WO2019135266A1 - Dispositif de traitement d'images de cellule, procédé de traitement d'images de cellule et programme de traitement d'images de cellule - Google Patents

Dispositif de traitement d'images de cellule, procédé de traitement d'images de cellule et programme de traitement d'images de cellule Download PDF

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Publication number
WO2019135266A1
WO2019135266A1 PCT/JP2018/000021 JP2018000021W WO2019135266A1 WO 2019135266 A1 WO2019135266 A1 WO 2019135266A1 JP 2018000021 W JP2018000021 W JP 2018000021W WO 2019135266 A1 WO2019135266 A1 WO 2019135266A1
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cell
rate
inflow
image
region
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PCT/JP2018/000021
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English (en)
Japanese (ja)
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朗 松下
良介 村田
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オリンパス株式会社
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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
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • 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
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the present invention relates to a culture image processing apparatus, a cell image processing method, and a cell image processing program.
  • the present invention has been made in view of the above-described circumstances, and a cell image processing device, a cell image processing method, and a cell image capable of accurately detecting cell proliferation and accurately managing cell culture.
  • the purpose is to provide a processing program.
  • a plurality of images obtained by capturing cells at time intervals are input, and any two of the input images are processed to obtain the images
  • a cell number change rate calculation unit that calculates a cell number change rate that is a change in the cell number per unit time in the first area of the first area, and any two of the images in the input image
  • a cell inflow rate calculating unit that calculates a cell inflow rate, which is the number of cells inflow per unit time into the second region of the image, and any two of the images in the input image
  • a cell flow rate calculation unit that calculates a cell flow rate, which is the number of cell flows per unit time from within the second region; the cell number change rate calculated by the cell number change rate calculation unit;
  • a proliferation rate calculation unit that calculates a cell proliferation rate per unit time in the third region of the image based on the cell efflux rate calculated by the cell efflux rate calculation unit.
  • any two images of the input images are processed by the cell number change rate calculation unit And the cell number change rate in the first region is calculated.
  • any two images in the input image are processed by the cell inflow rate calculating unit to calculate the cell inflow rate into the second region.
  • any two images in the input image are processed by the cell efflux rate calculation unit to calculate the cell efflux rate from within the second region.
  • the growth rate calculation unit calculates the cell growth rate based on the cell number change rate, the cell inflow rate, and the cell outflow rate. Since the cell proliferation rate can be determined not only in terms of the cell number change rate but also the cell inflow rate and the cell outflow rate in two time-spaced images, the degree of cell proliferation due to cell division is more accurate It can be detected well and the cell culture can be managed precisely.
  • the calculation of the cell number change rate, the cell inflow rate and the cell outflow rate may use the same two images or different images. That is, the time interval between two images for calculating the cell number change rate, the cell inflow rate and the cell outflow rate may be different from each other.
  • the cell number change rate may be calculated for all two adjacent images, and the cell inflow rate and the cell outflow rate may be calculated periodically or aperiodically without calculating for all images. It may be calculated for the two selected images.
  • the first region, the second region, and the third region may be the same or different.
  • the growth rate calculation unit calculates a correction change rate, which is a change rate of the number of cells in the third region, based on the cell number change rate, and the third change rate is calculated based on the cell inflow rate.
  • the corrected inflow rate which is the inflow rate into the area, is calculated
  • the corrected outflow rate which is the outflow rate from the third area, is calculated based on the cell outflow rate, and the corrected inflow rate is calculated from the corrected change rate.
  • the cell proliferation rate may be calculated by subtraction and addition of the corrected outflow rate.
  • a cell mortality rate calculation part which computes a cell death rate which is a death number of the above-mentioned cell per unit time in the 3rd field, and the above-mentioned growth rate calculation part
  • the cell proliferation rate may be calculated by subtracting the corrected inflow rate, adding the corrected outflow rate, and subtracting the cell mortality rate.
  • the cell mortality calculation unit calculates the cell mortality in the third region, and the corrected inflow rate and the cell mortality are subtracted from the corrected change rate. By adding the corrected outflow rate, the cell proliferation rate can be detected more accurately.
  • the cell mortality calculation unit may count the number of cells suspended per unit time in the third region. By doing this, when observing cells that adhere to and grow on the bottom of the culture vessel, the number of cell death is easily calculated by counting the number of suspended cells in the third region. be able to.
