Patent Publication Number: US-9841414-B2

Title: Aggregated cell evaluation method and aggregated cell evaluation device

Description:
TECHNICAL FIELD 
     The present invention relates to an aggregated cell evaluation method and an aggregated cell evaluation apparatus. 
     BACKGROUND ART 
     The conventional evaluation of cardiac muscle cells was carried out, for example, in such a manner that cardiac muscle cells were isolated and extracted from a cardiac organ of a laboratory animal, they were cultured on a laboratory dish or the like to prepare primary cultured cells, and the primary cultured cells were used to evaluate the cardiac muscle cells. During the primary culture, cells grow horizontally in a monolayer state (in a sheet shape) in a certain period and the cardiac muscle cells come to beat (contract and relax) in synchronization. However, such primary cultured cells are not human cells and thus are not suitable for use in evaluation of cardiotoxicity or the like on human cardiac muscle cells because of the difference of species. 
     In recent years, with progress of technologies to culture stem cells such as iPS cells or ES cells, it became feasible to artificially create aggregated cells being a three-dimensional aggregate of cells and evaluation of the aggregated cells (e.g., evaluation of change of the aggregated cells upon administration of a drug) has been becoming important. To conduct evaluation of drug efficacy using the cardiac muscle cells prepared from human iPS cells or human ES cells as a specimen is extremely important in evaluation of drug efficacy and safety because it can be done by use of cells of human origin. A cardiac muscle tissue, which is a three-dimensional aggregate of cardiac muscle cells created from human iPS cells or human ES cells, is a tissue in which not only the cardiac muscle cells but also fibroblast cells and others for holding the peripheries of the cells are cultured in mixture, and thus it is feasible to perform the evaluation under a condition similar to a human heart. 
     Here, Non Patent Document 1 suggests the evaluation method for sheet-shaped cells being a two-dimensional aggregate of cells. Since the sheet-shaped cardiac muscle is obtained by sampling the cardiac muscle cells nearly 100% and culturing them, the evaluation of the sheet-shaped cardiac muscle is evaluation in a state different from the real heart. Further, since the sheet-shaped cells adhere individually to a base plate such as the bottom of a laboratory dish, the adhesion inhibits change in motion to be caused by drug effect and thus such cells do not allow accurate evaluation of motion. 
     In contrast to it, aggregated cells are less affected by the inhibition effect as to motion of cells located in regions away from the base plate, though motion of cells near the base plate is inhibited by adhesion to the base plate. Therefore, the aggregated cells allow more accurate evaluation of motion than the sheet-shaped cells, and are more likely to physiologically reflect the change in motion to be caused by drug effect, the aggregated cells are thus in a favorable state for evaluation of cardiotoxicity or the like in drug discovery. 
     CITATION LIST 
     Non Patent Literature 
     
         
         Non Patent Document 1: Hayakawa T, “Noninvasive evaluation of contractile behavior of cardiomyocyte monolayers based on motion vector analysis”, Tissue Engineering Part C, Vol. 18, No. 1, pp. 21-32, 2012 
         Non Patent Document 2: Peterson D W, Griffith D W Jr, Napolitano C A., “Decreased myocardial contractility in papillary muscles from atherosclerotic rabbits”, Circ Res, 1979 September; 45(3), pp. 338-346 
         Non Patent Document 3: Ruri Chihara, “The Role of Rho Kinase: Third Kinase System in the Regulation of Excitation—Contraction Coupling of Cardiac Muscle” J Saitama Med School Vol, 31 No, 2 Apr. 2004 pp. 103-113 
       
