WO2019135267A1 - Dispositif de traitement d'image de cellules, procédé de traitement d'image de cellules, et programme de traitement d'image de cellules - Google Patents

Dispositif de traitement d'image de cellules, procédé de traitement d'image de cellules, et programme de traitement d'image de cellules Download PDF

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Publication number
WO2019135267A1
WO2019135267A1 PCT/JP2018/000022 JP2018000022W WO2019135267A1 WO 2019135267 A1 WO2019135267 A1 WO 2019135267A1 JP 2018000022 W JP2018000022 W JP 2018000022W WO 2019135267 A1 WO2019135267 A1 WO 2019135267A1
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WIPO (PCT)
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cell
parameter
size
migration
area
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PCT/JP2018/000022
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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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Priority to PCT/JP2018/000022 priority Critical patent/WO2019135267A1/fr
Priority to JP2019563718A priority patent/JP6963033B2/ja
Publication of WO2019135267A1 publication Critical patent/WO2019135267A1/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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis

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 acquired by capturing cells at time intervals are input, and any two evaluation images of the input images are processed to perform the evaluation.
  • a growth parameter calculating unit for calculating a growth parameter representing a change in the amount of cells per unit time in a predetermined counting area of an image, an inflow of the cells per unit time of the counting area of each evaluation image or
  • a migration parameter calculation unit that calculates a migration parameter representing an outflow amount, a region size calculation unit that calculates the size of the counting region based on the past growth parameter and the past migration parameter, and the region size calculation unit Growth rate calculation for calculating a cell growth rate based on the growth parameter and the migration parameter calculated for the count area having a size calculated by It is a cell image processing apparatus and a part.
  • any two evaluation images of the input images are processed by the proliferation parameter calculation unit. Growth parameters within the counting area are then calculated. Also, the evaluation image is processed by the migration parameter calculation unit to calculate the migration parameter of the counting area.
  • the size of the counting area is calculated by the area size calculating unit based on the past growth parameters and the past migration parameters, and the proliferation is calculated based on the growth parameters and the migration parameters calculated for the calculated counting area.
  • the cell growth rate is calculated by processing by the rate calculation unit.
  • the cell proliferation rate can be determined not only by proliferative parameters indicating changes in cell mass in two time-spaced images but also by migration parameters representing cell influx and cell efflux, so The degree of cell proliferation can be detected more accurately, and cell culture can be managed with high accuracy.
  • the proliferation parameter is proportional to the area of the counting area and the migration parameter is proportional to the perimeter of the counting area
  • the smaller the counting area the larger the influence of the migration parameter in the calculation of the cell proliferation rate and the error Becomes larger. Therefore, the cell proliferation rate can be accurately calculated by calculating the cell proliferation rate based on the proliferation parameter and the migration parameter calculated for the counting area set to an appropriate size.
  • the evaluation image and the proliferation measurement image may use the same image or different images. That is, the time interval between two evaluation images and the time interval between two proliferation measurement images may be the same or different. For example, for the evaluation image, all two adjacent images may be used, or two images selected periodically or non-periodically may be used.
  • the growth parameter calculation unit may calculate the number of cells increased in the counting area per unit time as the growth parameter. By doing this, two evaluation images obtained at intervals of time are processed, the number of cells present in each counting area is counted, and the difference is divided by the interval of time to simplify the process. Growth parameters can be calculated.
  • the migration parameter calculation unit may calculate, as the migration parameter, a difference between the number of cells flowing in from the outer edge of the counting area per unit time and the number of cells flowing out. In this way, two evaluation images obtained at intervals are processed to count the number of cells inflowing and the number of cells inflowing beyond the outer edge of each counting area, and the difference is calculated by time. By dividing by the interval, the migration parameter can be easily calculated.
  • the region size calculating unit calculates the lower limit of the size of the counting region by comparing the ratio of the migration parameter in the past to the past growth parameter and the predetermined tolerance. It is also good. By doing this, the growth rate calculation unit can calculate the cell growth rate with high accuracy less than the allowable error.
  • the counting area may be rectangular. By doing so, the proliferation parameter, the migration parameter and the cell proliferation rate can be easily calculated.
