Abstract:
A cell sorting chip and a cell sorting technology are to be established which can positively detect and sort a specified cell for cell separation and detection using micro flow paths formed on a substrate, whereby a cell analyzing/sorting device is provided which uses an inexpensive disposable chip replaceable for each sample. To this end, micro flow paths formed on the substrate are formed, and cells are roughly sorted in a first stage and then finely sorted in a second stage. More specifically, cells are sorted roughly by using scattered light or according to intensity of luminescence in the first stage. In the second stage, the roughly sorted cells are sorted with high precision using image recognition.

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention relates to a cell sorter configured on a substrate.  
       BACKGROUND OF THE INVENTION  
       [0002]     An anatomy of a multicellular organism retains a harmonious function as a whole by each cell taking a different complementary role. Once a part of the multicellular organism becomes cancerous (hereinafter referred to as a cancer, including a tumor), the cells in the part grow into neoplasm different from its peripheral region. However, the cancerous region and a normal tissue region away therefrom may not necessarily be distinguished by a certain borderline and the region surrounding the cancer is affected in some way. Therefore, in order to analyze a function of an organ tissue, it is necessary to pick up a small number of cells present in a small region.  
         [0003]     Otherwise, in the medical field, in order to examine a region suspected of cancer in the normal tissue, it is necessary to sort the region suspected of cancer from a piece of tissue acquired by biopsy. For separation and collection of such specific cells, it is common to fix the cells, perform various cell stainings, and cut out a target part. Recently for this purpose, a method called laser microdissection to get cells only from a target region subjected to the laser has been developed.  
         [0004]     Otherwise, in the field of regeneration medicine, there is an endeavor to separate and purify a stem cell from the tissue, cultivate the stem cell, and conduct the differentiation induction to regenerate the target tissue, and furthermore an organ.  
         [0005]     To classify, identify or purify cells, it is necessary to distinguish the different cells according to a certain reference. Common methods of distinguishing cells include the following;  
         [0006]     1) Visualized cell classification based on morphology: an examination for a bladder cancer, an urethral cancer and the like by detection of an atypical cell present in urine, and a cancer screening by a classification of the atypical cells in blood or a cytological diagnosis in the tissue can be taken as examples.  
         [0007]     2) Cell classification based on the cell surface antigen (marker) stained by the fluorescent specific antibody test: this is to stain a cell surface antigen, generally called as a CD marker, with a fluorescent labeling antibody specific thereto, and used for cancer screenings by a cell purification using a cell sorter, a flow cytometer, or tissue staining. These techniques are frequently used not only in the medical field but also for the cytophysiological study and the industrial use of the cells.  
         [0008]     3) Separation of the stem cells using fluorescent pigments taken into cells as reporters: The target stem cell is purified by separating a differentiated target stem cell from roughly separated stem cells and by actually re-cultivating the differentiated stem cell afterward. That is to say, since an effective marker for the stem cell has not yet been established, the target cell is selected by their differentiated characteristics of cells after their cultivation.  
         [0009]     Separating and retrieving a specific cell in a culture fluid in this way is an important technique for biological and medical analyses.  
         [0010]     When cells are separated based on a difference in the specific gravity of the cells, the target cells can be purified by the velocity sedimentation method. However, when there is little difference in the specific gravity of the cells enough to differentiate a non-sensitized cell from a sensitized cell, it is necessary to separate the cells one by one based on information from staining with the fluorescent antibody marker or other visual information. This technique may be represented by, for instance, a cell sorter.  
         [0011]     The conventional cell sorter employs a technique to drop the fluorescence-stained cells in a charged droplet as isolated in the unit of cell into the air after obtainment of information on the presence of the fluorescence and scattered light of the cell, and applying a high electric field in any direction on the plane perpendicular to the dropping direction in the process of the droplet dropping, whereby the dropping direction of the droplet is controlled by the applied voltage, based on the optical measurement of the presence and localization of the fluorescence in the cell in the droplet and the intensity of the light scattering diffraction, to fractionate and retrieve the droplet in a plurality of containers placed at the bottom (Non-patent document 1: Kamarck, M. E., Methods Enzymol. Vol. 151, p 150-165 (1987)).  
         [0012]     However, this technique involves the following problems: the system is expensive; the system is large; a high electric field of some thousand volts is required; a large number of samples are required; cells may be damaged during generation of the droplets; the sample cannot be directly observed.  
         [0013]     To solve these problems, a cell sorter has been recently developed which generates fine flow paths using the microfabrication technology and sorts the cells flowing through the laminar flow in the flow path while directly observing them under a microscope (Non-patent document 2: Micro Total Analysis, 98, pp. 77-80 (Kluwer Academic Publishers, 1998)), (Non-patent document 3: Analytical Chemistry, 70, pp. 1909-1915 (1998)).  
         [0014]     However, since the cell sorter which generates the fine flow paths using the microfabrication technology is slow in the response speed of the sample sorting with respect to the observation unit, another processing method that does not damage the sample and is faster in response is required in order to put the cell sorter into practical use.  
         [0015]     In order to solve the problems, the present inventors have filed the applications for a cell analyzer/sorter capable of fractionating the samples based on the fine optical image of the sample and the distribution and localization of the fluorescence in the sample utilizing the microfabrication technology and easily analyzing/sorting the sample cells without damaging the samples retrieved (patent documents 1 to 3). This apparatus is a substantially useful cell sorter for use in a laboratory, but for practical industrial/medical use, new techniques are required for the microfluidic pathway, cell transportation, retrieving method, and sample preparation.  
