を帯びた分子(カルシウム、ナトリウムなど)の分泌物の量を、電荷量として測定する技術である。 Electrochemical measurement is a technique that measures the amount of secreted charged molecules (calcium, sodium, etc.) produced by cells placed on an electrode, as the amount of charge.
　ＳＰＲ測定は、金薄膜近傍２００ｎｍの屈折率を標識なしに測定できる技術である。金薄膜近傍に細胞を設置することで、細胞膜内に存在する細胞骨格の動きに呼応した測定領域に存在する細胞集団の変化をリアルタイムに観測することができる。 SPR measurement is a technique that allows the refractive index at 200 nm near the gold thin film to be measured without a label. By placing cells near the gold thin film, it is possible to observe in real time changes in the cell population present in the measurement region in response to the movement of the cytoskeleton within the cell membrane.
　しかしながら、上述した従来技術では、以下に示す問題がある。 However, the above-mentioned conventional technology has the following problems.
　まず、蛍光標識観察では、長時間の測定は細胞活性に影響を与える光毒性をもつという問題がある。また、蛍光標識観察では、標識対象ごとの染色作業、標識物質の非特異吸着を防ぐためのブロッキング作業、および洗浄作業を必要とするため、標識工程に手間を要し、さらに標識工程により細胞にストレスが加わるという問題がある。 First, fluorescent label observation has the problem that long-term measurements have phototoxicity that affects cell activity. In addition, fluorescent label observation requires staining work for each labeled target, blocking work to prevent non-specific adsorption of labeling substances, and washing work, so the labeling process is labor-intensive, and the labeling process also requires staining work for each target to be labeled. The problem is that it adds stress.
　次に、位相差顕微鏡では、細胞の形状の情報だけしか観察することができず、また、初期の細胞応答の検出が困難であり、反応の解像度が低いという問題がある。 Next, with a phase contrast microscope, only information about the shape of cells can be observed, and it is difficult to detect early cell responses, resulting in low response resolution.
　電気化学測定については、電気化学だけで細胞の形状変化を一度に測定することは難しく、また、電荷を帯びない、もしくは弱い分子を増感なしに測定することが難しいという問題がある。 Regarding electrochemical measurements, there are problems in that it is difficult to measure cell shape changes all at once using electrochemistry alone, and it is also difficult to measure uncharged or weak molecules without sensitization.
　また、従来のＳＰＲ測定では、測定領域の合算された変化値を測定しているため、単一細胞の測定のためには大型の装置が必要になるという問題があった。また、ＬＳＰＲ測定は解像度が低いという問題があった。 In addition, in conventional SPR measurement, since the total change value of the measurement area is measured, there is a problem in that a large device is required to measure a single cell. Additionally, LSPR measurement has a problem of low resolution.
　上述したように、従来の技術は、細胞の活性評価などのための測定が、測定対象の細胞にストレスなどを与えることなく、高い解像度で容易に実施することができないという問題があった。 As described above, the conventional technology has a problem in that measurements for evaluating cell activity cannot be easily carried out at high resolution without stressing the cells to be measured.
　本発明は、以上のような問題点を解消するためになされたものであり、細胞の活性評価などのための測定が、測定対象の細胞にストレスなどを与えることなく、高い解像度で容易に実施できるようにすることを目的とする。 The present invention has been made to solve the above-mentioned problems, and enables measurements for evaluating cell activity to be easily performed with high resolution without causing stress to the cells being measured. The purpose is to make it possible.
　本発明に係る細胞評価方法は、一部にハイドロゲルの層を備えて表面プラズモン共鳴法による測定を実施する測定領域に測定対象の細胞を配置し、測定領域におけるハイドロゲルの層が未形成の箇所の表面プラズモン共鳴法による第１測定結果と、測定領域におけるハイドロゲルの層が形成されている箇所の表面プラズモン共鳴法による第２測定結果とを得る測定ステップと、第１測定結果により、細胞の細胞膜の変化を評価する第１評価ステップと、第１測定結果と第２測定結果との比較により、細胞から発生する物質のハイドロゲルにおける拡散速度を評価する第２評価ステップとを備える。 In the cell evaluation method according to the present invention, cells to be measured are placed in a measurement area that partially includes a hydrogel layer and is used for measurement by surface plasmon resonance, A measurement step of obtaining a first measurement result by surface plasmon resonance at a location and a second measurement result by surface plasmon resonance at a location where a hydrogel layer is formed in the measurement region; The method includes a first evaluation step of evaluating changes in the cell membrane of the cell membrane, and a second evaluation step of evaluating the diffusion rate of a substance generated from the cells in the hydrogel by comparing the first measurement result and the second measurement result.
　また、本発明に係る細胞評価システムは、一部にハイドロゲルの層を備えて表面プラズモン共鳴法による測定を実施する測定領域と、表面プラズモン共鳴法により、測定領域におけるハイドロゲルの層が未形成の箇所における第１測定と、測定領域におけるハイドロゲルの層が形成されている箇所における第２測定とを実施する測定装置とを備える。 In addition, the cell evaluation system according to the present invention has a measurement region that partially includes a hydrogel layer and performs measurement by surface plasmon resonance method, and a measurement region in which a hydrogel layer is not formed in the measurement region by surface plasmon resonance method. and a measuring device that performs a first measurement at a location where a hydrogel layer is formed in the measurement region and a second measurement at a location where a hydrogel layer is formed in the measurement region.
　以上説明したように、本発明によれば、ハイドロゲルの層が未形成の箇所の表面プラズモン共鳴法による第１測定結果と、測定領域におけるハイドロゲルの層が形成されている箇所の表面プラズモン共鳴法による第２測定結果とを得るので、細胞の活性評価などのための測定が、測定対象の細胞にストレスなどを与えることなく、高い解像度で容易に実施できる。 As explained above, according to the present invention, the first measurement result by the surface plasmon resonance method at a location where a hydrogel layer is not formed and the surface plasmon resonance method at a location where a hydrogel layer is formed in the measurement region. Since the second measurement result is obtained by the method, measurements for cell activity evaluation etc. can be easily carried out with high resolution without applying stress to the cells to be measured.
