# US 2019/0244349 A1 · Method for Analyzing State of Cells in Spheroid > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0244349 A1 - Document ID: US20190244349A1 - Status: Published application - Kind: A1 - Application number: 16/306,615 - Filed: 2016-06-16 - Published: 2019-08-08 - Earliest claimed: 2016-06-16 - Assignees: Hitachi High-Technologies Corporation - Applicants: Hitachi High-Technologies Corporation - Inventors: Naoko SENDA; Toshinari SAKURAI - Web page: https://patentgrep.com/patent/US20190244349A1 ## CPC - G01N 15/1433: using image recognition - G06T 7/0012: {Biomedical image inspection} - C12M 1/34: Measuring or testing with condition measuring or sensing means, e.g. colony counters - C12M 41/14: {Incubators; Climatic chambers (per se B01L1/00)} - C12M 41/36: {of biomass, e.g. colony counters or by turbidity measurements (electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404)} - C12Q 1/02: involving viable microorganisms - G01N 21/27: using photo-electric detection (G01N21/31 takes precedence){; circuits for computing concentration (logarithmic circuits G06G7/24; photometric circuits in general G01J)} - G06T 5/92: based on global image properties - G06V 20/695: {Preprocessing, e.g. image segmentation} - G06V 20/698: {Matching; Classification} - G01N 2015/1006: {for cytology} - G01N 2015/1445: {Three-dimensional imaging, imaging in different image planes, e.g. under different angles or at different depths, e.g. by a relative motion of sample and detector, for instance by tomography} - G01N 2015/1452: {Adjustment of focus; Alignment} - G01N 2015/1497: {Particle shape} - G06T 2207/10101: Optical tomography; Optical coherence tomography [OCT] - G06T 2207/30024: Cell structures in vitro; Tissue sections in vitro - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL - 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} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G06V: IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING ## Abstract The present invention provides a method and an apparatus for analyzing a cell state, cell death in particular, in an interior of a spheroid non-invasively and quantitatively when the spheroid is cultured. More specifically, the present invention provides a method and an apparatus for analyzing a cell state by implementing optical imaging of a spheroid by using an optical instrument characterized by a high resolution and analyzing the internal structure of the spheroid. ## Claims ### Claim 1 A cell analysis method of analyzing a cell state in the interior of a spheroid, comprising: a step of irradiating the spheroid with light; a step of acquiring a plurality of spheroid cross-sectional images having different distances from a light irradiation position; and a step of correcting brightness of the images in consideration of signal strength attenuation. ### Claim 2 (depends on 1) The cell analysis method according to claim 1, wherein the signal strength attenuation depends on a three-dimensional shape of the spheroid and/or a distance from the light irradiation position to a measurement position. ### Claim 3 (depends on 1) The cell analysis method according to claim 1, wherein the spheroid is a spheroid formed by agglomerating cells in the vicinity of a culture surface, and a plurality of spheroid cross-sectional images having different distances from the culture surface as the irradiation position are acquired. ### Claim 4 (depends on 1) The cell analysis method according to claim 1, further comprising steps of partitioning each of the images into pieces containing a plurality of pixels of a grid pattern and determining an average brightness for each of the pieces. ### Claim 5 (depends on 1) The cell analysis method according to claim 1, further comprising a step of measuring an external shape of the spheroid. ### Claim 6 (depends on 1) The cell analysis method according to claim 1, further comprising a step of correcting the spheroid position on the basis of external XYZ coordinates of the spheroid. ### Claim 7 (depends on 1) The cell analysis method according to claim 1, further comprising, after the steps according to claim 1 are implemented: a step of irradiating the spheroid with light; a step of acquiring a second cross-sectional image of the spheroid; and a step of comparing the brightness of the second image with brightness acquired through the steps according to claim 1. ### Claim 8 A cell analysis apparatus for analyzing a cell state in the interior of a spheroid, comprising: a light source, a light condensation optical system configured to irradiate cells on a culture surface with light from the light source, and a detection optical system configured to detect light from the cells; and an analysis unit configured to analyze an image based on information acquired from the detection optical system, wherein the analysis unit has: an image acquisition unit configured to acquire a plurality of cross-sectional images having different distances from the culture surface in the vertical direction; a brightness measurement unit configured to measure brightness of the plurality of cross-sectional images; and a state analysis unit configured to correct the brightness in consideration of signal strength attenuation and analyze a cell state in the spheroid interior. ### Claim 9 (depends on 8) The cell analysis apparatus according to claim 8, wherein the light source, the light condensation optical system, and the detection optical system are configured as an optical coherence tomograph (OCT). ### Claim 10 (depends on 8) The cell analysis apparatus according to claim 8, further comprising a memory unit configured to store signal strength attenuation data and/or brightness data, which is related to a specific cell state in the interior of a spheroid. ### Claim 11 (depends on 8) The cell analysis apparatus according to claim 8, wherein the analysis unit further has a measurement unit configured to measure an external shape of the spheroid. ### Claim 12 A cell culture apparatus comprising a culture unit configured to culture a spheroid, an analysis unit configured to analyze a cell state in the interior of the spheroid by using light, and a control unit configured to control culture and analysis of the spheroid, wherein (1) the analysis unit has: a light source, a light condensation optical system configured to irradiate cells on a culture surface with light from the light source, a detection optical system configured to detect light from the cells; and an image analysis unit configured to analyze an image based on information acquired from the detection optical system, and the image analysis unit has: an image acquisition unit configured to acquire a plurality of cross-sectional images having different distances from the culture surface in the vertical direction; a brightness measurement unit configured to measure brightness of the plurality of cross-sectional images; and a state analysis unit configured to analyze a cell state in the spheroid interior on the basis of the brightness, and (2) the control unit controls at least one of supply of a cell solution, supply of a culture medium, disposal of a culture medium, culture of cells, irradiation of light, detection of light, acquisition of an image, measurement of brightness, and analysis of a cell state. ### Claim 13 (depends on 12) The cell culture apparatus according to claim 12, wherein the culture unit has: a thermostatic chamber; a culture vessel to be arranged in the thermostatic chamber, which is configured to culture a spheroid; a cell bottle being connected to the culture vessel, which is configured to supply a cell solution; a culture medium bottle being connected to the culture vessel, which is configured to supply a culture medium; and a waste fluid bottle being connected to the culture vessel, which is configured to store a culture medium discarded from the culture vessel. ### Claim 14 (depends on 12) The cell culture apparatus according to claim 12, wherein the state analysis unit is configured to correct the brightness in consideration of signal strength attenuation and analyze a cell state in the spheroid interior. ### Claim 15 (depends on 12) The cell culture apparatus according to claim 12, wherein the control unit is configured to control the culture of the spheroid in the culture unit on the basis of an output from the state analysis unit.