    
     SUMMARY OF INVENTION 
     Technical Problem 
     The method described in Non Patent Document 1 is intended for evaluation of the sheet-shaped cells in two-dimensional motion, and it is difficult to apply it to evaluation of aggregated cells in three-dimensional motion. 
     The present invention has been accomplished in order to solve the above problem, and it is an object of the present invention to provide a method and an apparatus capable of readily evaluating the motion of aggregated cells. 
     Solution to Problem 
     An aggregated cell evaluation method of the present invention comprises: (1) a speckle image acquisition step of irradiating aggregated cells with laser light to capture speckle images by forward scattered light generated in the aggregated cells by irradiation with the laser light, at respective times t 1  to t N  in time series; (2) an SC calculation step of calculating a speckle contrast value K n  of the speckle image at each time t n  out of the times t 1  to t N  acquired in the speckle image acquisition step, determining a maximum value K max  among the speckle contrast values K 1  to K N , and normalizing each speckle contrast value K n  by the maximum value K max  to obtain a normalized speckle contrast value K n ′; and (3) an evaluation step of evaluating motion of the aggregated cells, based on the normalized speckle contrast value K n ′ at each time t n  obtained in the SC calculation step or based on a correlation time; or a speed V n  obtained therefrom. 
     An aggregated cell evaluation apparatus of the present invention comprises: (1) a laser light source for outputting laser light; (2) a speckle image acquisition unit for capturing speckle images by forward scattered light generated in aggregated cells by irradiation of the aggregated cells with the laser light output from the laser light source, at respective times t 1  to t N  in time series; (3) an SC calculation unit for calculating a speckle contrast value K n  of the speckle image at each time t n  out of the times t 1  to t N  acquired by the speckle image acquisition unit, determining a maximum value K max  among the speckle contrast values K 1  to K N , and normalizing each speckle contrast value K n  by the maximum value K max  to obtain a normalized speckle contrast value K n ′; and (4) an evaluation unit for evaluating motion of the aggregated cells, based on the normalized speckle contrast value K n ′ at each time t n  obtained by the SC calculation unit or based on a correlation time τ n  or a speed V n  obtained therefrom. 
     Advantageous Effects of Invention 
     The present invention has made it feasible to readily evaluate the motion of aggregated cells. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a drawing showing a configuration of an aggregated cell evaluation apparatus  1  according to an embodiment. 
         FIG. 2  includes photographs of speckle images acquired in an example. 
         FIG. 3  is a graph showing speeds V n  at respective times t n  obtained in an example. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     An embodiment of the present invention will be described below in detail with reference to the accompanying drawings. In the description of the drawings the same elements will be denoted by the same reference symbols, without redundant description. 
       FIG. 1  is a drawing showing the configuration of the aggregated cell evaluation apparatus  1  of the present embodiment.  FIG. 1  shows a configuration of an inverted microscope. The aggregated cell evaluation apparatus  1  includes a laser light source  10 , a speckle image acquisition unit  20 , an SC calculation unit  30 , an evaluation unit  40 , and a memory unit  50 . The aggregated cell evaluation apparatus  1  is an apparatus that evaluates motion of aggregated cells  90  being a three-dimensional aggregate of cells. The aggregated cells  90 , together with a culture medium  91 , are put in a laboratory dish  92 , and this laboratory dish  92  is placed on a stage  93 . The aggregated cells  90  are, for example, a cardiac muscle tissue which is a three-dimensional aggregate of cardiac muscle cells created from human iPS cells or human ES cells. The cardiac muscle tissue is a tissue in which not only the cardiac muscle cells but also fibroblast cells and others are cultured in mixture. 
     The laser light source  10  is provided above the stage  93 . The laser light source  10  outputs laser light to irradiate the aggregated cells  90  in the laboratory dish  92  with the laser light. The laser light source  10  to be used herein can be any laser light source. The laser light output from the laser light source  10  may be applied to the aggregated cells  90  after its beam diameter is increased by a beam expander. 
     The speckle image acquisition unit  20  acquires a two-dimensional speckle image by forward scattered light generated in the aggregated cells  90  by irradiation of the aggregated cells  90  with the laser light output from the laser light source  10 . The speckle image acquisition unit  20  acquires such speckle images at respective times t 1  to t N  in time series. The speckle image acquisition unit  20  includes an objective lens  21 , a mirror  22 , an imaging lens  23 , and an image pickup unit  24 . 