  • the other aspect of this invention is provided with the processor by which several sheets of images acquired by imaging a cell at intervals of time are input,
  • This processor is a unit time in the past predetermined
  • the size of the counting area is calculated based on the growth parameter representing the change in the amount of cells per cell and the migration parameter representing the inflow or outflow of the cells per unit time in the past in the counting area Processing the image of any two of the input images using the counting area of the determined size to calculate the proliferation parameter and the migration parameter in the counting area of the image It is an image processing device.
  • a plurality of images acquired by capturing cells at time intervals are input, and any two of the input images are processed.
  • Calculating a growth parameter representing a change in the amount of cells per unit time within a predetermined counting area of the image, and an inflow of the cells per unit time of the counting area of each image Alternatively, a migration parameter calculating step of calculating a migration parameter representing an outflow amount, a region size calculating step of calculating the size of the counting region based on the past growth parameter and the past migration parameter, and the region size calculation Cells based on the growth parameter and the migration parameter calculated for the counting area of the size calculated by the step; It is a cell image processing method comprising a growth rate calculation step of calculating a ⁇ .
  • a plurality of images acquired by capturing cells at time intervals are input, and any two of the input images are processed.
  • Calculating a growth parameter representing a change in the amount of cells per unit time within a predetermined counting area of the image, and an inflow of the cells per unit time of the counting area of each image Alternatively, a migration parameter calculating step of calculating a migration parameter representing an outflow amount, a region size calculating step of calculating the size of the counting region based on the past growth parameter and the past migration parameter, and the region size calculation Cells based on the growth parameter and the migration parameter calculated for the counting area of the size calculated by the step; Is a cell image processing program for executing the growth rate calculation step of calculating a computer ⁇ .
  • 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 image.
  • the processing unit 3 processes the images P n and P n + 1 stored in the memory 2 and changes the number of cells per unit time in a predetermined counting area R in the images (evaluation images) P n and P n + 1 .
  • Cell number change rate calculation unit (growth parameter calculation unit) 4 for calculating the cell number change rate (growth parameter) which is a rate, and per unit time into the counting area R in the images (evaluation images) P n and P n + 1 Cell inflow rate calculation unit (migration parameter calculation unit) 5 that calculates the cell inflow rate (migration parameter), which is the inflow number of cells X, and the counting area R in the images (evaluation images) P n and P n + 1 Cell flow rate calculation unit (migration parameter calculation unit) 6 that calculates the cell flow rate (migration parameter), which is the number of cell X flows per unit time, and the calculated cell number change rate, cell inflow rate, and cell flow rate Growth measurement image from And a growth rate calculation unit 7 that calculates the cell growth rate in the count region R.
  • the cell image processing apparatus 1 includes an area size calculating unit 8 that sets the size of the counting area R.
  • the cell number change rate calculation unit 4 calculates the number N n of cells in the counting area R in the two images P n and P n + 1 acquired with a time interval of the first time ⁇ t 1. the n n + 1 counted respectively, each image P n, counted cell number n n in P n + 1, by dividing the difference between n n + 1 in the first hour .DELTA.t1, per unit time the number of cells n n, the n n + 1
  • the cell number change rate ⁇ N / ⁇ t which is the change rate, 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 count in the P n + 1
  • the cell inflow rate ⁇ N MOVEIN / ⁇ t 2 which is the inflow number ⁇ N MOVEIN of the cell X is calculated.
  • Cells outflow rate calculation part 6 also, as shown in FIG. 3, the second time ⁇ t2 only the image P n of the two that was acquired at time intervals, P n + 1 images P n on the basis of the counting of the P n + 1 Cells per unit time by counting the number of outflows ⁇ N MOVEOUTn + 1 of cells X that have exited across the boundary from the region R to the adjacent region and dividing the counted number of outflows of cells X ⁇ N MOVEOUTn + 1 by the second time ⁇ t 2
  • the cell outflow rate ⁇ N MOVEOUT / ⁇ t 2 which is the number of outflows ⁇ N MOVEOUT of X is calculated.
  • the second time ⁇ t2 for calculating the inflow of cells X into the counting area R and the outflow from the counting area R satisfy the conditional expression (1) below.
  • 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 counting area R. Also, as shown in FIG. 5, using the threshold E of the length to determine entry or exit, it is judged that the cell X has entered by the threshold E from the boundary between the counting region R and the adjacent region, and entry is made from the boundary When the cell X comes out of the counting area R to the adjacent area by the threshold E, it may be judged to be an exit.