         [0016]     [Non-patent document 1] Kamarck, M. E., Methods Enzymol. Vol. 151, p 150-165 (1987)  
         [0017]     [Non-patent document 2] Micro Total Analysis, 98, pp. 77-80 (Kluwer Academic Publishers, 1998)  
         [0018]     [Non-patent document 3] Analytical Chemistry, 70, pp. 1909-1915 (1998)  
         [0019]     [Patent document 1] JP-A-2003-107099  
         [0020]     [Patent document 2] JP-A-2004-85323  
         [0021]     [Patent document 3] PCT Patent Publication No. WO2004/101731  
       SUMMARY OF THE INVENTION  
       [0022]     It is an object of the present invention to establish a cell sorting chip and a cell sorting technique for positively detecting and sorting a predetermined cell for the purpose of cell sorting or detection using a micro flow path formed on a substrate, and to provide a cell analyzer/sorter using a chip inexpensive and replaceable for each sample.  
         [0023]     When a micro flow path is formed on a substrate and fluid flows therethrough, the fluid flowing therethrough generally becomes a laminar flow. A cell sorter system using a micro flow path formed on a substrate also uses the sheath flow technique to array the cells in line, and the image recognition technique is used to extract the cells and sort a specific cell. While this technique allows for sorting and retrieving the cells with a high degree of precision, the throughput is slower than that of a conventional cell sorter as described above, which does not use a substrate but recognizes and sorts the cells contained in a droplet based on the scattered light and fluorescent light intensity.  
         [0024]     Therefore, it is an object of the present invention to develop a sell sorter chip having the throughput of sorting the cells increased as much as that of the conventional cell sorter and to establish a sorting algorism.  
         [0025]     The cells assumed in the present invention ranges from a bacteria at the smallest to an animal cell (a cancer cell) at the largest. Therefore, the size (diameter) of the cell ranges approximately from 0.5 micrometers to 30 micrometers φ. To perform the cell sorting using a micro flow path incorporated in a substrate, the first problem is the width of the flow path (cross-sectional dimension). The micro flow path is assumed to be formed in a space of approximately 10 to 100 micrometers in the thickness direction of the substrate substantially in a two-dimensional plane. Based on the size of the cell, the suitable size of the micro flow path will be 5 to 10 micrometers for the bacteria, and 10 to 50 micrometers for the animal cell.  
         [0026]     To process all the cells flowing through the micro flow path by image recognition, the throughput depends on the speed of recognizing the image, namely on the frame rate of the camera taking the image in and the speed of sequential image processing of the image taken in. For instance, when a high-speed camera capable of 500 frames/second is used, it is necessary to process one frame of image in less than 1/500 second. Even if there are no more than a few cell images in each frame, a technique of extracting dimensional features with each cell linked between frames is feasible once it is intended. The inventors of the present invention actually realized the processing of 2000 cells/second by developing the high-speed camera capable of 500 frames/second and a dedicated image processing chip.  
         [0027]     This numeral value enables processing equivalent substantially to cell sorting processing of 60,000 to 80,000 cells/second (in fact the range of 2000 to 5000 cells/second is most commonly used to secure the purity and recovery rate) by the conventional cell sorter. It is difficult to achieve a further improvement of processing the cells only by the image recognition with the current technology.  
         [0028]     Therefore, the present invention provides a step of identifying/sorting cells with scattered light or fluorescent light intensity before cell image recognition. That is to say, a rough sorting is performed in a first step, and a finer cell sorting is performed in a second step. More specifically, the cells are roughly sorted by the scattered light or fluorescent light intensity in the first step. In this step, the rough sorting is performed so that the cells to be collected are not lost even if not all unnecessary cells are removed. Next, the roughly-sorted cells are re-sorted more finely using the image recognition in the second step. The two-step sorting is formed on one chip in a cascaded state. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0029]      FIG. 1  is a conceptual diagram of a cell sorting system showing the configuration of elemental functions of the cell sorter chip separating the cells in the second step of the embodiment and the devices accompanying each elemental function;  
         [0030]      FIG. 2  is a plan view schematically showing an example of the configuration of the cell sorter chip according to the embodiment;  
         [0031]      FIG. 3  is a cross-sectional view of a chip substrate  101  viewed in the direction of the arrowhead at the position of A-A crossing the centers of holes  201 ,  202 , and  203  in the region of a reservoir  210 ;  
         [0032]      FIGS. 4A, 4B , and  4 C are partial cross-sectional views of the chip substrate  101  focused on the holes  202 ,  203  and a filter  230  in the region of the reservoir  210  to explain the artifice in the introducing section for the sample cells;  
         [0033]      FIG. 5  illustrates the detailed structure in the vicinity of a first cell sorting region  262 ;  
         [0034]      FIG. 6  is a chart explaining the cell distribution in the micro flow path  221  after the confluence as a result of the fact that a buffer fluid flowing down a micro flow path  221  is pushed to the center by the buffer fluid flowing down micro flow paths  224 ,  224 ′;  
         [0035]      FIG. 7  illustrates the detailed structure in the vicinity of a second cell sorting region  320 ;  
         [0036]      FIG. 8  is a diagram explaining a scattered light detecting section in the case where a first cell detecting region  261  obtaining information used for sorting the cells in the first cell sorting region  262  obtains the information of the cell from the forward scattering and the attenuation of the transmitted light;  
         [0037]      FIG. 9  is a diagram explaining a side-scattered light detecting section in the case where the first cell detecting region  261  providing information used for sorting the cells in the first cell sorting region  262  obtains the information of the cell from the side-scattered light;  
         [0038]      FIG. 10  is a diagram explaining an example of the configuration of an image detecting section where a second cell detecting region  310  providing information used for sorting the cells in the second cell sorting region  320  obtains the cell information in the form of image information;  
         [0039]      FIG. 11  is a diagram explaining an example of the configuration of the image detecting section where a second cell detecting region  310  providing information for sorting the cells in the second cell sorting region  320  obtains the cell information in the form of the fluorescent image of the cell; and  
         [0040]      FIG. 12  is a diagram explaining an example of the configuration of the optical system performing the cell detection based on the fluorescence intensity of the cells fluorescence-labeled in advance. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
     Embodiment  
       [0041]      FIG. 1  is a conceptual diagram of a cell sorting system showing the configuration of elemental functions of the cell sorter chip separating the cells in the second step of the embodiment and the devices accompanying each elemental function.  