     The objective lens  21  is provided below the stage  93 . The objective lens  21  receives the forward scattered light generated in the aggregated cells  90  by irradiation of the aggregated cells  90  with the laser light output from the laser light source  10 . This forward scattered light travels via the objective lens  21 , mirror  22 , and imaging lens  23  to reach an imaging plane of the image pickup unit  24 . The image pickup unit  24  is configured, for example, by a CCD camera or a CMOS camera. 
     An angle between the direction of incidence of the laser light to the aggregated cells  90  and the optical axis direction of the objective lens  21  is preferably appropriately set so as to prevent light not scattered by the aggregated cells  90  out of the laser light output from the laser light source  10 , from entering the objective lens  21 . This setup allows the imaging plane of the image pickup unit  24  to receive light consisting primarily of the forward scattered light generated in the aggregated cells  90 , thereby enabling acquisition of clear speckle images. 
     The evaluation apparatus  1  may have, instead of the configuration of the inverted microscope, a configuration of an upright microscope. In the latter case, the laser light source  10  is disposed below the stage  93  and the objective lens  21  above the stage  93 . 
     The aggregated cells  90  are a three-dimensional aggregate of cells each having the size of about 10 μm and have the total size of several hundred μm. When the whole aggregated cells  90  are desired to be observed, the magnification of the objective lens  21  is preferably low enough to secure an observation field. The magnification of the objective lens  21  is, for example, 4× or 10×. In another case, where the whole aggregated cells  90  cannot fall within the image field, a partial region of the aggregated cells  90  may be set in the image field. 
     The aggregated cells  90  have the thickness in the optical axis direction of the objective lens  21  and cannot fit in the depth of focus of the objective lens  21  in some cases. However, speckle images have a property of being formed not only on the focal plane but also on any plane off the focal plane, and positions in the optical axis direction of the objective lens  21  is not significant. It is sufficient that the focal plane of the objective lens  21  be located inside the aggregated cells  90 . 
     An exposure time for acquisition of each speckle image by the speckle image acquisition unit  20  affects contrast of the speckle image of the aggregated cells  90  in motion and thus is preferably set to an appropriate duration of time in the range of about 1 ms to 30 ms to allow acquisition of clear speckle images. In the present embodiment, the exposure time is an important parameter. The major reason for variation in speckle contrast due to motion of a specimen is that the light intensity of a speckle image repetitively becomes bright and dark in the exposure time in conjunction with the motion of the specimen whereby the intensity is averaged in terms of time. Therefore, there is no variation in speckle contrast if the exposure time is sufficiently short with respect to the motion of the specimen. On the other hand, there is no variation in speckle contrast if the exposure time is sufficiently long with respect to the motion of the specimen. Therefore, the exposure time needs to be set to an appropriate duration of time. 
     In the case of the cardiac muscle and papillary muscle of animals, according to Non Patent Documents 2 and 3, the time to arrival at a contraction peak is approximately 200 to 400 msec and the time necessary for relaxation approximately 400 to 800 msec. When evaluation of drug efficacy to strengthen or weaken pulsation is considered, it is necessary to assume the speeds ranging from approximately one tenth to ten times the foregoing ranges, and thus the time range to be taken into consideration in analysis of contraction and relaxation is from 20 to 8000 msec. However, the beat rate differs depending upon species, e.g., between animals and a human, and therefore the time range for analysis of contraction and relaxation should be further expanded; it is considered herein that the time range to be taken into consideration is from 1 to 10000 msec. 
     The exposure time Δt should be considered in view of the following two conditions. The first condition is that the exposure time Δt needs to satisfy f S &gt;f B , where f B  represents the frequency of heart beats of the cardiac muscle tissue and f S  the frame rate (Hz) of the camera. At frame rates of the camera not satisfying this relational expression, it is impossible to separate speeds in contraction and relaxation of the cardiac muscle tissue. Since there is the relation of f S &lt;1/Δt between the frame rate f S  of the camera and the exposure time Δt, the exposure time Δt satisfying f S &gt;f B  can be determined. 
     The second condition is that, in conjunction with the sampling theorem, a phase change amount (2π/λ)VΔt of light due to speed change needs at least to be not more than π, where V represents a speed of motion of the cardiac muscle tissue and Δ an exposure time by which a speckle image is obtained without blur. Namely, it is necessary to satisfy the condition of Δt&lt;λ/V. For example, when the illumination light used is HeNe laser light with the center wavelength of 0.633 μm and the speed of the cardiac muscle tissue is V=10μ/s, the exposure time is estimated to be about Δt=63 ms. In practice the preferred exposure time is a value ranging from a half to one tenth of the thus-obtained value. 