図１は、本発明の実施の形態に係る細胞評価方法を説明するためのフローチャートである。FIG. 1 is a flowchart for explaining a cell evaluation method according to an embodiment of the present invention. 図２Ａは、本発明の実施の形態に係る細胞評価システムの構成を示す構成図である。FIG. 2A is a configuration diagram showing the configuration of a cell evaluation system according to an embodiment of the present invention. 図２Ｂは、測定チップ１００の構成を示す構成図である。FIG. 2B is a configuration diagram showing the configuration of the measurement chip 100. 図２Ｃは、測定チップ１００の他の構成を示す構成図である。FIG. 2C is a configuration diagram showing another configuration of the measurement chip 100. 図２Ｄは、測定チップ１００の他の構成を示す構成図である。FIG. 2D is a configuration diagram showing another configuration of the measurement chip 100. 図２Ｅは、測定システムの一部構成を示す斜視図である。FIG. 2E is a perspective view showing a partial configuration of the measurement system. 図３Ａは、培養３日後の第１箇所の細胞の位相差顕微鏡による観察結果を示す写真である。FIG. 3A is a photograph showing the observation results of cells at the first location after 3 days of culture using a phase contrast microscope. 図３Ｂは、培養３日後の第２箇所の細胞の位相差顕微鏡による観察結果を示す写真である。FIG. 3B is a photograph showing the observation results of cells at the second location after 3 days of culture using a phase contrast microscope. 図４Ａは、対象の細胞を搭載した測定チップを用いた表面プラズモン共鳴法による測定結果を示す特性図である。FIG. 4A is a characteristic diagram showing measurement results by surface plasmon resonance method using a measurement chip loaded with target cells. 図４Ｂは、対象の細胞を搭載した測定チップを用いた表面プラズモン共鳴法による、測定Ｔ秒後のＳＰＲ測定位置によるＳＰＲ角度測定を示す特性図である。FIG. 4B is a characteristic diagram showing SPR angle measurement based on the SPR measurement position after T seconds of measurement, using a surface plasmon resonance method using a measurement chip loaded with target cells. 図４Ｃは、対象の細胞を搭載した測定チップを用いた表面プラズモン共鳴法による、細胞が付着しているＰポイントについて、時間経過を観察した結果を示す特性図である。FIG. 4C is a characteristic diagram showing the results of observing the passage of time for the P point to which the cells are attached, using a surface plasmon resonance method using a measurement chip loaded with target cells. 図５Ａは、複数の観測ポイントをまたがる大きさの細胞の、各測定ポイントで計測されるＳＰＲ角度を示す特性図である。FIG. 5A is a characteristic diagram showing the SPR angle measured at each measurement point of a cell whose size spans multiple observation points. 図５Ｂは、複数の観測ポイントをまたがる大きさの細胞の、各測定ポイントで計測されるＳＰＲ角度ベースラインを観察位置毎にプロットした結果を示す特性図である。FIG. 5B is a characteristic diagram showing the results of plotting the SPR angle baseline measured at each measurement point for each observation position of a cell whose size spans a plurality of observation points. 図６Ａは、ハイドロゲル層が形成されていない第１箇所の細胞が付着していないリファレンス領域のＳＰＲ測定における、ある時間のＳＰＲ角度測定結果を示す特性図である。FIG. 6A is a characteristic diagram showing the SPR angle measurement results at a certain time in SPR measurement of a reference region to which no cells are attached at a first location where a hydrogel layer is not formed. 図６Ｂは、ハイドロゲル層が形成されていない第１箇所の細胞が付着していないリファレンス領域のＳＰＲ測定における、純水を供給した後のＳＰＲ角度測定結果を示す特性図である。FIG. 6B is a characteristic diagram showing the SPR angle measurement results after supplying pure water in SPR measurement of the reference region to which no cells are attached at the first location where no hydrogel layer is formed. 図６Ｃは、ハイドロゲル層が形成されていない第１箇所の細胞が付着している領域のＳＰＲ測定における、純水を供給した後のＳＰＲ角度測定結果を示す特性図である。FIG. 6C is a characteristic diagram showing the SPR angle measurement results after supplying pure water in SPR measurement of the region where cells are attached at the first location where no hydrogel layer is formed.
　以下、本発明の実施の形態に係る細胞評価方法について図１を参照して説明する。まず、ステップＳ１０１で、測定領域に測定対象の細胞を配置する。測定領域は、例えば、表面プラズモン共鳴（surface plasmon resonance；ＳＰＲ）測定システムに装着して用いられる測定チップに形成されている。測定領域は、一部にハイドロゲルの層を備える。測定領域は、表面プラズモン共鳴法による測定が実施される領域である。 Hereinafter, a cell evaluation method according to an embodiment of the present invention will be described with reference to FIG. First, in step S101, cells to be measured are placed in a measurement area. The measurement region is formed, for example, on a measurement chip used by being attached to a surface plasmon resonance (SPR) measurement system. The measurement area partially includes a layer of hydrogel. The measurement area is an area where measurement by surface plasmon resonance is performed.
　次に、ステップＳ１０２で、測定領域におけるハイドロゲルの層が未形成の箇所の表面プラズモン共鳴法による第１測定結果と、測定領域におけるハイドロゲルの層が形成されている箇所の表面プラズモン共鳴法による第２測定結果とを得る（測定ステップ）。 Next, in step S102, the first measurement result by the surface plasmon resonance method of a portion in the measurement region where the hydrogel layer is not formed, and the first measurement result by the surface plasmon resonance method of the portion in the measurement region where the hydrogel layer is formed. and obtain a second measurement result (measurement step).
　ＳＰＲ測定システムにより、対象の細胞が配置されているハイドロゲルの層が未形成の箇所、およびハイドロゲルの層が形成されている各箇所における屈折率（ＳＰＲ角度）を時系列的に測定し、各々における屈折率変化（ＳＰＲ角度の時間変化）を取得する。ハイドロゲルの層が未形成の箇所で測定される屈折率変化（ＳＰＲ角度の時間変化）が、第１測定結果である。また、ハイドロゲルの層が形成されている箇所で測定される屈折率変化が、第２測定結果である。 Using an SPR measurement system, the refractive index (SPR angle) is measured over time at locations where the target cells are located where no hydrogel layer has been formed, and at each location where a hydrogel layer has been formed. The refractive index change (temporal change in SPR angle) in each is acquired. The first measurement result is the refractive index change (temporal change in SPR angle) measured at a location where the hydrogel layer is not formed. Moreover, the refractive index change measured at the location where the hydrogel layer is formed is the second measurement result.
　次に、ステップＳ１０３で、第１測定結果により、細胞の細胞膜の変化を評価する（第１評価ステップ）。このステップでは、配置されている細胞に刺激を与えることによる第１測定結果より、細胞の細胞膜の変化を評価する。 Next, in step S103, changes in the cell membrane of the cells are evaluated based on the first measurement results (first evaluation step). In this step, changes in the cell membrane of the cells are evaluated based on the first measurement results obtained by applying stimulation to the placed cells.
　また、ステップＳ１０４で、第１測定結果と第２測定結果との比較により、細胞から発生する物質のハイドロゲルにおける拡散速度を評価する（第２評価ステップ）。このステップでは、配置されている細胞に刺激を与えることによる第１測定結果と第２測定結果との比較により、細胞から発生する物質のハイドロゲルにおける拡散速度を評価する。 Furthermore, in step S104, the diffusion rate of the substance generated from the cells in the hydrogel is evaluated by comparing the first measurement result and the second measurement result (second evaluation step). In this step, the diffusion rate of the substance generated from the cells in the hydrogel is evaluated by comparing the first measurement result and the second measurement result obtained by stimulating the placed cells.
　次に、本発明の実施の形態に係る細胞評価システムについて、図２Ａ、図２Ｂ、図２Ｃ、図２Ｄ、図２Ｅを参照して説明する。細胞評価システムは、測定チップ１００、測定装置１３０を備える。また、細胞評価システムは、刺激印加装置１４０を備えることができる。 Next, a cell evaluation system according to an embodiment of the present invention will be described with reference to FIGS. 2A, 2B, 2C, 2D, and 2E. The cell evaluation system includes a measurement chip 100 and a measurement device 130. Further, the cell evaluation system can include a stimulation application device 140.
　測定チップ１００は、図２Ｂ、図２Ｃに示すように、透明基板１０１、金属層１０２、ハイドロゲル層１０３を備える。また、第１箇所２０２の金属層１０２の表面、および第２箇所２０３のハイドロゲル層１０３の各々の表面に、測定対象の細胞１０５を接着（固定）するための細胞接着層１０４を形成することができる。 The measurement chip 100 includes a transparent substrate 101, a metal layer 102, and a hydrogel layer 103, as shown in FIGS. 2B and 2C. Further, a cell adhesion layer 104 for adhering (fixing) the cells 105 to be measured is formed on each surface of the metal layer 102 at the first location 202 and the hydrogel layer 103 at the second location 203. I can do it.