         [0042]     Reference numeral  100  denotes the cell sorter chip. Reference numeral  1  denotes a cell suspension storing section which stores the cell suspension to be sorted. A scattered light detecting section  2  irradiates the cells included in the sell suspension flowing down from the cell suspension storing section  1  with a laser beam using a laser light source  15 . A light detector  11  detects the scattered light of the laser light scattered by the cells. Information of the scattered light detected by the light detector  11  is transmitted to a personal computer  10  to compute the size of the cell emitting the scattered light. The cell suspension passed by the scattered light detecting section  2  reaches a first sorting section  3 . In the first sorting section  3 , when a cell having a forward scattered light intensity equivalent to or less than a certain scattered light intensity (for instance, a cell with the size of approximately 5 μm or less) flows down based on the computed result of the personal computer  10 , a power supply  13  is operated by a command from the personal computer  10  to move the cell to a waste reservoir  4  as a cell in a first waste group. On the other hand, the first sorting section  3  lets a cell with higher scattered light intensity (for instance, a cell with the size (diameter) exceeding approximately 5 μm) flow down as it is as a cell in a first refined cell group. The cell suspension corrected from the cells in the first waste group, namely the cell suspension including the target cells in the first refined cell group, reaches an image detecting section  5 . In the image detecting section  5 , the cell suspension is irradiated with light from a prespecified light source  16 , and an image data of the cell obtained by a image processing device  12  is transmitted to the personal computer  10 , which evaluates the image parameter. The cell suspension that has passed the image detecting section  5  reaches a second sorting section  6 . In the second sorting section  6 , when a cell having the result of evaluation by the personal computer  10  is within a prespecified condition (for instance, a cell with the longer diameter and the shorter diameter equivalent to or less than a prespecified value) flow down, a power supply  14  is operated by a command from the personal computer  10  to move the cell to a waste reservoir  7  as a cell in a second waste group. On the other hand, the second sorting section  6  lets a cell under a prespecified condition (for instance, a cell with the longer diameter and the shorter diameter exceeding a prespecified value) flow down as it is as a cell in a second refined cell group and is retrieved into a sorting reservoir  8 .  
         [0043]     The image parameter to be evaluated by the personal computer  10  is more specifically described below. For the information of the scattered light detected by the light detector  11 , a method of determining the cell to be sorted by the parameter depending on the forward scattering depending on the cell size, or a method of determining the cell to be sorted by the parameter of the side-scattering depending on the scattering of minute particles in the cell may be used, or attenuation in the amount of transmitted light through scattering may be simply used. As for the image parameter for the cell obtained by the image processing device  12 , a random combination of the long diameter and short diameter of the cell, the area projected on the image, the shape, the permeability, and the distribution of the transparency in the cell can be used as the image parameter.  
         [0044]      FIG. 2  is a plan view schematically showing an example of the configuration of the cell sorter chip according to the embodiment. The cell sorter chip  100  includes a substrate  101 . The substrate  101  is provided with a micro flow path at the bottom plane and an opening on the top plane in communication with the micro flow path. This opening serves as a supply port for the samples or necessary buffer fluid. A reservoir is also provided for supplying a sufficient amount of the buffer fluid and for controlling the flow rate of the buffer fluid in each micro flow path. The micro flow path may be formed by the injection molding, which pours plastics such as PMMA into a mold tool. The general size of the chip substrate  101  is 20×40×1 mm (t).  
         [0045]     In order to make the groove carved in the bottom plane of the chip substrate  101  and the through-hole in the substrate into the form of a micro flow path and a well, respectively, a 0.1-mm-thick laminate film is thermo-compression-bonded on the bottom formed with the groove. Cells flowing through the micro flow path can be observed through the 0.1-mm-thick laminate film using an objective lens with 1.4 numerical aperture and ×100 magnification. A lens with lower magnification naturally allows for observation without a problem.  
         [0046]     The chip substrate  101  is provided on the top surface thereof with a hole  201  for introduction of the sample buffer fluid including the cells into the micro flow path, holes  202 ,  203 ,  204 ,  205 ,  205 ′,  206  and  206 ′ for introduction of the buffer fluid excluding the cells, and a reservoir  210  including all the holes mentioned above. A wall  211  is provided around the hole  201  used for introduction of the sample buffer fluid including the samples to prevent the sample buffer fluid including the cells from spreading. The wall  211  is lower than the wall of the reservoir  210 . The holes  201 ,  202 ,  203 ,  204 ,  205 ,  205 ′,  206  and  206 ′ are each in communication with a corresponding one of micro flow paths  221 ,  222 ,  223 ,  224 ,  224 ′,  225 , and  225 ′. Therefore, when the reservoir  210  is supplied with the sufficient buffer fluid to the level higher than the wall  211 , the holes  201 ,  202 ,  203 ,  204 ,  205 ,  205 ′,  206  and  206 ′ communicate with one another through the buffer fluid. The buffer fluid also flows into the micro flow paths  221 ,  222 ,  223 ,  224 ,  224 ′,  225 , and  225 ′ each in communication with the corresponding one of these holes.  