     The foregoing appropriate exposure time may be determined as follows: images are taken once with a sufficiently short exposure time (or at a sufficiently fast frame rate) for blur-less imaging of speckle images generated from a moving object, the time-series speckle images are stored in a memory of a computer, an adequate number of frames are then accumulated on the computer, and, based thereon the exposure time is adjusted to a quasi-appropriate value. 
     The speckle contrast varies depending upon the exposure time. Therefore, when a speed is quantitatively determined from speckle contrasts, it is preferable to normalize speckle contrast values by the exposure time T. It is noted that if the exposure time is sufficiently short with respect to the motion of the specimen or if the exposure time is sufficiently long with respect to the motion of the specimen, an accurate speed cannot be obtained even with the normalization of speckle contrast values by the exposure time T. 
     The memory unit  50  stores a speckle image I n (x, y) acquired at each time t n  out of times t 1  to t N  by the speckle image acquisition unit  20 . x, y are coordinate values indicating a position in a two-dimensional speckle image, and when the image pickup unit  24  has a two-dimensional pixel structure as in the case of a CCD, x, y are coordinate values indicating a pixel position. The memory unit  50  also stores the result obtained by the SC calculation unit  30  as described below. 
     The SC calculation unit  30  calculates a speckle contrast value K n  of the speckle image I n  at each time t n  stored by the memory unit  50 . The SC calculation unit  30  determines a maximum value K max  among these speckle contrast values K 1  to K N . The SC calculation unit  30  normalizes the speckle contrast value K each time t n  by the maximum value K max  to obtain a normalized speckle contrast value K n ′. Then the evaluation unit  40  evaluates the motion of the aggregated cells  90 , based on the normalized speckle contrast value K n ′ at each time t n  obtained by the SC calculation unit  30 . 
     Here, there is mutual dependence among the three parameters, the normalized speckle contrast value K n ′ at each time t n , correlation time τ n , and speed V n , as described below. Therefore, the evaluation of the motion of the aggregated cells  90  may be made based on any one of the normalized speckle contrast value K n ′ at each time t n , correlation time τ n , and speed V n . 
     The apparatus is preferably provided with a drug administration means, as schematically illustrated by a drug administration unit  95  in  FIG. 1 . This drug administration means administers a drug to the aggregated cells  90  prior to or in the middle of the acquisition of speckle images by the speckle image acquisition unit  20 . At this time, the evaluation unit  40  evaluates influence of the drug administered by the drug administration means on the aggregated cells  90 , based on the evaluation result of the motion of the aggregated cells  90 . 
     The aggregated cell evaluation method of the present embodiment can be carried out with use of the aggregated cell evaluation apparatus  1 . The aggregated cell evaluation method of the present embodiment includes a speckle image acquisition step, an SC calculation step, and an evaluation step which are carried out in order. 
     In the speckle image acquisition step, the laser light source  10  irradiates the aggregated cells  90  with the laser light. Then the speckle image acquisition unit  20  acquires speckle images by forward scattered light generated in the aggregated cells  90  by the irradiation with the laser light, at respective times t 1  to t N  in time series. The speckle image I n (x, y) acquired at each time t n  is stored in the memory unit  50 . 
     In the SC calculation step, the SC calculation unit  30  calculates the speckle contrast value K n  of the speckle image at each time t n , determines the maximum value K max  among these speckle contrast values K 1  to K N , and normalizes the speckle contrast value K n  at each time t n  by the maximum value K max  to obtain the normalized speckle contrast value K n ′. In the evaluation step, the evaluation unit  40  then evaluates the motion of the aggregated cells, based on the normalized speckle contrast value K n ′ at each time t n . 
     The method preferably further comprises a drug administration step of administering a drug to the aggregated cells  90  by the drug administration means prior to or in the middle of the speckle image acquisition step. In this case, the evaluation step is configured so that the evaluation unit  40  evaluates the influence of the drug on the aggregated cells, based on the evaluation result of the motion of the aggregated cells. 
     Next, the contents of the respective processes by the SC calculation unit  30  and the evaluation unit  40  (the respective processes of the speckle image acquisition step and the SC calculation step) will be described in more detail. 
     First, a mean value I mean  and a standard deviation σ are determined from intensities of (2 W+1) 2  pixels in a region with a window size 2 W+1 centered at each position (x, y) of the speckle image I n (x, y). The speckle contrast value K n  at each position (x, y) is calculated from these mean value I mean  and standard deviation a by the following formula (1). The speckle contrast value K n  is determined for all pixels of the speckle image I n (x, y) at each time t n , thereby calculating a speckle contrast image K n (x, y) for the speckle image I n (x, y). 
     