　透明基板１０１は、例えば、後述するＳＰＲ測定装置のプリズムと屈折率のガラス（ＢＫ７ガラス、石英ガラス）やプラスチック（アクリル）などから構成することができる。ハイドロゲル層１０３は、例えば、透明基板１０１の表面から２００ｎｍ以内に感度を持つＳＰＲの観測領域に対して十分厚い（＞１μｍ）厚さに形成する。 The transparent substrate 101 can be made of, for example, a prism and refractive index glass (BK7 glass, quartz glass), plastic (acrylic), etc. of the SPR measuring device described later. The hydrogel layer 103 is formed to have a thickness sufficiently thick (>1 μm) for the SPR observation region having sensitivity within 200 nm from the surface of the transparent substrate 101, for example.
　金属層１０２は、例えば、Ａｕから構成し、厚さ５０ｎｍ程度とすることができる。金属層１０２は、例えば、スパッタ法などの堆積技術により形成することができる。透明基板１０１の金属層１０２が形成されている領域が、表面プラズモン共鳴法による測定領域２０１となる。 The metal layer 102 is made of, for example, Au and can have a thickness of about 50 nm. The metal layer 102 can be formed, for example, by a deposition technique such as a sputtering method. The region of the transparent substrate 101 where the metal layer 102 is formed becomes the measurement region 201 by the surface plasmon resonance method.
　また、透明基板１０１の測定領域２０１の途中に、ハイドロゲル層１０３が形成されている。ハイドロゲルは、例えば、モノマー架橋が可能なアクリルアミドゲルである。ハイドロゲル層１０３は、例えば、平面視で、２ｍｍ×１．５ｍｍの矩形とし、厚さ８０μｍとすることができる。ハイドロゲルは、金属層１０２に物理的または化学的に吸着し、送液時に金属層から剥離しないものであれば良く、アクリルアミドゲルに限定されない。また、ハイドロゲルの編目の寸法（メッシュサイズ）や膨潤度についても限定はない。ハイドロゲルは、ポリマー同士の「TetraPEG」、天然由来のマドリゲルなどとすることができる。また、細胞から発生する物質の大きさによって、フィルタを実施したい場合、ハイドロゲルのメッシュサイズを、対象とする物質（分子）の大きさに合わせて選択することにより分子透過によるフィルタリング効果が得られる。 Additionally, a hydrogel layer 103 is formed in the middle of the measurement region 201 of the transparent substrate 101. Hydrogels are, for example, acrylamide gels capable of monomer crosslinking. For example, the hydrogel layer 103 can have a rectangular shape of 2 mm x 1.5 mm and a thickness of 80 μm in plan view. The hydrogel is not limited to acrylamide gel, as long as it is physically or chemically adsorbed to the metal layer 102 and does not peel off from the metal layer during liquid feeding. Furthermore, there are no limitations on the mesh size (mesh size) or degree of swelling of the hydrogel. The hydrogel can be a polymer-polymer "TetraPEG", a naturally derived madrigel, etc. In addition, if you want to perform filtering depending on the size of the substance generated from cells, you can obtain a filtering effect by molecular permeation by selecting the mesh size of the hydrogel according to the size of the target substance (molecule). .
　例えば、ハイドロゲル層１０３を形成する箇所に開口を有するリフトオフマスクを用い、透明基板１０１の所定箇所にリフトオフマスクを配置し、ハイドロゲルの原料を塗布し、紫外線を照射してゲル化反応を実施してハイドロゲルとする。リフトオフマスクとしては、エレグリップテープを用いることができる。この後、リフトオフマスクを除去（リフトオフ）することで、透明基板１０１の透明基板１０１となる領域の所定箇所に、ハイドロゲル層１０３が形成できる。 For example, a lift-off mask having an opening at the location where the hydrogel layer 103 is to be formed is used, the lift-off mask is placed at a predetermined location on the transparent substrate 101, the hydrogel raw material is applied, and ultraviolet rays are irradiated to perform the gelation reaction. to make a hydrogel. Elegrip tape can be used as a lift-off mask. Thereafter, by removing (lifting off) the lift-off mask, the hydrogel layer 103 can be formed at a predetermined location in the region of the transparent substrate 101 that will become the transparent substrate 101.
　また、ハイドロゲルをアクリルアミドゲルとし、金属層１０２をＡｕから構成する場合、金属層１０２の表面を、bis(2-methacryloyl)oxyethyl disulfide（Bis-thiol）などのジチオールとアクリル基を有する化合物によって修飾することで、ハイドロゲルとの接着性を向上させることができる。 In addition, when the hydrogel is an acrylamide gel and the metal layer 102 is made of Au, the surface of the metal layer 102 is modified with a compound having a dithiol and an acrylic group, such as bis(2-methacryloyl)oxyethyl disulfide (Bis-thiol). By doing so, the adhesiveness with the hydrogel can be improved.
　また、細胞接着層１０４は、コラーゲンなどの細胞接着分子（接着因子）を含む材料から構成することができる。 Furthermore, the cell adhesion layer 104 can be made of a material containing cell adhesion molecules (adhesion factors) such as collagen.
　ここで、測定領域２０１におけるハイドロゲル層１０３が未形成の第１箇所２０２が、第１測定が実施される領域となる。また、測定領域２０１におけるハイドロゲル層１０３が形成されている第２箇所２０３が、第２測定が実施される領域となる。 Here, the first location 202 in the measurement region 201 where the hydrogel layer 103 is not formed becomes the region where the first measurement is performed. Furthermore, the second location 203 where the hydrogel layer 103 is formed in the measurement area 201 becomes the area where the second measurement is performed.
　第２箇所２０３では、第１箇所２０２と同等のＳＰＲ測定結果が得られる。このため、例えば、第１箇所２０２における細胞から発生する物質の到達時間と、第２箇所２０３おける細胞から発生する物質の到達時間との違いから、ハイドロゲルが形成されている第２箇所２０３における上記物質の到達時間を求めることができる。ここで求められる第２箇所２０３の上記物質の到達時間を利用して、ＳＰＲ角変化曲線の時間原点を補正することで、上記物質がハイドロゲル層１０３の上面からハイドロゲルの厚さ分だけ拡散するのに要する時間が算出できる。 At the second location 203, an SPR measurement result equivalent to that at the first location 202 is obtained. For this reason, for example, due to the difference in the arrival time of substances generated from cells at the first location 202 and the arrival time of substances generated from cells at the second location 203, the arrival time of substances generated from cells at the second location 203 where the hydrogel is formed is determined. The arrival time of the above substance can be determined. By correcting the time origin of the SPR angle change curve using the arrival time of the substance at the second location 203 determined here, the substance diffuses from the top surface of the hydrogel layer 103 by the thickness of the hydrogel. The time required to do this can be calculated.
　例えば、図２Ｃに示すように、測定チップ１００は、流路１１３の中に配置することができる。流路１１３は、ガラス基板１１１に、流路１１３となる溝部，導入口１１４，および排出口１１５を備える流路基板１１２に貼り合わせることで形成することができる。測定チップ１００に配置（搭載）した測定対象の細胞に刺激を与える溶液を、導入口１１４から導入し、流路１１３で輸送することで、測定チップ１００に搭載した細胞に接触（作用）させることができる。 For example, as shown in FIG. 2C, the measurement chip 100 can be placed in the flow path 113. The flow path 113 can be formed by bonding the glass substrate 111 to a flow path substrate 112 that includes a groove that becomes the flow path 113, an inlet 114, and an outlet 115. A solution that stimulates the cells to be measured placed (mounted) on the measurement chip 100 is introduced from the inlet 114 and transported through the flow channel 113 to contact (act on) the cells mounted on the measurement chip 100. I can do it.