  • Region size calculation unit 8 is connected to memory 2 for storing the past cell proliferation rate ⁇ N BIRTH / ⁇ t, the past cell inflow rate ⁇ N MOVEIN / ⁇ t and the past cell outflow rate ⁇ N MOVEOUT / ⁇ t calculated for each cell X Based on the past cell proliferation rate ⁇ N BIRTH / ⁇ t, cell inflow rate ⁇ N MOVEIN / ⁇ t and cell outflow rate ⁇ N MOVEOUT / ⁇ t stored in the memory 2, the index of the error evaluation is less than the predetermined tolerance Ka
  • the size of the counting area R is determined as follows.
  • the memory 2 stores the past cell numbers A, B, C representing the past cell proliferation rate ⁇ N BIRTH / ⁇ t calculated in the past or determined for each cell X, and the tolerance Ka of counting.
  • Region size calculation unit 8 reads the number of cells A, B, C and tolerance Ka of the past from memory 2 and uses conditional numbers (A, B, C) and tolerance Ka read to obtain conditional expression (5). The length of one side of the counting area R is calculated using this.
  • the proliferation rate calculation unit 7 is configured to calculate a cell proliferation rate ⁇ N BIRTH / ⁇ t, which is the number of cell divisions per unit time in the counting area R.
  • the cell proliferation rate ⁇ N BIRTH / ⁇ t is obtained by subtracting the cell inflow rate ⁇ N MOVEIN / ⁇ t from the cell number change rate ⁇ N / ⁇ t by the following equation (6) and adding the cell outflow rate ⁇ N MOVEOUT / ⁇ t. Is calculated.
  • ⁇ N BIRTH / ⁇ t ⁇ N / ⁇ t ⁇ N MOVEIN / ⁇ t + ⁇ N MOVEOUT / ⁇ t (6)
  • 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 number of cells A, B, C and the tolerance Ka are read from the memory 2 (step S2).
  • the length L of one side of the counting area R is calculated using the read cell numbers A, B, C and the tolerance error Ka (area size calculation step S3).
  • the image P n and the image P n + 1 are used, and the number of cells ⁇ N MOVEIN n + 1 entering the counting area R from outside the counting area R and the number of cells exiting the counting area R from outside the counting area R ⁇ N MOVEOUTn + 1 is calculated (migration parameter calculation step S7, S8).
  • the proliferation rate calculation unit 7 the cell proliferation rate ⁇ N BIRTH n + 1 / ⁇ t per unit time is calculated by the following equation (7) (proliferation rate calculation step S9).
  • 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 (8).
  • ⁇ N BIRTH n + 1 (N n + 1 -N n ) - ⁇ N MOVEIN n + 1 + ⁇ N MOVEOUT n + 1 (8)
  • the cell proliferation rate ⁇ N BIRTHn + 1 / ⁇ t is 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 due to cell division is detected more accurately There is an advantage that the cell culture can be managed precisely.
  • the second time ⁇ t2 for calculating the cell inflow rate ⁇ N MOVEIN n + 1 / ⁇ t 2 and the cell outflow rate ⁇ N MOVEOUT n + 1 / ⁇ t 2 satisfies the conditional expression (1), and The entering cell X and the exiting cell X 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 / ⁇ t1 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 calculation unit 4, the cell inflow rate calculation unit 5, the cell outflow rate calculation unit 6, the proliferation rate calculation unit 7 and the region size calculation unit 8.
  • the processing unit 3 may be configured by an electric circuit or may be configured by one or more processors.
  • the present invention configures the proliferation parameter calculation steps S5 and S6, the migration parameter calculation steps S7 and S8, the proliferation rate calculation step S9, and the area size calculation step S3 by a computer-executable program May be
  • a cell mortality calculation unit that calculates the cell mortality ⁇ N DEATHn + 1 / ⁇ t, which is the number of deaths of the cell X per unit time in the counting region R. 9 may be further provided.
  • the growth rate calculation unit 7 may calculate the cell growth rate ⁇ N BIRTH n + 1 / ⁇ t by the following equation (9) (step S13).
  • ⁇ 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 (9)
  • the death number ⁇ N BIRTH n + 1 of the cell X counts the number N n + 1 of cells suspended without being able to adhere to the bottom surface. It can be easily detected by
  • 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 cell inflow number ⁇ N MOVEIN, and the cell X outflow number ⁇ N MOVEOUT may be displayed graphically.
  • the cell growth rate ⁇ N BIRTHn + 1 / ⁇ t is exemplified as the growth parameter, but instead, the change rate of confluency, which is the ratio of the area of the cell area to the area of the counting area R, is adopted.
  • the difference of cell inflow rate (DELTA) N MOVEINn + 1 / (DELTA) t and cell outflow rate (DELTA) N MOVEOUTn + 1 / (DELTA) t was illustrated as a migration parameter, it replaces with this and it is two images Pn and Pn + 1 which photoed count field R.
  • An image deformation amount which is a conversion parameter of projective transformation, or a target cell movement amount which is an average value of movement amounts of several target cells detected by image processing may be adopted.
  • the size of the counting area R is calculated based on the past growth parameters and migration parameters, and the tolerance error Ka, but the size of the counting area R is determined in advance. Alternatively, the magnitude of the error may be estimated.
  • the length L of one side of the square counting area R is calculated as the size of the counting area R, but instead, if the counting area R is circular, the radius r
  • the long side L1 of the rectangular counting area R having the long side L1 and the short side L2 may be defined, and the short side L2 may be calculated by the following equation (11).
  • L2 2 ⁇ (B ⁇ C) ⁇ L1 / (A ⁇ L1 ⁇ K + 2 ⁇ (C ⁇ B)) (11)