  • the cell inflow rate calculation unit counts the number of cells flowing into the second area across the boundary with another area adjacent to the second area per unit time, and
  • the outflow rate calculating unit may count the number of cells flowing out of the second area across the boundary per unit time. In this way, the cell inflow rate and the cell outflow rate can be calculated more accurately by monitoring the boundary between the second region and the other region and counting the number of cells moved across the boundary. Can.
  • the cell inflow rate calculating unit and the cell outflow rate calculating unit may use the image with an interval equal to or less than a time obtained by dividing the length of the cell by the speed of the cell. . In this way, cells located near the boundary in the second region, cells that have entered the second region from the adjacent region, or cells that have moved from the other locations in the second region to near the boundary And the cell inflow rate and the cell outflow rate can be accurately calculated.
  • another aspect of the present invention includes a processor into which a plurality of images acquired by capturing a cell at intervals of time are input, and the processor is any of the input images
  • the two images are processed to calculate a cell number change rate which is a change in the number of cells per unit time in the first region of the images, and any two images in the input image are calculated.
  • Processing to calculate a cell inflow rate which is the number of cells inflow per unit time into the second area of the image, and processing any two of the images in the input image to process the second
  • the cell outflow rate which is the number of cell outflows per unit time from the area, is calculated, and the third area of the image is calculated based on the calculated cell number change rate, the cell inflow rate, and the cell outflow rate. Calculate the cell growth rate per unit time in Providing a cell image processing apparatus.
  • a plurality of images acquired by capturing cells at time intervals are input, and any two of the input images are processed.
  • Calculating a cell number change rate calculating a cell number change rate which is a change in the cell number per unit time in the first region of the image, and processing any two images in the input image
  • Calculating a cell inflow rate which is the number of cells inflow per unit time into the second region of the image, and any two of the images in the input image.
  • Cell flow rate calculation step of processing and calculating a cell flow rate which is the number of cell flows per unit time from within the second region, and the cell number change rate calculated by the cell number change rate calculation step Calculating the cell inflow rate Cell proliferation rate per unit time in the third region of the image based on the cell inflow rate calculated by the cell flow rate and the cell outflow rate calculated by the cell outflow rate calculating step It is a cell image processing method including the calculation step.
  • a plurality of images acquired by capturing cells at time intervals are input, and any two of the input images are processed.
  • Calculating a cell number change rate calculating a cell number change rate which is a change in the cell number per unit time in the first region of the image, and processing any two images in the input image
  • Calculating a cell inflow rate which is the number of cells inflow per unit time into the second region of the image, and any two of the images in the input image.
  • Cell flow rate calculation step of processing and calculating a cell flow rate which is the number of cell flows per unit time from within the second region, and the cell number change rate calculated by the cell number change rate calculation step Calculating the cell inflow rate Cell proliferation rate per unit time in the third region of the image based on the cell inflow rate calculated by the cell flow rate and the cell outflow rate calculated by the cell outflow rate calculating step It is a cell image processing program which makes a computer execute a calculation step.
  • FIG. 1 is an overall configuration diagram showing a cell image processing apparatus according to an embodiment of the present invention. It is a figure explaining the effect
  • a cell image processing apparatus 1, a cell image processing method, and a cell image processing program according to an embodiment of the present invention will be described below with reference to the drawings.
  • a plurality of images P n and P n + 1 acquired by photographing with a time interval opened by an imaging unit 50 such as a microscope are input.
  • the cell image processing device 1 stores the image P n and P n + 1 input from the imaging unit 50 in a memory 2 that sequentially stores the image P n and P n + 1 in association with time information when the image P n and P n + 1 was captured. And a processing unit 3 for processing the captured images P n and P n + 1 .
  • the processing unit 3 processes the images P n and P n + 1 stored in the memory 2 to determine the number of cells per unit time of the number of cells in the first region R1 in the images P n and P n + 1 .