　また、図２Ｄに示すように、測定チップ１００は、基板１１６に形成した空間１１７に配置することができる。測定チップ１００に配置（搭載）した測定対象の細胞に刺激を与える溶液を、空間１１７の上部から滴下することで、測定チップ１００に搭載した細胞に接触（作用）させることができる。 Furthermore, as shown in FIG. 2D, the measurement chip 100 can be placed in a space 117 formed in the substrate 116. By dropping a solution that stimulates the cells to be measured placed on (mounted on) the measurement chip 100 from the upper part of the space 117, it can come into contact with (act on) the cells placed on the measurement chip 100.
　測定装置１３０は、図２Ｅに示すように、光源１３１、プリズム１３２、いわゆるＣＣＤイメージセンサなどの撮像素子よりなるセンサ１３４を備えるＳＰＲ装置である。ＳＰＲ装置としては、例えば、エヌ・ティ・ティ・アドバンステクノロジ株式会社製の「Ｓｍａｒｔ　ＳＰＲ」が挙げられる。 As shown in FIG. 2E, the measuring device 130 is an SPR device that includes a light source 131, a prism 132, and a sensor 134 made of an imaging device such as a so-called CCD image sensor. An example of the SPR device is "Smart SPR" manufactured by NTT Advanced Technology Corporation.
　測定装置１３０において、光源１３１から出射された光を集光してプリズム１３２に入射させ、プリズム１３２の測定面１３３に密着させている測定チップ１００の測定領域に照射する。測定チップ１００を構成する透明基板１０１の測定領域には金属層（不図示）が形成されており、金属層の裏面に、測定チップ１００を透過してきた集光光が照射される。 In the measurement device 130, the light emitted from the light source 131 is focused and made to enter the prism 132, and is irradiated onto the measurement region of the measurement chip 100 that is in close contact with the measurement surface 133 of the prism 132. A metal layer (not shown) is formed in the measurement region of the transparent substrate 101 constituting the measurement chip 100, and the back surface of the metal layer is irradiated with the condensed light that has passed through the measurement chip 100.
　このようにして照射された集光光は、対象の溶液が接触した金属層の裏面で反射し、センサ１３４で光電変換されて強度（光強度）が得られる。このようにして得られた光強度の変化により屈折率の変化（ＳＰＲ角度変化）が求められる。 The condensed light irradiated in this manner is reflected on the back surface of the metal layer with which the target solution came into contact, and is photoelectrically converted by the sensor 134 to obtain intensity (light intensity). A change in refractive index (SPR angle change) is determined from the change in light intensity obtained in this manner.
　このＳＰＲ角度の測定においては、第１箇所２０２、第２箇所２０３におけるＳＰＲ角度の変化を測定する。第１箇所２０２、第２箇所２０３には、センサ１３４の検出領域が対応している。センサ１３４の検出領域には、複数のフォトダイオード素子（画素）が、第１箇所２０２から第２箇所２０３への方向に並んで配置されており、第１箇所２０２、第２箇所２０３では、各フォトダイオード素子の位置（ピクセル位置）毎に、光強度の変化（ＳＰＲ角度）が測定される。例えば、センサ１３４の検出領域の測定領域２０１に対応する箇所には、４８０ピクセルのフォトダイオード素子が、１０μｍ間隔で一列に並んでいる。 In measuring this SPR angle, changes in the SPR angle at the first location 202 and the second location 203 are measured. The detection area of the sensor 134 corresponds to the first location 202 and the second location 203. In the detection area of the sensor 134, a plurality of photodiode elements (pixels) are arranged side by side in the direction from the first location 202 to the second location 203. A change in light intensity (SPR angle) is measured for each position of the photodiode element (pixel position). For example, in a portion of the detection area of the sensor 134 corresponding to the measurement area 201, 480 pixel photodiode elements are arranged in a line at intervals of 10 μm.
　なお、透明基板１０１の屈折率をｎ、金属層１０２の誘電率をεｍ、溶液の誘電率をεｓ、透明基板１０１と金属層１０２との界面に入射する光の入射角度をθとすると、「ｎ（ω/ｃ）ｓｉｎθ＝（ω/ｃ）［εｍ×εｓ/（εｍ＋εｓ）］1/2・・（１）」が成り立つ条件の時に、入射角度と、透明基板１０１と金属層１０２との界面に誘起されるプラズモンの共鳴が起こる。この角度θが、ＳＰＲ角度である。 Note that if the refractive index of the transparent substrate 101 is n, the dielectric constant of the metal layer 102 is εm, the dielectric constant of the solution is εs, and the angle of incidence of light incident on the interface between the transparent substrate 101 and the metal layer 102 is θ, then n(ω/c) sin θ=(ω/c) [εm×εs/(εm+εs)] 1/2 ...(1)'', the incident angle and the relationship between the transparent substrate 101 and the metal layer 102 are Resonance of plasmons induced at the interface occurs. This angle θ is the SPR angle.
　また、プラズモンの共鳴が起きると反射する光が減衰するため、この状態がセンサ１３４のいずれかのフォトダイオード素子の検出値の変化として現れる。従って、検出光強度が低下したフォトダイオード素子のピクセル位置（ピクセル値）により、ＳＰＲ角度が求められ、結果として屈折率が得られる。例えば、上記ピクセル値より、例えば、「屈折率値＝ピクセル値×１．２７３９×１０-4＋１．３１８８（光源波長７７０ｎｍ）」などの換算式により、屈折率値が得られる。 Further, when plasmon resonance occurs, the reflected light is attenuated, so this state appears as a change in the detected value of one of the photodiode elements of the sensor 134. Therefore, the SPR angle can be determined from the pixel position (pixel value) of the photodiode element where the detected light intensity has decreased, and the refractive index can be obtained as a result. For example, a refractive index value can be obtained from the above pixel value using a conversion formula such as "refractive index value=pixel value×1.2739×10 −4 +1.3188 (light source wavelength 770 nm)."
　測定装置１３０は、表面プラズモン共鳴法により、測定領域２０１におけるハイドロゲルの層が未形成の第１箇所２０２（第２箇所２０３）における第１測定と、測定領域２０１におけるハイドロゲルの層が形成されている第２箇所２０３における第２測定とを実施する。測定装置１３０の測定による、第１測定による第１測定結果と、第２測定による第２測定結果との比較によるハイドロゲルにおける、細胞から発生する物質（分子）の拡散速度の評価は、例えば、コンピュータ機器を用いて実施することができる。コンピュータ機器を用い、所定のプログラムを動作させることで、上述した評価が実施できる。 The measuring device 130 performs a first measurement at a first location 202 (second location 203) where a hydrogel layer is not formed in the measurement region 201 and a hydrogel layer is formed in the measurement region 201 by surface plasmon resonance method. A second measurement at a second location 203 is performed. The evaluation of the diffusion rate of substances (molecules) generated from cells in the hydrogel by comparing the first measurement result of the first measurement and the second measurement result of the second measurement by measurement by the measuring device 130 can be performed by, for example, It can be performed using computer equipment. The above-mentioned evaluation can be carried out by using computer equipment and running a predetermined program.
　なお、測定領域２０１におけるハイドロゲル層１０３が未形成の第１箇所２０２の表面プラズモン共鳴法による測定で得られたＳＰＲ角度の時間変化を第１測定結果とする。また、測定領域２０１におけるハイドロゲル層１０３が形成されている第２箇所２０３の表面プラズモン共鳴法による測定で得られたＳＰＲ角度の時間変化を第２測定結果とする。 Note that the time change in the SPR angle obtained by the measurement using the surface plasmon resonance method at the first location 202 where the hydrogel layer 103 is not formed in the measurement region 201 is defined as the first measurement result. Moreover, the time change of the SPR angle obtained by the measurement using the surface plasmon resonance method of the second location 203 where the hydrogel layer 103 is formed in the measurement region 201 is defined as a second measurement result.