         [0047]     While more details will be explained later, the sample buffer fluid including the cells introduced into the hole  201  flows down the micro flow path  221 , the cells are evaluated with a first parameter in a first cell detecting region  261 , and based on the result thereof, the cells are sorted in a first cell sorting region  262 . One of the sorted parties flows down a micro flow path  219  into a retrieving hole  271 . The other sorted party flows down a micro flow path  218 , the cells are evaluated with a 12th parameter in a second cell detecting region  310 , and based on the result thereof, the cells are sorted in a second cell sorting region  320 . One of the sorted parties flows down a micro flow path  330  into a retrieving hole  272 . The other sorted party flows down a micro flow path  331  into a retrieving hole  273 . The retrieving holes are each surrounded by a corresponding one of reservoirs  281 ,  282 , and  283  to prevent the sample buffer fluid including the retrieved cells from spreading, and a reservoir  284  including the reservoirs  281 ,  282  and  283  is further provided. The reservoir  284  is higher than the wall  271  to prevent the sample buffer fluid including the retrieved cells from spreading and walls  282  and  283  described later, and the buffer fluid is filled to the level higher than the walls  281 ,  282 ,  283  before the sorting operation. However, this height is assumed to be lower than the level of the buffer fluid filled in the reservoir  210 .  
         [0048]      FIG. 3  is a cross-sectional view of the chip substrate  101  viewed in the direction of the arrowhead at the position of A-A crossing the centers of the holes  201 ,  202 , and  203  in the region of the reservoir  210 . The grooves carved in the bottom plane of the chip substrate  101  are covered by a laminate film  410  to provide the respective micro flow paths  221 ,  222  and  223 . Similarly, the holes  201 ,  203 , and  204  bored in the chip substrate  101  are covered by the laminate film  410  to provide respective wells open on the top plane of the substrate  101 . The holes  201 ,  203 , and  204  communicate with the micro flow paths  221 ,  222  and  223 , respectively. The hole  201  is surrounded by the reservoir  211  provided on the upper surface of the substrate  101  and is formed as a cone-shaped hollow. In addition, a membrane filter  411  is provided on the top plane of the hole  201 . This configuration is provided in order to positively introduce the cells in the sample buffer fluid including the cells into the micro flow path  221  and to prevent large dust from flowing into the micro flow path  221 . The relationship between the other holes and the micro flow paths are the same although not shown.  
         [0049]     As shown in  FIG. 3 , since the reservoir  210  is supplied with a sufficient amount of a buffer fluid  200 , the micro flow paths  221 ,  222 ,  223 ,  224 ,  224 ′,  225 , and  225 ′ in communication with the holes  201 ,  202 ,  203 ,  204 ,  205 ,  205 ′,  206 , and  206 ′, respectively, are supplied with the buffer fluid to the same level. Therefore, an equal hydraulic pressure is applied to the entrance of the hole  201  for introducing the sample buffer fluid including the cells into the micro flow path and the holes  202 ,  203 ,  204 ,  205 ,  205 ′,  206 , and  206 ′ for introducing the buffer fluid excluding the cells into the micro flow path. Accordingly, if having the same width (assuming that they have the same height) or the same cross-sectional area, and the same length, then both micro flow paths will have substantially the same flow rate. For instance, the micro flow paths  224  and  224 ′ associated with the holes  205  and  205 ′, respectively, are supplied with the buffer fluid to the same level to equalize the flow rate of the buffer fluid flowing through the micro flow paths  224  and  224 ′. Specific examples of dimensions of each section are shown in the description below.  
         [0050]      FIGS. 4A, 4B , and  4 C are partial cross-sectional views of the chip substrate  101  focused on the holes  202 ,  201  and a filter  230  in the region of the reservoir  210  to explain the artifice in the introducing section for the sample cells. As shown in  FIG. 4A , the micro flow path  221  (20 μm wide, 15 μm deep) is connected to the hole  202  located upstream of the hole  201 . Therefore, as shown in  FIG. 4B , the buffer fluid including a sample cell  501  introduced into the hole  201  flows down the micro flow path  221  along with the buffer fluid excluding the cells supplied from the hole  202 . The flow of the cell solution supplied from the hole  201  and the flow of the buffer fluid supplied from the hole  202  make a laminar flow in locations downstream of the hole  201 . Since a layer of the cell solution supplied from the hole  201  is formed on the layer of the flow of the buffer fluid supplied from the hole  202 , therefore, the cells flow smoothly down in the micro flow path  221  without any contact with the bottom thereof. The filter  230  incorporated directly in the chip as a fine structure is disposed downstream of the hole  201  in the micro flow path  221  to prevent the micro flow path  221  from clogging.  FIG. 4C  schematically shows a state in which cells flowing into the flow path  221  from the opening  201  come into contact with the laminate film  410  causing accumulation. When one of the cells contacts the laminate film  410  to stay there, other cells get stuck with the cell to accumulate there one after another, consequently stopping the flow of the cells.  
         [0051]     The sample buffer fluid including the cells that has passed through the filter  230  flows down the micro flow path  221  and is gathered with two side flows of sheath buffer excluding cells supplied from the two micro flow paths  224 ,  224 ′ (12 μm wide, 15 μm deep) connected to two buffer reservoir holes  205 , and  205 ′ in upper steams, respectively. A micro flow path (20 μm wide, 15 μm deep)  240  is the confluent pathway of above three pathways, a part of which is also used as the first cell detecting region  261 . The reason for placing the first cell detecting region  261  in the micro flow path  240  on which the micro flow path  221  and micro flow paths  224 ,  224 ′ converge will be described later with reference to  FIG. 5 .  
         [0052]     At the lower reach of the stream from the first cell detection region  261 , the micro flow path  240  is gathered with the micro flow path  222  (20 μm wide, 15 μm deep) through which the buffer fluid excluding the cells supplied from the hole  203 . Reference numeral  241  denotes a micro flow path (40 μm wide, 15 μm deep) after the confluence of two micro path ways  240  and  222 , a part of which is used for the first cell sorting region  262 . The confluent micro flow path  241  forks into the micro flow paths  218  (20 μm wide, 15 μm deep) and  219  (20 μm wide, 15 μm deep) at the lower reach of the stream from the first cell sorting region  262 . A pair of gel electrodes are in contact with the buffer fluid at the first cell sorting region  262  flowing down the micro flow path  241 . When voltage is applied to the gel electrodes, the cells are sorted by a synthetic vector of the electrophoretic force working on cells and a force applied by the buffer fluid flowing through the micro flow path  241 . The configuration of the first cell sorting region  262  and the force to sort the cells are also explained with reference to  FIG. 5 .  