  • Cell number change rate calculation unit 4 that calculates change rates
  • cell inflow rate calculation that calculates the cell inflow rate, which is the number of cell X inflows per unit time into the second region R2 in the images P n and P n + 1
  • Cell flow rate calculation unit 6 that calculates the cell flow rate, which is the number of cell X flows out per unit time from the second region R2 in the image P n and P n + 1 , and the calculated cell number change
  • the proliferation rate calculating unit 7 is provided to calculate the cell proliferation rate in the counting area (third area) R3 from the rate, the cell inflow rate and the cell outflow rate.
  • the cell number change rate calculation unit 4 calculates the number N n of cells in the first region R1 in the two images P n and P n + 1 acquired with a time interval of the first time ⁇ t1. , N n + 1 are respectively counted, and the difference between the cell numbers N n and N n + 1 counted in the respective images P n and P n + 1 is divided by the first time ⁇ t 1 to obtain the cell numbers N n and N n + 1 per unit time.
  • the cell number change rate ⁇ N / ⁇ t which is the change rate of is calculated.
  • Cellular influx rate calculating unit 5 images P n on the basis of the image P n of the two obtained at a just time interval second time .DELTA.t2, to P n + 1, the in P n + 1 Per unit time by counting the inflow number ⁇ N MOVEINn + 1 of cells X entering the two regions R2 across the border from the adjacent region and dividing the counted inflow number ⁇ N MOVEINn + 1 of the cell X by the second time ⁇ t 2
  • the cell inflow rate ⁇ N MOVEIN which is the inflow number ⁇ N MOVEIN of the cells X, is to be calculated.
  • Cells outflow rate calculation part 6 also, as shown in FIG. 3, images P n on the basis of the image P n of the two obtained at a just time interval second time .DELTA.t2, to P n + 1, the in P n + 1
  • P n + 1 By counting the number of outflows ⁇ N MOVEOUTn + 1 of the cells X which exited across the boundary from the two regions R2 to the adjacent region, and dividing the counted number of outflows of the cells X ⁇ N MOVEOUTn + 1 by the second time ⁇ t2,
  • the cell outflow rate ⁇ N MOVEOUT / ⁇ t 2 which is the outflow number ⁇ N MOVEOUT of the cell X is calculated.
  • the second time ⁇ t2 for calculating the inflow of the cell X into the second region R2 and the outflow of the second region R2 preferably satisfies the following conditional expression (1).
  • D is the length of cell X
  • V is the migration rate of cell X.
  • the length D of the cell X and the migration rate V may be set to a reference value at the time of past counting, or may be set to an average value in the second region R2.
  • the threshold value E of the length to determine entry or exit, it is determined that the cell X enters as the threshold E from the boundary between the second region R2 and the adjacent region, and the boundary From the second region R2 to the adjacent region, the cell X may be determined to be withdrawn.
  • the growth rate calculation unit 7 is configured to calculate a cell growth rate ⁇ N BIRTH / ⁇ t, which is the number of cell divisions per unit time in the third region R3 which is a counting region. Specifically, the cell number change rate calculated for the first region R1 is converted to the corrected change rate ⁇ N / ⁇ t for the third region R3, and the cell inflow rate and the cell outflow rate calculated for the second region R2 are calculated as the third
  • the corrected inflow rate ⁇ N MOVEIN / ⁇ t is converted into the corrected inflow rate ⁇ N MOVEIN / ⁇ t and the corrected outflow rate ⁇ N MOVEOUT / ⁇ t for the region R3, and the corrected inflow rate ⁇ N MOVEIN / ⁇ t is subtracted from the corrected change rate ⁇ N / ⁇ t converted by the following equation (2)
  • the cell proliferation rate ⁇ N BIRTH / ⁇ t is calculated by adding the corrected outflow rate ⁇ N MOVEOUT / ⁇ t.
  • Conversion of the cell number change rate calculated for the first region R1 to the corrected change rate ⁇ N / ⁇ t for the third region R3 is, for example, an area ratio of the third region R3 to the first region R1 It is done by multiplying by. Also, conversion to the corrected inflow rate ⁇ N MOVEIN / ⁇ t and the corrected outflow rate ⁇ N MOVEOUT / ⁇ t for the third area R3 of the cell inflow rate ⁇ N MOVEIN / ⁇ t and the cell outflow rate ⁇ N MOVEOUT / ⁇ t calculated for the second area R2 For example, the area ratio between the third region R3 and the second region R2 is multiplied by the cell inflow rate ⁇ N MOVEIN / ⁇ t and the cell outflow rate ⁇ N MOVEOUT / ⁇ t.