　刺激印加装置１４０は、測定チップ１００の測定領域２０１に配置された細胞に対して刺激を与える。刺激印加装置１４０は、例えば、所定の薬物を測定チップ１００の測定領域２０１に配置された細胞に対して供給する。また、刺激印加装置１４０は、測定チップ１００の測定領域２０１に配置された細胞に対して、例えば、光を照射する。刺激印加装置１４０は、測定チップ１００の測定領域２０１に配置された細胞に対して、例えば、地場を印加する。また、刺激印加装置１４０は、測定チップ１００の測定領域２０１に配置された細胞に対して、例えば、電波を照射する。 The stimulation applying device 140 applies stimulation to cells arranged in the measurement region 201 of the measurement chip 100. The stimulation application device 140 supplies, for example, a predetermined drug to cells arranged in the measurement region 201 of the measurement chip 100. Furthermore, the stimulation application device 140 irradiates, for example, light onto the cells arranged in the measurement region 201 of the measurement chip 100. The stimulation applying device 140 applies, for example, a local voltage to the cells arranged in the measurement region 201 of the measurement chip 100. Further, the stimulation application device 140 irradiates, for example, radio waves to cells arranged in the measurement region 201 of the measurement chip 100.
　この細胞評価システムを用いた細胞評価方法において、表面プラズモン共鳴法による測定（ＳＰＲ信号が観測）が可能な領域は、第１箇所２０２および第２箇所２０３の２種類に分かれている。まず、ハイドロゲルのない第１箇所２０２では、例えば刺激を受けた細胞から発生する物質が、直接、ＳＰＲ測定領域（金属層１０２の表面から高さ２００ｎｍ程度までの領域）に到達する。金属層１０２の表面に到達する細胞から発生する物質の濃度の経時的な変化は自由拡散（分子拡散）に準じ、自由拡散（分子拡散）の分散に応じたＳＰＲ信号の経時変化が観測される。 In the cell evaluation method using this cell evaluation system, the areas where measurement by surface plasmon resonance (SPR signals are observed) are possible are divided into two types: a first location 202 and a second location 203. First, at the first location 202 where there is no hydrogel, substances generated from stimulated cells, for example, directly reach the SPR measurement region (an area up to a height of about 200 nm from the surface of the metal layer 102). Changes over time in the concentration of substances generated from cells that reach the surface of the metal layer 102 follow free diffusion (molecular diffusion), and changes over time in the SPR signal according to the dispersion of free diffusion (molecular diffusion) are observed. .
　第１箇所２０２の位置でのＳＰＲ信号の変化から、（１）刺激を受けた細胞から発生する物質による自由拡散（分子拡散）の影響、（２）第１箇所２０２における刺激を受けた細胞から発生する物質の最大信号強度、を決定することができる。この、第１箇所２０２でのＳＰＲ信号の経時変化を参照曲線として測定する。 Changes in the SPR signal at the first location 202 reveal (1) the influence of free diffusion (molecular diffusion) due to substances generated from the stimulated cells, and (2) the effect of free diffusion (molecular diffusion) from the stimulated cells at the first location 202. The maximum signal intensity of the generated substance can be determined. This change over time of the SPR signal at the first location 202 is measured as a reference curve.
　次に，ハイドロゲル層１０３が形成されている第２箇所２０３では、刺激を受けた細胞から発生する物質は、ハイドロゲル層１０３の上部表面に到達した後、ハイドロゲル層１０３の中を下方向へと拡散して、ＳＰＲ測定領域まで到達する。 Next, at the second location 203 where the hydrogel layer 103 is formed, substances generated from the stimulated cells reach the upper surface of the hydrogel layer 103 and then move downward in the hydrogel layer 103. and reaches the SPR measurement area.
　測定装置１３０では、測定領域２０１の延在方向と平行なゲルの表面からも細胞発生物質が供給され、測定結果に影響を及ぼす可能性がある。しかし、ハイドロゲル層１０３のサイズを、ゲルの厚さ８０μｍに対して１．５ｍｍと十分大きくとること、および、測定領域２０１の延在方向と平行なゲルの表面をスペーサと密着させることで、上述した問題となる状態の可能性を無視できるように設計している。 In the measurement device 130, cell-generating substances are also supplied from the surface of the gel parallel to the extending direction of the measurement region 201, which may affect the measurement results. However, by setting the size of the hydrogel layer 103 to be sufficiently large at 1.5 mm relative to the gel thickness of 80 μm, and by bringing the surface of the gel parallel to the extending direction of the measurement region 201 into close contact with the spacer, The design is such that the possibility of the problematic conditions mentioned above can be ignored.
　ハイドロゲル層１０３中を拡散するため、刺激を受けた細胞から発生する物質がＳＰＲ測定領域に到達するまでの時間には、第１箇所２０２での結果に比べて遅延が生じる。この時間遅延は、ハイドロゲル中の細胞から発生する物質の拡散特性を表すものであり、ハイドロゲルのメッシュサイズやゲルの化学修飾による細胞から発生する物質の吸着に応じて変化する。従って、第１測定結果と、第２測定結果とを比較することで、ハイドロゲル中の細胞から発生する物質の拡散特性を評価することができる。 Because it diffuses through the hydrogel layer 103, there is a delay in the time it takes for substances generated from stimulated cells to reach the SPR measurement area compared to the results at the first location 202. This time delay represents the diffusion characteristics of substances generated from cells in the hydrogel, and changes depending on the mesh size of the hydrogel and the adsorption of substances generated from cells due to chemical modification of the gel. Therefore, by comparing the first measurement result and the second measurement result, the diffusion characteristics of the substance generated from the cells in the hydrogel can be evaluated.
　次に、実施例を用いてより詳細に説明する。 Next, it will be explained in more detail using examples.
［実施例］
　まず、測定チップの作製について説明する。はじめに、ガラス製の基板を用意し、基板の上にスパッタ法または蒸着法により金を堆積し、基板表面に金属層を形成した。次に、形成した金属層の表面のハイドロゲルの層を形成する箇所（第２箇所）以外にブルーシートを貼ってマスクする。 [Example]
First, the production of the measurement chip will be explained. First, a glass substrate was prepared, and gold was deposited on the substrate by sputtering or vapor deposition to form a metal layer on the substrate surface. Next, a blue sheet is pasted on the surface of the formed metal layer other than the area where the hydrogel layer is to be formed (the second area) to mask it.
　次いで、マスクされていない金属層の表面を、bis(2-methacryloyl)oxyethyl disulfide（Bis-thiol）などのジチオールとアクリル基を有する化合物によって修飾する。次いで、修飾した金属層の表面にアクリルアミドゲル溶液を滴下し、カバーガラスでシールし、窒素パージ下で紫外線を照射して重合反応を起こさせる。十分に重合させた後、カバーガラスを取り除く。この状態で、ブルーシートと同じ厚さのハイドロゲル層が、金属層の一部（第２箇所）に形成される。 Next, the surface of the unmasked metal layer is modified with a compound having a dithiol and an acrylic group, such as bis(2-methacryloyl)oxyethyl disulfide (Bis-thiol). Next, an acrylamide gel solution is dropped onto the surface of the modified metal layer, sealed with a cover glass, and irradiated with ultraviolet light under a nitrogen purge to cause a polymerization reaction. After sufficient polymerization, remove the cover glass. In this state, a hydrogel layer having the same thickness as the blue sheet is formed on a part of the metal layer (second location).
　次に、マスクとしてのブルーシートを貼り付けたままで、ハイドロゲル層の表面のみに、「Sulfo-SANPAH」等のクロスリンカー剤を滴下することで、ハイドロゲル層の表面に、ＮＨＳ基を修飾する。 Next, with the blue sheet as a mask attached, a cross-linker agent such as "Sulfo-SANPAH" is dropped only on the surface of the hydrogel layer to modify the surface of the hydrogel layer with NHS groups. .
