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WO2023238375A1 - Procédé et système d'évaluation de cellules - Google Patents

Procédé et système d'évaluation de cellules Download PDF

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Publication number
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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WIPO (PCT)
Prior art keywords
measurement
cell
cells
location
hydrogel layer
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Ceased
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PCT/JP2022/023435
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English (en)
Japanese (ja)
Inventor
鈴代 井上
友海 村井
あや 田中
陸 高橋
倫子 瀬山
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Nippon Telegraph and Telephone Corp
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Nippon Telegraph and Telephone Corp
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Priority to PCT/JP2022/023435 priority Critical patent/WO2023238375A1/fr
Publication of WO2023238375A1 publication Critical patent/WO2023238375A1/fr
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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

Definitions

  • 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.

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

À l'étape S101, les cellules à mesurer sont situées dans une zone de mesure. À l'étape S102, un premier résultat de mesure est obtenu par le procédé de résonance plasmonique de surface à un endroit de la zone de mesure où une couche d'hydrogel n'est pas constituée, et un second résultat de mesure est obtenu par le procédé de résonance plasmonique de surface à un endroit de la zone de mesure où la couche d'hydrogel est constituée (étape de mesure). À l'étape S103, une modification de la membrane cellulaire des cellules est évaluée en fonction du premier résultat de mesure (première étape d'évaluation). À l'étape S104, la vitesse de diffusion d'une substance générée à partir des cellules dans l'hydrogel est évaluée en comparant le premier résultat de mesure avec le deuxième résultat de mesure (deuxième étape d'évaluation).
PCT/JP2022/023435 2022-06-10 2022-06-10 Procédé et système d'évaluation de cellules Ceased WO2023238375A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025186861A1 (fr) * 2024-03-04 2025-09-12 Ｎｔｔ株式会社 Composite gel-métal et procédé de production de composite gel-métal
WO2025224858A1 (fr) * 2024-04-24 2025-10-30 Ｎｔｔ株式会社 Procédé de fabrication de composite gel-métal

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009510423A (ja) * 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） 表面プラズモン共鳴（ｓｐｒ）検出用の新規チップ
JP2013257154A (ja) * 2012-06-11 2013-12-26 Nippon Telegr & Teleph Corp <Ntt> 測定チップ

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009510423A (ja) * 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） 表面プラズモン共鳴（ｓｐｒ）検出用の新規チップ
JP2013257154A (ja) * 2012-06-11 2013-12-26 Nippon Telegr & Teleph Corp <Ntt> 測定チップ

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN YUN-CHU, CHEN JING-JIE, HSIAO YANG-JYUN, XIE CHENG-ZHE, PENG CHIEN-CHUNG, TUNG YI-CHUNG, CHEN YIH-FAN: "Plasmonic gel films for time-lapse LSPR detection of hydrogen peroxide secreted from living cells", SENSORS AND ACTUATORS B: CHEMICAL, ELSEVIER BV, NL, vol. 336, 1 June 2021 (2021-06-01), NL , pages 129725, XP093113028, ISSN: 0925-4005, DOI: 10.1016/j.snb.2021.129725 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025186861A1 (fr) * 2024-03-04 2025-09-12 Ｎｔｔ株式会社 Composite gel-métal et procédé de production de composite gel-métal
WO2025224858A1 (fr) * 2024-04-24 2025-10-30 Ｎｔｔ株式会社 Procédé de fabrication de composite gel-métal

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