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Abstract

L'invention concerne un dispositif de traitement d'image de cellules (1) qui comprend : une partie calcul de paramètre de prolifération (4) qui reçoit comme entrée une pluralité d'images qui ont été acquises par photographie de cellules à intervalles de temps et traite l'une quelconque de deux images d'évaluation parmi les images entrées pour calculer un paramètre de prolifération qui exprime le changement, par unité de temps, dans la quantité de cellules dans une région de comptage prescrite des images d'évaluation; une partie calcul de paramètre de migration (6) qui calcule un paramètre de migration qui exprime la vitesse d'entrée ou de sortie, par unité de temps, de cellules dans/à partir de la région de comptage des images d'évaluation; une partie calcul de taille de région (8) qui calcule la taille de la région de comptage sur la base d'un paramètre de prolifération antérieure et d'un paramètre de migration antérieure; et une partie calcul de taux de prolifération (7) qui calcule un taux de prolifération cellulaire sur la base du paramètre de prolifération et du paramètre de migration calculés pour une région de comptage de la taille calculée par la partie calcul de taille de région (8).
PCT/JP2018/000022 2018-01-04 2018-01-04 Dispositif de traitement d'image de cellules, procédé de traitement d'image de cellules, et programme de traitement d'image de cellules WO2019135267A1 (fr)

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PCT/JP2018/000022 WO2019135267A1 (fr) 2018-01-04 2018-01-04 Dispositif de traitement d'image de cellules, procédé de traitement d'image de cellules, et programme de traitement d'image de cellules
JP2019563718A JP6963033B2 (ja) 2018-01-04 2018-01-04 細胞画像処理装置、細胞画像処理方法および細胞画像処理プログラム

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007327843A (ja) * 2006-06-07 2007-12-20 Olympus Corp 画像処理装置および画像処理プログラム
JP2009022276A (ja) * 2007-06-19 2009-02-05 Kuraray Co Ltd 細胞評価方法及び細胞評価システム
JP2014085950A (ja) * 2012-10-25 2014-05-12 Dainippon Printing Co Ltd 細胞挙動解析装置、細胞挙動解析方法、及びプログラム
JP2014083042A (ja) * 2012-10-26 2014-05-12 Ehime Univ 細胞の評価方法およびその利用
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
JP2007327843A (ja) * 2006-06-07 2007-12-20 Olympus Corp 画像処理装置および画像処理プログラム
JP2009022276A (ja) * 2007-06-19 2009-02-05 Kuraray Co Ltd 細胞評価方法及び細胞評価システム
JP2014085950A (ja) * 2012-10-25 2014-05-12 Dainippon Printing Co Ltd 細胞挙動解析装置、細胞挙動解析方法、及びプログラム
JP2014083042A (ja) * 2012-10-26 2014-05-12 Ehime Univ 細胞の評価方法およびその利用
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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