       
         
           
             
               
                 
                   
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     A mean value (mean speckle contrast value) K n   _   mean  is calculated from the speckle contrast values of all the pixels in the speckle contrast image K n (x, y) at each time t n . A maximum value K max  is determined among the N mean speckle contrast values K 1   _   mean  to K N   _   mean . Then the mean speckle contrast value K n   _   mean  at each time t n  is normalized by the maximum value K max  to obtain the normalized speckle contrast value K n ′(=K n   _   mean /K max ). 
     The reason for this normalization is as follows. Namely, it is known that, theoretically, the contrast value is 1 for a fully developed speckle obtained from a still specimen under an ideal condition. It is also known that speckle contrast values are generally not more than 1. On the other hand, actual conditions even with the specimen at a standstill are different from the ideal condition. In practice, there are cases where contrast values of speckles obtained from the still specimen are not 1, for example, because of stray light or coherence length of laser light. This difference between the theoretical value and the actually measured value would raise a problem in the evaluation of motion of the specimen. Therefore, the normalization as described above is carried out. 
     Then, the correlation time τ n  is determined from the normalized speckle contrast value K n ′ at each time t n  by the following formula (2) and the speed V n  is determined from the correlation time τ n  at each time t n  by the following formula (3). It is noted that T represents the exposure time in acquisition of each speckle image and λ the wavelength of the laser light. There are also known relational expressions other than the formula (2) as relations between the speckle contrast value and correlation time. 
     
       
         
           
             
               
                 