  • a cell image processing method using the cell image processing apparatus 1 according to the present embodiment configured as described above will be described below.
  • the start time t1 of the count, the end time t m and the time interval ⁇ t of the counting is set (step S1).
  • the image P n + 1 obtained at time t n + 1 from the time t n has passed by the time interval ⁇ t is the number of cells N n + 1 in the count region R3 is processed in cell number change rate calculating section 4 is counted (number of cells Change rate calculation step S4).
  • the image P n and the image P n + 1 are used, and the number of cells ⁇ N MOVEIN n + 1 having entered into the counting area R1, R2, R3 from outside the counting area R1, R2, R3 and the counting area R1, R2, The number of cells ⁇ N MOVEOUT n + 1 having exited from the inside of R3 out of the counting areas R1, R2 and R3 is calculated (cell inflow rate calculating step S5, cell outflow rate calculating step S6). Then, in the growth rate calculation unit 7, the cell growth rate ⁇ N BIRTH n + 1 / ⁇ t per unit time is calculated by the following equation (growth rate calculation step S7).
  • the cell number change rate ⁇ N / ⁇ t, the cell inflow rate ⁇ N MOVEINn + 1 / ⁇ t, the cell outflow rate ⁇ N MOVEOUTn + 1 / ⁇ t, and the cell proliferation rate ⁇ N BIRTHn + 1 / ⁇ t are all calculated based on the same adjacent image.
  • ⁇ t exists in the denominator. Therefore, all terms can be multiplied by ⁇ t to be equivalently converted to the following conditional expression (4).
  • cell number change rate ⁇ N / ⁇ t cell inflow rate ⁇ N MOVEINn + 1 / ⁇ t
  • cell outflow rate ⁇ N MOVEOUTn + 1 / ⁇ t cell proliferation rate ⁇ N BIRTHn + 1 / ⁇ t are all calculated for the same counting areas R1, R2 and R3. So there is no need for conversion.
  • ⁇ N BIRTH n + 1 (N n + 1 -N n ) - ⁇ N MOVEIN n + 1 + ⁇ N MOVEOUT n + 1 (4)
  • the cell proliferation rate ⁇ N BIRTHn + 1 / ⁇ t corresponds to two images P n and P n + 1 with a time interval ⁇ t. Not only the cell number change rate ⁇ N / ⁇ t but also the cell inflow rate ⁇ N MOVEINn + 1 / ⁇ t and the cell outflow rate ⁇ N MOVEOUTn + 1 / ⁇ t are taken into consideration, so the degree of proliferation of the cell X by cell division is detected more accurately There is an advantage that the cell culture can be managed with high accuracy.
  • the counting area R1,1 is satisfied by satisfying the conditional expression (1).
  • Cells X entering and exiting R2 and R3 can be properly detected. That is, if ⁇ t2 is too short, it is necessary to calculate cell inflow rate ⁇ N MOVEINn + 1 / ⁇ t2 and cell outflow rate ⁇ N MOVEOUTn + 1 / ⁇ t2 for many images P n and P n + 1 one by one, the data amount increases and the load of counting increases.
  • the time ⁇ t1 for calculating the cell number change rate ⁇ N / ⁇ t is set shortest
  • the time ⁇ t2 for calculating the cell inflow rate ⁇ N MOVEINn + 1 / ⁇ t2 and the cell outflow rate ⁇ N MOVEOUTn + 1 / ⁇ t2 is larger than the time ⁇ t1 It is preferable to set to the appropriate time which satisfy
  • the processing unit 3 includes the cell number change rate calculating unit 4, the cell inflow rate calculating unit 5, the cell outflow rate calculating unit 6, and the proliferation rate calculating unit 7. It may be constituted by an electric circuit or may be constituted by one or more processors.
  • the present invention configures the cell number change rate calculation step S4, the cell inflow rate calculation step S5, the cell outflow rate calculation step S6, and the proliferation rate calculation step S7 by a cell image processing program that can be executed by a computer. May be
  • the cell death to calculate the cell mortality rate ⁇ N DEATHn + 1 / ⁇ t which is the death number ⁇ N DEATHn + 1 of the cell X per unit time in the third region R3.