　次に、ブルーシートを剥がし、ハイドロゲル層が形成されていない領域（第１箇所）の金属層の表面を露出させる。次いで、コラーゲンを始めとする接着因子溶液を塗布し、金属層表面、およびハイドロゲル層上面に細胞接着層を形成する。 Next, the blue sheet is peeled off to expose the surface of the metal layer in the region (first location) where the hydrogel layer is not formed. Next, an adhesion factor solution including collagen is applied to form a cell adhesion layer on the surface of the metal layer and the upper surface of the hydrogel layer.
　次に、上述したように、金属層、ハイドロゲル層、細胞接着層を形成した測定チップを、ヒト血管内皮細胞ＨＵＶＥＣを含有した細胞培地に浸漬し、３日間培養した。培養は、ペトリシャーレの中で実施した。３日間の培養の後、リン酸緩衝液 (phosphate-buffered saline：ＰＢＳ）でリンスすることで、金属層（第１箇所）およびハイドロゲル層（第２箇所）に細胞を搭載した測定チップを得た。なお、バックグラウンド値の測定のため、第１箇所および第２箇所の各々に、対象となる細胞を配置しないリファレンス領域を設ける。例えば、第１箇所、第２箇所の各々において、表面側と基板裏面側とに滅菌したネオジム磁石をとりつけて、細胞を搭載させない領域を設ける方法が挙げられる。 Next, as described above, the measurement chip on which the metal layer, hydrogel layer, and cell adhesion layer were formed was immersed in a cell culture medium containing human vascular endothelial cells HUVEC and cultured for 3 days. Cultivation was performed in Petri dishes. After 3 days of culture, a measurement chip with cells mounted on the metal layer (first location) and hydrogel layer (second location) was obtained by rinsing with phosphate-buffered saline (PBS). Ta. Note that in order to measure the background value, a reference region in which no target cells are placed is provided at each of the first location and the second location. For example, in each of the first and second locations, a sterilized neodymium magnet may be attached to the front side and the back side of the substrate to provide an area where cells are not mounted.
　培養３日後の第１箇所の細胞の状態を図３Ａに示し、培養３日後の第２箇所の細胞の状態を図３Ｂに示す。これらは、位相差顕微鏡による観察結果（写真）である。 The state of the cells at the first location after 3 days of culture is shown in FIG. 3A, and the state of the cells at the second location after 3 days of culture is shown in FIG. 3B. These are the observation results (photographs) using a phase contrast microscope.
　上述したように培養することで、対象の細胞を搭載した測定チップを用いた表面プラズモン共鳴法による測定結果を、図４Ａに示す。ＳＰＲ装置により、１０μｍ毎のＳＰＲ角度変化、つまり屈折率変化を一定時間ごとに測定した。また、測定において、ＰＢＳを測定チップ上に注入した。 By culturing as described above, the measurement results by surface plasmon resonance method using a measurement chip loaded with target cells are shown in FIG. 4A. Using an SPR device, changes in the SPR angle every 10 μm, that is, changes in the refractive index, were measured at regular intervals. In addition, in the measurement, PBS was injected onto the measurement chip.
　測定Ｔ秒後のＳＰＲ測定位置によるＳＰＲ角度測定を見ると、細胞が付着している位置では、細胞が付着していない部分と比較して、ＳＰＲ角度が大きく観測される（図４Ｂ）。これは、ＳＰＲの測定領域に存在する細胞本体に由来する細胞骨格などの屈折率を反映している。この観測結果により、細胞が付着している観察位置の部分を特定することができる。 Looking at the SPR angle measurement at the SPR measurement position after T seconds of measurement, the SPR angle is observed to be larger at the position where cells are attached compared to the area where cells are not attached (FIG. 4B). This reflects the refractive index of the cytoskeleton derived from the cell body present in the SPR measurement region. Based on this observation result, it is possible to specify the part of the observation position where cells are attached.
　細胞が付着しているある点（Ｐポイント）について、時間経過を観察した結果を図４Ｃに示す。観測されたＳＰＲ角度は、周期的に大きなピークを形成する様子が観測され、これは、刺激を受けた生細胞の細胞骨格の収縮により引き起こされる事象と考えられる。このように、刺激を受けた細胞の活動によって引き起こされる細胞骨格の経時的な動きを、付着している細胞ごとに測定することができる。 Figure 4C shows the results of observing the time course at a certain point (P point) where cells are attached. The observed SPR angle was observed to periodically form large peaks, which is considered to be an event caused by contraction of the cytoskeleton of the stimulated living cells. In this way, the movement of the cytoskeleton over time caused by stimulated cell activity can be measured for each attached cell.
　細胞が複数の観測ポイント（フォトダイオード素子のピクセル位置）をまたがる大きさの場合、各測定ポイントで計測されるＳＰＲ角度ベースライン（ピークを除いたＳＰＲ角度の平均値）を観察位置毎にプロットすることで、ＳＰＲ観察ポイント上に存在するＳＰＲ角度（屈折率）に依存した細胞の形状を類推することができる（図５Ａ，図５Ｂ）。したがって、ＳＰＲ装置のセンサを構成する撮像素子の画素密度をより高くすることで、より詳細に細胞の形状を評価することが可能となる。 If the cell is large enough to span multiple observation points (pixel positions of photodiode elements), plot the SPR angle baseline (average value of SPR angles excluding peaks) measured at each measurement point for each observation position. By doing so, it is possible to infer the shape of the cell depending on the SPR angle (refractive index) existing on the SPR observation point (FIGS. 5A and 5B). Therefore, by increasing the pixel density of the image sensor that constitutes the sensor of the SPR device, it becomes possible to evaluate the shape of cells in more detail.
　また、各位置の骨格密度とともに、その細胞骨格密度の動きも分かるため、細胞の位置分布と各位置に対する細胞骨格密度の時間分布を両方同時に測定することができる。例えば図５Ａ，図５Ｂの場合、Ｅ点とＦ点で鋭いピークを持つＳＰＲ角度の時間変移が起きている事がわかる。つまり、上記で類推した細胞形状の中で、特異的なＳＰＲ角度変化が発生する部位を局所的に把握することができる。 Furthermore, since the movement of the cytoskeletal density as well as the skeletal density at each position is known, both the cell position distribution and the time distribution of the cytoskeletal density for each position can be measured simultaneously. For example, in the case of FIGS. 5A and 5B, it can be seen that the SPR angle changes over time with sharp peaks at points E and F. In other words, it is possible to locally grasp the region where a specific SPR angle change occurs in the cell shape analogized above.
　ハイドロゲル層が形成されている第２箇所でのＳＰＲ測定における、ある時間のＳＰＲ角度測定をみると（図６Ａ）、測定領域に細胞が付着していても、ハイドロゲル層の圧さがＳＰＲ観察領域（基板表面から＜２００ｎｍ）よりも十分高いため、ハイドロゲル層が未形成の第１箇所のＳＰＲ測定に見られていたような、細胞本体による位置依存的なＳＰＲ角度変化は観察されない。つまりＳＰＲ測定で直接測定できる金属層の表面近傍に細胞が存在しない状態となる厚さにハイドロゲル層を形成することで、細胞骨格の存在または細胞骨格の変化に伴う大きな屈折率変化の影響を受けることがないことがわかる。 Looking at the SPR angle measurement at a certain time in the SPR measurement at the second location where the hydrogel layer is formed (Figure 6A), even if cells are attached to the measurement area, the pressure of the hydrogel layer is Since it is sufficiently higher than the observation area (<200 nm from the substrate surface), position-dependent SPR angle changes due to the cell body are not observed, as was seen in the SPR measurement at the first location where no hydrogel layer was formed. In other words, by forming a hydrogel layer at a thickness that eliminates cells near the surface of the metal layer, which can be directly measured by SPR measurement, we can eliminate the effects of large refractive index changes due to the presence of a cytoskeleton or changes in the cytoskeleton. I know that I will not receive it.