         [0053]      FIG. 5  is a diagram showing the detailed structure in the vicinity of the first cell sorting region  262 . Because of the sample buffer fluid flowing down the micro flow path  221  is gathered with two micro flow paths  224  and  224 ′ from both sides, as shown at the top of  FIG. 5 , cells  501  flowing down jumblingly through the micro flow path  221  are lined up and spaced adequately at the center of the micro flow path  240  after the confluence. The reason for this line-up effect is explained with reference to  FIG. 6 .  
         [0054]      FIG. 6  is a chart showing the cell distribution in the micro flow path  221  after the confluence as a result of the fact that a buffer fluid flowing down the micro flow path  221  is concentrated to the center by the push of two side buffer fluids from micro flow paths  224 ,  224 ′. Reference numeral  259  in the chart denotes a side wall of the flow path. More specifically, the chart shows the following state by indicating the location of the micro flow path  221  on the horizontal axis and the frequency of appearance of the cells on the longitudinal axis: the flow of the buffer fluid including the cells flowing down the 20 -μm-wide micro flow path  221  is concentrated to the center of the 20-μm-wide micro flow path  221  by the push of two side flows of the buffer fluid flowing down the 12-micrometer wide micro flow paths  224  and  224 ′. A curve  301  indicates that the cells are distributed in the width of approximately 10 μm at the center of the micro flow path  221  in the following case. That is to say, each buffer fluid flowing down each of the micro flow paths  224  and  224 ′ is roughly half of the volume of the buffer fluid including the cells flowing down the micro flow path  221 . In other words, the cross-sectional area of each of the micro flow paths  224  and  224 ′ is roughly half of that of the micro flow path  221 . A curve  302  indicates the cell distribution when the width of each of the micro flow paths  224  and  224 ′ is narrower, and a curve  303  indicates the cell distribution when the micro flow paths  224  and  224 ′ are not provided. As obvious from the curve  301 , setting a suitable width of the micro flow paths  224  and  224 ′ enables the cells flow substantially away from the wall of the flow path to prevent the cells from reaching the wall.  
         [0055]     Returning to  FIG. 5 , the explanation continues. Since the cells flowing down the micro flow path  221  thus pass in the center of the micro flow path  221  in an orderly manner at the first cell detecting region  261 , each cell can be detected enabling to evaluate parameters of the cell more accurately.  
         [0056]     At the downstream of the first cell detecting region  261 , the micro flow path  222  (20 μm wide, 15 μm deep) joins the micro flow path  240  made by joining the micro flow paths  224  and  224 ′ into the micro flow path  221  from both sides, forming the new confluent micro flow path  241  (40 μm wide, 15 μm deep). The buffer fluid excluding the cells flows into the micro flow path  222  from the hole  203 . The micro flow path  240  and the micro flow path  222  are assumed to have the same width and the width of the micro flow path  241  is assumed to be two times wider than the former width; therefore, the buffer fluids flowing down the confluent micro flow path  240  and the micro flow path  222  flow down while substantially keeping the boundary of two layers of each laminar flow in the micro flow path  241 . Thus, though the cell distribution curve  301  shown in  FIG. 6  tends to slightly expand toward the entrance of the micro flow path  222 , there is not so much difference.  
         [0057]     From the confluent point of the micro flow path  240  and the micro flow path  222  to the micro flow path  241  after the confluence is used for the first cell sorting region  262 . In this region, conjunction sections  255  and  256  are formed in the bottom plane of the substrate  101  as with the micro flow path. The conjunction sections  255 ,  256  have a liquid junction structure of approximately 15 μm wide (length along the micro flow path), 15 μm deep and 20 μm long filled internally with gel including an electrolyte. In addition, they are connected with the micro flow path  240  and the micro flow path  222  through the walls thereof, respectively, so that the gel including the electrolyte directly comes into contact with the buffer fluid flowing down the micro flow paths. The area of contact between the gel and the buffer fluid flowing down the micro flow path is 15 μm 2 . The conjunction sections  255  and  256  are disposed, as shown in  FIG. 5 , so that the conjunction section  255  is downstream of the conjunction section  256 . The other ends of the conjunction sections  255  and  256  are similarly connected with bending sections of micro structures  253  and  254 , respectively, which have 200 μm wide and 15 μm high and are formed on the bottom plane of the substrate  101 . The micro structures  253  and  254  are provided at the ends thereof with holes  251 ,  251 ′ and  252 ,  252 ′ (2 mm in diameter), respectively, connected to the top plane of the substrate  101 . The holes  251 ,  251 ′ and  252 ,  252 ′ are used to introduce the gel including the electrolyte therein. The gel is inserted into the holes  251  and  252  until the gel comes out of the holes  251 ′ and  252 ′, whereby the micro structures  253  and  254  on the bottom plane of the substrate  101  and the conjunction sections  255  and  256  of the liquid junction structure are filled with the gel including the electrolyte.  
         [0058]     Electrodes  257  and  258  denoted by black circles are connected to the holes  251  and  252 , respectively, for introducing the gel and are connected with the power supply  13  explained with reference to  FIG. 1 . At the right moment when the cells detected in the first cell detecting region  261  flow down between the conjunction sections  255 ,  256 , voltage is applied between the electrodes and thus to the buffer fluid in response to a signal given by the personal computer  10 .  