  • the rate calculating unit 8 may be further provided.
  • the growth rate calculation unit 7 may calculate the cell growth rate ⁇ N BIRTHn + 1 / ⁇ t by the following equation (5) (step S11).
  • ⁇ N BIRTHn + 1 / ⁇ t (N n + 1 -N n ) / ⁇ t- ⁇ N MOVEIN n + 1 / ⁇ t + ⁇ N MOVEOUT n + 1 / ⁇ t + ⁇ N DEATH n + 1 / ⁇ t (5)
  • the death number ⁇ N DEATHn + 1 of the cell X counts the number N of cells suspended without being able to adhere to the bottom. It can be detected easily.
  • the time change of the counted cell number N and the calculated proliferating cell number ⁇ N BIRTH is graphically displayed on the display unit 60, instead of or in addition to this, Alternatively, at least one of the death number ⁇ N DEATH of the cell X, the inflow number ⁇ N MOVEIN of the cell X, and the outflow number ⁇ N MOVEOUT of the cell X may be displayed graphically.

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Abstract

L'invention concerne un dispositif de traitement d'images de cellule (1), comprenant : une unité de calcul de taux de changement de nombre de cellules (4), qui traite deux images quelconques, parmi une pluralité d'images acquises par des cellules d'imagerie à des intervalles de temps et entrées dans ces dernières, et qui calcule un taux de changement de nombre de cellules, qui signifie un changement du nombre de cellules dans une première zone par unité de temps; une unité de calcul de taux d'entrée de cellules (5), qui traite deux images quelconques et qui calcule un taux d'entrée de cellules, qui signifie le nombre de cellules s'écoulant dans une deuxième zone par unité de temps; une unité de calcul de taux de sortie de cellules (6), qui traite deux images quelconques et qui calcule un taux de sortie de cellules, qui signifie le nombre de cellules s'écoulant hors d'une deuxième zone par unité de temps; et une unité de calcul de taux de prolifération (7), qui calcule un taux de prolifération de cellules dans une troisième zone dans chaque image par unité de temps, sur la base du taux de changement de nombre de cellules, du taux d'entrée de cellules et du taux de sortie de cellules calculés ci-dessus.
PCT/JP2018/000021 2018-01-04 2018-01-04 Dispositif de traitement d'images de cellule, procédé de traitement d'images de cellule et programme de traitement d'images de cellule WO2019135266A1 (fr)

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PCT/JP2018/000021 WO2019135266A1 (fr) 2018-01-04 2018-01-04 Dispositif de traitement d'images de cellule, procédé de traitement d'images de cellule et programme de traitement d'images de cellule

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006120924A1 (fr) * 2005-05-10 2006-11-16 Olympus Corporation Appareil d’observation cellulaire, procede d’observation cellulaire et programme d’observation
JP2014082957A (ja) * 2012-10-19 2014-05-12 Olympus Corp 細胞計数装置及び細胞計数プログラム
JP2015001859A (ja) * 2013-06-17 2015-01-05 大日本印刷株式会社 情報処理装置、情報処理システム及びプログラム
WO2015182382A1 (fr) * 2014-05-30 2015-12-03 富士フイルム株式会社 Dispositif, procédé, et programme d'évaluation de cellules
JP2015223174A (ja) * 2014-05-30 2015-12-14 富士フイルム株式会社 細胞判定装置および方法並びにプログラム

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006120924A1 (fr) * 2005-05-10 2006-11-16 Olympus Corporation Appareil d’observation cellulaire, procede d’observation cellulaire et programme d’observation
JP2014082957A (ja) * 2012-10-19 2014-05-12 Olympus Corp 細胞計数装置及び細胞計数プログラム
JP2015001859A (ja) * 2013-06-17 2015-01-05 大日本印刷株式会社 情報処理装置、情報処理システム及びプログラム
WO2015182382A1 (fr) * 2014-05-30 2015-12-03 富士フイルム株式会社 Dispositif, procédé, et programme d'évaluation de cellules
JP2015223174A (ja) * 2014-05-30 2015-12-14 富士フイルム株式会社 細胞判定装置および方法並びにプログラム

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