                   
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     The arithmetic operation to determine the mean speckle contrast value K n   _   mean  from the speckle contrast image K n (x, y) at each time t n  may be performed after the formula (2). In that case, a mean correlation time τ n   _   mean  is calculated from a correlation time image τ n (x, y). Furthermore, the above mean calculation may be performed after the formula (3) and in this case, a speed image V n (x, y) is obtained from the correlation time image τ n (x, y) and thereafter a mean speed V n  may be calculated. 
     The SC calculation unit  30  may divide the resultant speckle image I n (x, y) into a plurality of segment images I n   (m)  each including an appropriate number of pixels. In this case, subsequently, K n , K n ′, τ n , and speed V n  are calculated for each of the segment images I n   (m)  to obtain two-dimensional mapping images of these parameters to evaluate the motion of the aggregated cells  90 . It is noted that m in I n   (m)  represents a segment number. 
     As indicated by the formula (2) and the formula (3), there is mutual dependence among the three values of the normalized speckle contrast value K n ′, the correlation time τ n , and the speed V n  at each time t n . Therefore, the normalized speckle contrast value K n ′, the correlation time τ n  and the speed V n  at each time t all are equivalent in the evaluation of the motion of the aggregated cells  90 , and can be used each as an index for the evaluation of the motion of the aggregated cells  90 . 
     Example 1 
     Next, the below will describe an example of the aggregated cell evaluation apparatus and aggregated cell evaluation method of the present embodiment. In the present example, the aggregated cells  90  as an evaluation object were a cardiac muscle tissue in the size of about 600 μm. The laser light source  10  was a HeNe laser light source to output the laser light at the wavelength of 633 nm. The magnification of the objective lens  21  was 10×. The image pickup unit  24  was a CCD camera having the number of pixels of 512×512 and the size of one pixel was 16×16 μm. The exposure time in imaging by the image pickup unit  24  was 10 ms and the frame rate 100 fps. 
       FIG. 2  includes photographs of speckle images acquired in the present example. The size of the field of view of each of the speckle images A to C shown in  FIG. 2  is 0.82×0.82 μm. The speckle image A is an image of the cardiac muscle tissue in a stop period. The speckle image B is an image of the cardiac muscle tissue in a contraction period. The speckle image C is an image of the cardiac muscle tissue in a relaxation period. 
     In the speckle image A of the cardiac muscle tissue in the stop period, a speckle pattern (bright-/dark-spot pattern) is clearly observed. In contrast to it, the speckle image B of the cardiac muscle tissue in the contraction period demonstrates reduction in the speckle pattern. The speckle pattern shows repetitions of brightness and darkness in conjunction with beats (expansion and contraction) of the cardiac muscle tissue. Since the speckle pattern becomes repetitively bright and dark sufficiently quickly with respect to the exposure time, the speckle pattern looks blurred in the speckle image B of the cardiac muscle tissue in the contraction period and, in other words, it suffers degradation of contrast. In this manner, there is correlation between the contrast of the speckle pattern and the speed of motion of the specimen. Based on this correlation, the correlation time τ n  can be determined from the speckle contrast K n ′ and the speed V n  can be further determined. 
       FIG. 3  is a graph showing the speed V n  at each time t n  obtained in the present example. In  FIG. 3 , peaks  1 ,  3 ,  5 ,  7 , and  9  indicate the speeds in maximum acceleration during contraction periods of the cardiac muscle tissue, and peaks  2 ,  4 ,  6 ,  8 , and  10  the speeds in maximum acceleration during relaxation periods of the cardiac muscle tissue. The times when the respective speckle images A to C in  FIG. 2  were acquired are indicated by A to C in  FIG. 3 . From this  FIG. 3 , the motion of the cardiac muscle tissue (e.g., intervals of appearance of the respective peaks, magnitudes of the peaks in the contraction periods, magnitudes of the peaks in the relaxation periods, and differences or ratios between the respective magnitudes of the peaks in the contraction periods and the peaks in the relaxation periods) can be evaluated. Furthermore, influence of a drug on the cardiac muscle tissue can be evaluated based on the evaluation result of the motion of the cardiac muscle tissue. 
     The aggregated cell evaluation method and aggregated cell evaluation apparatus according to the present invention are not limited to the above-described embodiment and configuration examples but can be modified in many ways. 
     The aggregated cell evaluation method according to the above embodiment is configured to comprise: (1) a speckle image acquisition step of irradiating aggregated cells with laser light to capture speckle images generated by forward scattered light generated in the aggregated cells by irradiation with the laser light, at respective times t 1  to t N  in time series; (2) an SC calculation step of calculating a speckle contrast value K n  of the speckle image at each time t n  out of the times t 1  to t N  acquired in the speckle image acquisition step, determining a maximum value K max  among the speckle contrast values K 1  to K N , and normalizing each speckle contrast value K n  by the maximum value K max  to obtain a normalized speckle contrast value K n ′; and (3) an evaluation step of evaluating motion of the aggregated cells, based on the normalized speckle contrast value K n ′ at each time t n  obtained in the SC calculation step or based on a correlation time τ n  or a speed V n  obtained therefrom. 
     The aggregated cell evaluation apparatus according to the above embodiment is configured to comprise: (1) a laser light source for outputting laser light; (2) a speckle image acquisition unit for capturing speckle images generated by forward scattered light generated in aggregated cells by irradiation of the aggregated cells with the laser light output from the laser light source, at respective times t 1  to t N  in time series; (3) an SC calculation unit for calculating a speckle contrast value K n  of the speckle image at each time t n  out of the times t 1  to t N  acquired by the speckle image acquisition unit, determining a maximum value K max  among the speckle contrast values K 1  to K N , and normalizing each speckle contrast value K n  by the maximum value K n , to obtain a normalized speckle contrast value K n ′; and (4) an evaluation unit for evaluating motion of the aggregated cells, based on the normalized speckle contrast value K n ′ at each time t n  obtained by the SC calculation unit or based on a con-elation time τ n  or a speed V n  obtained therefrom. 
     The aggregated cell evaluation method of the above configuration preferably further comprises; a drug administration step of administering a drug to the aggregated cells prior to or in the middle of the speckle image acquisition step, and in this case, the evaluation step preferably comprises evaluating influence of the drug on the aggregated cells, based on the evaluation result of the motion of the aggregated cells. 
     The aggregated cell evaluation apparatus of the above configuration preferably further comprises: drug administration means for administering a drug to the aggregated cells prior to or in the middle of acquisition of the speckle images by the speckle image acquisition unit, and in this case, the evaluation unit preferably evaluates influence of the drug on the aggregated cells, based on the evaluation result of the motion of the aggregated cells. 
     In the aggregated cell evaluation method of the above configuration, the SC calculation step preferably comprises dividing the speckle image at each time t, into a plurality of segment images each including a plurality of pixels, and calculating the speckle contrast value K n  for each of the segment images. In the aggregated cell evaluation apparatus of the above configuration, the SC calculation unit preferably divides the speckle image at each time t n  into a plurality of segment images each including a plurality of pixels, and calculates the speckle contrast value K n  for each of the segment images. In this case, thereafter, K n ′, τ n , and speed V n  are calculated for each of the segment images to obtain two-dimensional mapping images of these parameters, and the motion of the aggregated cells is evaluated based thereon. 
     INDUSTRIAL APPLICABILITY 
     The present invention is applicable as a method and an apparatus capable of readily evaluating the motion of aggregated cells. 
     REFERENCE SIGNS LIST 
     
         
         
           
               1 —aggregated cell evaluation apparatus,  10 —laser light source,  20 —speckle image acquisition unit,  21 —objective lens,  22 —mirror,  23 —imaging lens,  24 —image pickup unit,  30 —SC calculation unit,  40 —evaluation unit,  50 —memory unit,  90 —aggregated cells,  91 —culture medium,  92 —laboratory dish,  93 —stage.