         [0059]     As describe above, the conjunction sections  255 ,  256  where the buffer fluid comes into contact with the gel in the first cell sorting section  262  are configured such that the conjunction section  256  is arranged at the upstream of the conjunction section  255 . When positive voltage is applied to the electrode  258  (anode) inserted in the hole  252  and negative voltage to the electrode  257  (cathode) inserted in the hole  251 , the cells flowing down the micro flow path  240  can be effectively moved to the micro flow path  218 . This is because an electrophoretic force works on a negatively charged cell to move to the positive electrode (anode)  258  when current is applied and a synthetic vector is formed by the vector received from this force and the buffer fluid flowing through the micro flow path and the vector of the electrophoresis. This configuration allows for more effective use of the electric field compared with a configuration forming the liquid junction sections  255  and  256  at the same points relative to the flow of the micro flow path (the opposite position with respect to the flow line), and the cells can move to the micro flow path  218  or the micro flow path  219  under a stable state with lower voltage. A retrieving hole  271  for the cells sorted in the first cell sorting region  262  is disposed downstream of the micro flow path  219 . A wall  281  is provided for the hole  271  to prevent the sample buffer fluid including the retrieved cells from spreading.  
         [0060]     The explanation continues with reference to  FIG. 2  again. The cells moved to the micro flow path  218  in the first cell sorting region  262  flow down to the second cell detecting region  310 . In this process, as with the micro flow path  240  described above, the micro flow paths  225  and  225 ′ (12 μm wide, 15 μm deep) which are two bypasses supplying the buffer fluid excluding the cells flowing from the holes  206  and  206 ′, respectively, provided in the reservoir  210  flow into the micro flow path  218 . As a result, a micro flow path  300  after the confluence allows the cells to flow in an even more orderly manner as with the micro flow path  240 , and is used as the second cell detecting region  310 . Further, a cell sorting region  320  is provided downstream of the second cell detecting region  310 , where the micro flow path  223  (20 μm wide, 15 μm deep) supplying the buffer fluid excluding the cells flowing from the hole  204  provided in the reservoir  210  joins the micro flow path  300 , as in the first cell sorting region  262 , to form a micro flow path  340  (40 μm wide, 15 μm deep).  
         [0061]     The second cell sorting region  320 , similarly to the first cell sorting region  262 , divides into the two micro flow paths  330  (20 μm wide, 15 μm deep) and  331  (20 μm wide, 15 μm deep) at the exit of the confluent micro flow path  340 . Also here, the second cell sorting region  320  includes conjunction sections  355  and  356  formed in the bottom plane of the substrate  101  as with the micro flow path and having a liquid junction structure of approximately 15 μm wide (length along the micro flow path), 15 μm deep and 20 μm long filled internally with gel including an electrolyte. In addition, the conjunction sections  355  and  356  communicate with the micro flow path  300  and the micro flow path  223  through the walls thereof, respectively. Consequently, the gel including the electrolyte directly comes into contact with the buffer fluid flowing down the micro flow path.  
         [0062]      FIG. 7  is a diagram showing the detailed structure in the vicinity of the second cell sorting region  320 , which is substantially the same as the structure of the first cell sorting region  262  shown in  FIG. 5 . Specifically, holes  351 ,  351 ′ and  352 ,  352 ′ are used for introduction of the gel including the electrolyte. The gel is inserted into the holes  351  and  352  until the gel comes out of the holes  351 ′ and  352 ′, respectively. Thus, micro structures  353  and  354  on the bottom plane of the substrate  101  and the conjunction sections  355  and  356  of the liquid junction structure are filled with the gel including the electrolyte. The bending sections of micro structures  353  and  354  are the conjunction sections  355  and  356 , respectively, having a liquid junction structure of approximately 20 μm long between the micro flow path  300  and the vicinity of the border of the micro flow paths  300  and  223 . In the cell sorting region  320 , the gel can directly contact the buffer fluid flowing near the micro flow path  340  formed by the micro flow path  340  and the micro flow path  223  flowing into each other. The area of contact between the gel and the buffer fluid is 15 μm (length along the micro flow path)×15 μm (height). Electrodes  357  and  358  denoted by black circles are inserted into the holes  351  and  352 , respectively, for introducing the gel. Voltage is applied to the buffer fluid between the electrodes in response to the signals provided by the personal computer  10  at the right moment when the cells detected in the second cell detecting region  310  flow down between the conjunction sections  355  and  356 .  
         [0063]     The conjunction sections  355  and  356  where the gel contacts the buffer fluid flowing through the micro flow path  340  in the second cell sorting region  320  is, as in the first cell sorting region  262 , configured so that the conjunction section  356  is located upstream of the micro flow path. When positive voltage is applied to the electrode  358  (anode) in the hole  352  and negative voltage to the electrode  357  (cathode) in the hole  351 , the cells flowing down the micro flow path  300  can be effectively moved to the micro flow path  331 . Specifically, this is because an electrophoretic force works on a negatively charged cell to move to the positive electrode (anode)  358  when current is applied and a synthetic vector is formed by the vector received from this force and the buffer fluid flowing through the micro flow path and the vector of the electrophoresis. This configuration allows for more effective use of the electric field compared with a configuration forming the liquid junction sections  355  and  356  at the same points relative to the flow of the micro flow path (the opposite positions with respect to the flow line). The cells can move to the micro flow path  330  or the micro flow path  331  under a stable state with lower voltage.  
         [0064]     In the second cell sorting region  320 , the cells in the sample buffer fluid roughly sorted in the first cell sorting region  262  is evaluated in the second cell detecting region  310  by a parameter different from the parameter used in the first cell detecting region  261  and sorted. Therefore, the cells flowing down the micro flow paths  330  and  331  are, as shown in  FIG. 7 , more strictly sorted.  
         [0065]     As explained with reference to  FIG. 2 , the retrieving holes  272  and  273  for the sorted cells are bored in downstream sections of the micro flow paths  330  and  331 , respectively. Walls  282  and  283  are arranged on the circumference of the holes  272  and  273 , respectively, to prevent the sample buffer fluid including the retrieved cells from spreading by their heights. Along with the wall  281 , the walls  282  and  283  are surrounded by the reservoir  284  including the same. The height of the reservoir  284  is higher than the height of the walls  271 ,  282 , and  283  to prevent the sample buffer fluid including the cells from spreading. The buffer fluid is filled in the reservoir to the level higher than the walls  281 ,  282  and  283  before operation, but the level is lower than the height of the reservoir  210 .  