　一方、刺激を受けた細胞が、例えば、情報伝達物質であるカルシウムやアミノ酸を発生（分泌）すると、これら物質がＳＰＲ測定で観測される。前述したように、ハイドロゲル層が未形成の第１領域と、ハイドロゲル層が形成されている第２領域とでは、細胞が発生した物質が金属層の表面近傍に到達する時間が異なる。この差（時間遅延）が、上記物質の、ハイドロゲル中の拡散特性を表しており、ハイドロゲルのメッシュサイズやハイドロゲルの化学修飾による細胞から発生する物質の吸着に応じて変化する。 On the other hand, when stimulated cells generate (secrete) information transmitting substances such as calcium and amino acids, these substances are observed by SPR measurement. As described above, the time it takes for substances generated by cells to reach the vicinity of the surface of the metal layer is different between the first region where no hydrogel layer is formed and the second region where a hydrogel layer is formed. This difference (time delay) represents the diffusion characteristics of the substance in the hydrogel, and changes depending on the mesh size of the hydrogel and the adsorption of substances generated from cells due to chemical modification of the hydrogel.
　従って、第１領域における第１測定結果と、第２領域における第２測定結果とを比較することで、細胞から発生する物質の、ハイドロゲル中の拡散特性を評価することができる。言い換えると、特定のメッシュサイズ、または特定の化学修飾が施されているハイドロゲルにおける、拡散特性が既知の物質の拡散特性との比較により、測定により得られた拡散特性を有する物質の定性・定量が可能となる。 Therefore, by comparing the first measurement result in the first region and the second measurement result in the second region, it is possible to evaluate the diffusion characteristics of substances generated from cells in the hydrogel. In other words, by comparing the diffusion properties of a substance with known diffusion properties in a hydrogel with a specific mesh size or a specific chemical modification, the qualitative and quantitative determination of a substance with the diffusion properties obtained by measurement is performed. becomes possible.
　次に、測定チップに配置（付着）した細胞に対して、純水を滴下した場合のＳＰＲ測定結果について説明する。測定チップに付着した細胞に対して、純水を滴下すると、大きな浸透圧負荷がかかり細胞膜が破裂して細胞内の分子が放出される。細胞が付着している測定チップは、初期状態においてＰＢＳでぬれている状態であるため、細胞が付着していないリファレンス領域では、純水を滴下していない状態（図６Ａ）に対し、純水混入により屈折率の低下がみられる（図６Ｂ）。一方、細胞が付着している領域のＳＰＲ角度変化をみると、屈折率の上昇がみられる（図６Ｃ）。リファレンス領域からの差分値をとることにより、細胞破壊によって放出された分子に起因する屈折率変化を測定することができる。 Next, the SPR measurement results when pure water is dropped onto cells placed (attached) on the measurement chip will be explained. When pure water is dropped onto cells attached to a measurement chip, a large osmotic pressure load is applied, causing the cell membrane to rupture and release molecules within the cell. Since the measurement chip to which cells are attached is initially wet with PBS, in the reference area where cells are not attached, pure water is not added (Fig. 6A). A decrease in the refractive index is observed due to the contamination (FIG. 6B). On the other hand, when looking at the SPR angle change in the region where cells are attached, an increase in the refractive index is seen (FIG. 6C). By taking the difference value from the reference region, it is possible to measure the change in refractive index caused by molecules released by cell destruction.
　実施の形態に係る細胞評価方法によれば、測定チップの測定領域に配置した細胞に供給する溶液を変えることにより、溶液に含まれる様々な分子に対する細胞の刺激応答性が測定可能である。例えば、薬物による細胞の刺激応答性などが、評価できる。屈折率変化は、薬物溶液中の薬物分子の流入によっても変化するが、リファレンス領域の屈折率を差し引くことによって細胞の刺激応答に依存した変化を求めることができる。 According to the cell evaluation method according to the embodiment, by changing the solution supplied to the cells placed in the measurement region of the measurement chip, it is possible to measure the stimulus responsiveness of cells to various molecules contained in the solution. For example, the responsiveness of cells to drug stimulation can be evaluated. The refractive index change also changes due to the influx of drug molecules in the drug solution, but by subtracting the refractive index of the reference region, changes depending on the stimulation response of the cells can be determined.
　以上に説明したように、本発明によれば、ハイドロゲルの層が未形成の箇所の表面プラズモン共鳴法による第１測定結果と、測定領域におけるハイドロゲルの層が形成されている箇所の表面プラズモン共鳴法による第２測定結果とを得るので、細胞の活性評価などのための測定が、測定対象の細胞にストレスなどを与えることなく、高い解像度で容易に実施できるようになる。 As explained above, according to the present invention, the first measurement result by the surface plasmon resonance method at a location where a hydrogel layer is not formed and the surface plasmon resonance at a location where a hydrogel layer is formed in the measurement region. Since the second measurement result is obtained by the resonance method, measurements for evaluating cell activity can be easily carried out with high resolution without applying stress to the cells to be measured.
　本発明によれば、細胞を配置した測定領域に分析対象の刺激分子を導入または滴下することで、細胞骨格の変形を始めとする細胞の物理的な振る舞い(細胞骨格等の変化)、細胞伝達分子の伝播を始めとする細胞の化学的な振る舞い(分子分泌)を、同一基板上で同時に測定することができ、より詳細な細胞応答機序を捉えることができる。例えば、化学的な振る舞いの測定により、初期の細胞応答のイベント発生を観察することができ、形質転換の初期を捉えることもできる。 According to the present invention, by introducing or dropping stimulating molecules to be analyzed into a measurement area where cells are placed, physical behavior of cells including deformation of the cytoskeleton (changes in the cytoskeleton, etc.), cell communication, etc. The chemical behavior of cells (molecular secretion), including the propagation of molecules, can be measured simultaneously on the same substrate, making it possible to understand more detailed cellular response mechanisms. For example, by measuring chemical behavior, it is possible to observe the occurrence of early cellular response events, and it is also possible to capture the early stages of transformation.
　ハイドロゲルは、細胞培養時の足場として採用されるなど、細胞の活動を妨げない物質であることから、細胞活性測定に影響を与えない。また、より生体組織の組成に近づけたＭＰＳとすることも可能である。ＳＰＲ測定では、測定に用いる入射光が、細胞に直接照射されないため、細胞活性への影響も少ない。 Hydrogel is a material that does not interfere with cell activity and is used as a scaffold during cell culture, so it does not affect cell activity measurements. Furthermore, it is also possible to create an MPS that has a composition closer to that of living tissue. In SPR measurement, the incident light used for measurement does not directly irradiate cells, so it has little effect on cell activity.
　測定領域に設けるハイドロゲルの層は、組成によって網目サイズや硬さを調整可能であり、作製するハイドロゲルの物性を制御することで、生体組織の病変組織モデルとすることができる。例えば、生体組織の線維化や瘢痕などによる、生体組織内部の局所的な物性変化を模擬することができる。また、フィルタとしての役割も果たすため、ハイドロゲルのメッシュサイズ設計により、不要な分子の侵入を抑え、検出したい分子郡だけ通すフィルタとしても作用させることができる。 The mesh size and hardness of the hydrogel layer provided in the measurement area can be adjusted depending on the composition, and by controlling the physical properties of the produced hydrogel, it can be used as a diseased tissue model of living tissue. For example, it is possible to simulate local changes in physical properties inside living tissue due to fibrosis or scarring of living tissue. In addition, since it also serves as a filter, by designing the mesh size of the hydrogel, it can act as a filter that prevents unnecessary molecules from entering and allows only the molecules that are desired to be detected to pass through.
　また、ＳＰＲ装置のセンサを構成する撮像素子の画素密度により、細胞の形状を算出することができるため、細胞の形状とハイドロゲルの厚さを踏まえて分子の拡散を推定することでターゲット分子群の定量も行うことができる。 In addition, since the shape of the cell can be calculated from the pixel density of the image sensor that constitutes the sensor of the SPR device, the target molecule group can be estimated by estimating the diffusion of molecules based on the shape of the cell and the thickness of the hydrogel. can also be quantified.
　測定領域には、１種類または複数種類のハイドロゲルを同時に配置することができため、分子量フィルタを変えた場合の分泌物質の拡散動態を評価することができる。 Since one type or multiple types of hydrogels can be placed simultaneously in the measurement area, it is possible to evaluate the diffusion dynamics of secreted substances when changing the molecular weight filter.
　本発明は、細胞培養や薬物刺激による細胞アッセイなどの分析に有用であり、薬理、組織工学や化学工学分野などに広く適応可能である。 The present invention is useful for analyzes such as cell culture and cell assays based on drug stimulation, and is widely applicable to fields such as pharmacology, tissue engineering, and chemical engineering.
　なお、本発明は以上に説明した実施の形態に限定されるものではなく、本発明の技術的思想内で、当分野において通常の知識を有する者により、多くの変形および組合わせが実施可能であることは明白である。 It should be noted that the present invention is not limited to the embodiments described above, and many modifications and combinations can be made within the technical idea of the present invention by those having ordinary knowledge in this field. One thing is clear.
　１００…測定チップ、１０１…透明基板、１０２…金属層、１０３…ハイドロゲル層、１０４…細胞接着層、１０５…細胞、１３０…測定装置、１４０…刺激印加装置、２０１…測定領域、２０２…第１箇所、２０３…第２箇所。 DESCRIPTION OF SYMBOLS 100...Measurement chip, 101...Transparent substrate, 102...Metal layer, 103...Hydrogel layer, 104...Cell adhesion layer, 105...Cell, 130...Measurement device, 140...Stimulation application device, 201...Measurement area, 202...No. 1 location, 203...2nd location.

Claims (6)

  1. 　一部にハイドロゲルの層を備えて表面プラズモン共鳴法による測定を実施する測定領域に測定対象の細胞を配置し、前記測定領域における前記ハイドロゲルの層が未形成の箇所の表面プラズモン共鳴法による第１測定結果と、前記測定領域における前記ハイドロゲルの層が形成されている箇所の表面プラズモン共鳴法による第２測定結果とを得る測定ステップと、
    　前記第１測定結果により、前記細胞の細胞膜の変化を評価する第１評価ステップと、
    　前記第１測定結果と前記第２測定結果との比較により、前記細胞から発生する物質の前記ハイドロゲルにおける拡散速度を評価する第２評価ステップと
    　を備える細胞評価方法。     Cells to be measured are placed in a measurement area that partially includes a hydrogel layer and measurements are performed using surface plasmon resonance, and surface plasmon resonance is performed on portions of the measurement area where the hydrogel layer is not formed. a measuring step of obtaining a first measurement result and a second measurement result by surface plasmon resonance of a portion of the measurement region where the hydrogel layer is formed;
    a first evaluation step of evaluating changes in the cell membrane of the cell based on the first measurement result;
    A cell evaluation method comprising: a second evaluation step of evaluating a diffusion rate of a substance generated from the cells in the hydrogel by comparing the first measurement result and the second measurement result.
  2. 　請求項１記載の細胞評価方法において、
    　前記第１評価ステップは、前記細胞に刺激を与えることによる前記第１測定結果より、前記細胞の細胞膜の変化を評価し、
    　前記第２評価ステップは、前記細胞に刺激を与えることによる前記第１測定結果と前記第２測定結果との比較により、前記物質の前記ハイドロゲルにおける拡散速度を評価する
    　ことを特徴とする細胞評価方法。     In the cell evaluation method according to claim 1,
    The first evaluation step evaluates changes in the cell membrane of the cell based on the first measurement result obtained by applying stimulation to the cell,
    Cell evaluation characterized in that the second evaluation step evaluates the diffusion rate of the substance in the hydrogel by comparing the first measurement result and the second measurement result by applying stimulation to the cell. Method.
  3. 　請求項１または２記載の細胞評価方法において、
    　前記測定領域における前記ハイドロゲルの層が未形成の箇所の表面プラズモン共鳴法による測定で得られたＳＰＲ角度の時間変化を前記第１測定結果とし、
    　前記測定領域における前記ハイドロゲルの層が形成されている箇所の表面プラズモン共鳴法による測定で得られたＳＰＲ角度の時間変化を前記第２測定結果とする
    　ことを特徴とする細胞評価方法。     In the cell evaluation method according to claim 1 or 2,
    The first measurement result is a time change in the SPR angle obtained by surface plasmon resonance measurement of a portion of the measurement region where the hydrogel layer is not formed;
    A cell evaluation method, characterized in that the second measurement result is a time change in the SPR angle obtained by surface plasmon resonance measurement at a location where the hydrogel layer is formed in the measurement region.
  4. 　一部にハイドロゲルの層を備えて表面プラズモン共鳴法による測定を実施する測定領域と、
    　表面プラズモン共鳴法により、前記測定領域における前記ハイドロゲルの層が未形成の箇所における第１測定と、前記測定領域における前記ハイドロゲルの層が形成されている箇所における第２測定とを実施する測定装置と
    　を備える細胞評価システム。     A measurement area that partially includes a hydrogel layer and performs measurements using surface plasmon resonance;
    A measurement in which a first measurement is performed at a location in the measurement region where the hydrogel layer is not formed, and a second measurement is performed at a location in the measurement region where the hydrogel layer is formed, using a surface plasmon resonance method. A cell evaluation system equipped with a device and .
  5. 　請求項４記載の細胞評価システムにおいて、
    　前記測定領域に配置される測定対象の細胞に刺激を与える刺激印加装置をさらに備えることを特徴とする細胞評価システム。     The cell evaluation system according to claim 4,
    A cell evaluation system further comprising a stimulation device for stimulating cells to be measured arranged in the measurement region.
  6. 　請求項４または５記載の細胞評価システムにおいて、
    　前記測定装置は、
    　前記測定領域における前記ハイドロゲルの層が未形成の箇所の表面プラズモン共鳴法による測定で得られたＳＰＲ角度の時間変化を前記第１測定による第１測定結果とし、
    　前記測定領域における前記ハイドロゲルの層が形成されている箇所の表面プラズモン共鳴法による測定で得られたＳＰＲ角度の時間変化を前記第２測定による第２測定結果とする
    　ことを特徴とする細胞評価システム。     The cell evaluation system according to claim 4 or 5,
    The measuring device includes:
    A time change in the SPR angle obtained by measurement using a surface plasmon resonance method at a location where the hydrogel layer is not formed in the measurement region is defined as a first measurement result of the first measurement,
    A cell evaluation characterized in that the time change in the SPR angle obtained by the surface plasmon resonance measurement of the portion where the hydrogel layer is formed in the measurement region is used as the second measurement result of the second measurement. system.
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JP2009510423A (en) * 2005-09-27 2009-03-12 サントル・ナシオナル・ドゥ・ラ・ルシェルシュ・シアンティフィーク（セーエヌエールエス） New chip for surface plasmon resonance (SPR) detection
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