         [0066]     A force for driving fluid flowing in each micro flow path is described below. In the present invention, the cell sorting chip is devised so that fluid can be fed in all of micro flow paths by itself only. In the present invention, fluid flow is fed by a difference of pressures between fluid levels in reservoirs having different heights according to Pascal&#39;s law. More specifically, a fluid level in the reservoir  210  is higher than that in the reservoir  284 , and this head generates a driving force caused by the difference of pressure for driving a buffer fluid flowing in each micro flow path and also produces a stable flow without pulsing. When a capacity of the reservoir  210  for a buffer fluid is sufficiently large, all of the sample buffer fluid containing cells introduced into the hole  201  can be allowed to flow into the micro flow path  221 . All of the fluid fed into the first cell sorting region  262  and into the second cell sorting region  320  is supplied from the reservoir  210 , and a driving force for feeding the fluid is generated due to a difference of fluid levels between the reservoir  210  and the reservoir  284 . Therefore, the same pressure is loaded to the inlet ports  201 ,  202 ,  203 ,  204 ,  205 ,  205 ′,  206 , and  206 ′ of the micro flow paths, which enables stable feed of fluid only with the cell sorting chip.  
         [0067]     In the embodiment described above, a two-stage cell sorting chip in which the first cell sorting region  262  and the second cell sorting region  320  are serially linked to each other is described, but the chip may have a multi-layered structure including three or more stages. In this case, a common reservoir for feeding fluid and also a common reservoir on the fluid recovery side are used to feed fluid, thereby making use of a head of fluid between respective fluid levels on the feed side and on the recovery side, which can realize a stable multi-staged cell sorter chip.  
         [0068]      FIG. 8  illustrates operations of a scattered light detecting section when the first cell detecting region  261  acquiring information for sorting cells in the first cell sorting region  262  obtains information by detecting forward scattering of light or attenuation of transmitted light. In  FIG. 8 , a laser beam emitted from a laser beam source  510  is directed, as a laser beam  513 , to a micro flow path  240  in the first cell detecting region  262  from a position above the substrate  101  via a optical fiber  511  and a collimate lens system  512 . The substrate  101  and the laminate film  410  can transmit the so-called visible light with a wavelength of 400 nm to 700 nm, and therefore the emitted laser beam  513  is scattered by cells flowing down the micro flow path  240 . The laser beam  513  going straight is shuttered by a stopper  514 , the scattered light is condensed by a condenser  516 , passed through a pinhole  517  with the background light removed, and is then detected by a photodiode  518 . The photodiode light detector which measures intensity of scattered light or a ring-formed photodiode array detector which measures an angle of scattered light may be used. The latter is better for measuring a size of each cell, but cell sorting performed in the first stage is for roughly sorting cells, and therefore a low cost photodiode available simply for measurement of intensity of scattered light is used.  
         [0069]      FIG. 9  is a diagram illustrating operations of the sideward scattered light detecting section when the first cell detecting region  261  acquiring information for sorting cells in the first cell sorting region  262  obtains information by detecting sideward scattering of light or attenuation of transmitted light. In this example, a YAG laser emitting a beam with a wavelength of 514 nm or an argon laser emitting a beam with a wavelength of 488 nm can be used. The laser beam is emitted from the rear side of the substrate  101  through a collimate lens  702 . The beam goes straight in the substrate  101  and is scattered by cells flowing down the micro flow path  240  in the first cell detecting region  261 . The scattered light obtained when no cells flow down the micro flow path  240  is used as a base. When a cell passes through the laser beam, scattered light reaches a photoelectron multiplier  705  via a condenser system  704 , and the intensity is measured.  
         [0070]     In the sideward scattered light measuring system as described above, smaller size particles can be measured, so that intensity of scattered light changes due to a difference of an internal structure of each cell. Therefore cells can be recognized and sorted according to a parameter different from that employed in measurement with forward scattered light.  
         [0071]      FIG. 10  is a view illustrating an example of configuration of an image detecting section in which the second cell detecting region  310  for providing information for sorting cells in the second cell sorting region  320  acquires cell information as image information. Light from a halogen lamp  520  with a cold mirror is incident onto cells flowing down the micro flow path  300  in the second cell detecting region  310  via a phase contrast ring  521  and a condenser lens  522 . The light transmitted a cell is focused by a phase contrast object lens  523  on an image pick-up element of a high-speed camera  524 . An image signal obtained by the image pick-up element of the high-speed camera  524  is sent to a personal computer.  
         [0072]      FIG. 11  is a diagram illustrating an example of configuration of an image detecting section in which the second cell detecting region  310  for providing information for sorting cells in the second cell sorting region  320  acquires cell information as a cell luminescence image. In this example, light from a mercury lamp  530  as a light source is converted by a dichroic mirror  531  to light with a wavelength in the excitation light band and is directed to cells flowing down the micro flow path  300  in the second cell detecting region  310  using the phase contrast object lens  523  for causing excitation of luminescence in each cell. The luminescence image emitted from the cell is condensed by the phase contrast object lens  523  and filtered by a filter  532  to obtain only the luminescence component, thereby picking up an image with the high speed camera  524 .  
         [0073]     Cell sorting in the second cell sorting region  320  is performed according to a cell form as a parameter. The second cell detecting region  310 , therefore, treats cells so that classification of cells can be performed with higher precision as compared to that in the first cell detecting region  261 , and cannot treat a large quantity of cells. In other words, the number of cells which can be treated in the second cell detecting region  310  depends on a frame rate of a camera and performance of a real time image processing device. However, by using, for instance, a CCD type camera capable of imaging real 500 frames per second as the high speed camera  524  used in the second cell detecting region  310  and also by using a device capable of treating 500 frames per second, it is possible to determine forms of 1000 or more cells per second. This figure is one-tenth less than the number of cells recognized in the first cell detecting region  261  based on scattered light. This means that the cell sorter chip having the two-stage configuration in which cells are roughly sorted in the first stage and are more precisely in the second stage has a greater merit.  
         [0074]     A large number of cells can be assessed in both of the cell detection based on the forward scattered light explained with reference to  FIG. 1  and the cell detection based on the sideward scattered light explained with reference to  FIG. 9  within a short period of time. Therefore, a large number of cells can efficiently be sorted with high precision by applying cell detection based on forward scattered light and cell detection based on sideward scattered light to the first cell detecting region  261  and the second cell detecting region  310  respectively.  
         [0075]     The rough cell sorting in the first stage may be performed not only by the method based on scattered light, but also by the method based on intensity of luminescence. To measure luminescence intensity, as a manner of course it is necessary to label cells with a luminescent material beforehand. Existent examples of labeling cells with a luminescent material include the nuclear staining method using a coloring matter such as DAPI and the cell surface antigen staining method using a luminescent antibody. The optical system shown in  FIG. 12  is used to detect the luminescent labels. In this case, light from a laser light source  830  is reflected by a dichroic mirror  831  and the reflected light is directed by a lens  823  onto cells flowing down the micro flow path  300  in the second cell detecting region  310  for exciting luminescence. The luminescence emitted from the cells is condensed by the lens  823  and passed through the dichroic mirror  831  and the filter  832  to obtain only the luminescence component for eliminating astray light, and the luminescence component is detected by a photoelectron multiplier  834 . After necessary cells are obtained, cell sorting in the second state is performed, and in this processing step, the image detecting method explained with reference to  FIG. 10  in which cell information is obtained as image information or the cell luminescence detecting method explained with reference to  FIG. 11  is employed for cell sorting with higher precision.  
       Example of Cell Sorting  
       [0076]     An example in which a mixed suspension of erythrocytes and cardiac cells as a sample is sorted is described below.  
         [0077]     Table 1 shows contents of cells obtained in each processing step from a mixture of erythrocytes and cardiac cells as a sample suspension.  
                                                                                 TABLE 1                                       Number of cells(cells/100 μl)                    Cardiac   Miscellaneous           Erythrocytes   cells   cells                        Sample cell suspension     1 × 10     1 × 10   2.1 × 10            First cell   Cells sorted   1.2 × 10   0.93 × 10   0.3 × 10       sorting   in the first       section 3   stage           Cells discarded   8.7 × 10   0.07 × 10   2.7 × 10           in the first           stage       Second cell   Cells sorted   0.01 × 10    0.78 × 10   0       sorting   in the second       section 6   stage           Cells discarded   1.1 × 10   0.11 × 10   1.3 × 1            in the second           stage                  
 
         [0078]     The sample suspension contains 1×10 5  erythrocytes/100 μl, 1×10 3  cardiac cells/100 μl, and 2.1×10 3  miscellaneous cells (not identified based on the forms or dust)/100 μl, and 50 μl of the suspension was put in the hole  201 . The cell detection based on forward scattered light described with reference to  FIG. 8  is applied to the first cell detecting region  261  and the cell detection based on image processing described with reference to FIG,  10  or  FIG. 11  is applied to the second cell detecting region  310  to perform sorting of cells contained in the sample suspension.  
         [0079]     Intensity of scattered light from flat and large-sized erythrocytes is high, while intensity of scattered light from spheric cultured cardiac cells is low, and therefore in the first cell sorting region  3 , by setting a threshold value for detection of scattered light in the first cell detecting region  261  so that most cardiac cells can be recovered, cardiac cells can be sorted from a mixture of erythrocytes and cardiac cells. As a result, in the first cell sorting section  3 , 1.2×10 4  erythrocytes/100 μl defined as a first refined cell group, 8.7×10 4  erythrocytes/100 μl as a first discard cell group, 0.93×10 3  cardiac cells/100 μl as a first refined cell group, 0.07×10 3  cardiac cells/100 μl as a first discarded cell group, 0.3×10 3  miscellaneous cells/100 μl as a first refined cell group, and 2.7×10 3  miscellaneous cells/100 μl as a first aborted cell group are obtained. In short, a mixture suspension containing 0.93×10 3  cardiac cells/100 μl, 1.2×10 4  erythrocytes/100 μl, and 0.3×10 3  miscellaneous cells/100 μl is obtained, and thus the cardiac cells are condensed.  
         [0080]     In the first cell sorting section  3 , cells are roughly sorted, so that a large number of erythrocytes is included in the resultant mixture suspension and also other miscellaneous cells are contained in the suspension. A ratio of cardiac cells to erythrocytes is heighten to a value about 8 times higher as compared with the original value, but still 13 times a larger number of erythrocytes remain.  
         [0081]     In the second cell sorting section  6 , the mixture suspension obtained from the first cell sorting section  3  is subjected to cell sorting by applying cell detection by image processing in the second cell detecting region  310 . As a result, 0.01×10 3  erythrocytes/100 μl as a second refined cell group, 1.1×10 4  erythrocytes/100 μl as a second discarded cell group, 0.78×10 3  cardiac cells/100 μl as a second refined cell group, 0.11×10 3  cardiac cells/100 μl as a second aborted cell group, zero miscellaneous cells/100 μl as a second refined cell group, and 1.3×10 3  miscellaneous cells/100 μl as a second aborted cell group are obtained. In short, contamination of cardiac cell by erythrocytes can be lowered to about 1%.  
         [0082]     As described above, with the present invention, it is possible to realize a disposable cell sorting chip capable of efficiently sorting a large number of cells with high precision.