Abstract:
A cell isolation instrument includes: a first container which has an opening in an upper portion; a filtration member which defines at least a part of a lower portion of the first container; an isolation member which is movably housed in the first container to collide with a tissue, thereby isolating cells; and a second container which houses the first container in a manner that the first container can be taken out.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    The present invention relates to a cell isolation instrument which is used in the field of clinics/research of regenerative medicine, or that of cell culture, and more particularly to a cell isolation instrument which is used for isolating cells from a tissue (body tissue) with a high survival rate. 
         [0002]    There is a technique in which, in order to obtain isolated living cells, a tissue is minced with a sharp blade, and the minced tissue is subjected to an enzyme process with collagenase or the like to digest the extracellular matrix. 
         [0003]    In the technique, procedures of the enzyme process and cell recovery must be repeatedly performed. Therefore, the technique requires labor and a long time period. The technique has a further problem in that the cell recovery rate is dispersed because of differences in the mincing process with using a blade and in the permeation rate in the enzyme process, etc. 
         [0004]    JP-T-2007-505631 discloses an apparatus in which the mincing process and the enzyme process are automatized. In a container disposed in the apparatus, a blade member which is rotatable about the central axis of the container, and a baffle which relatively provides a resistance to the movement of the blade member are placed. When a tissue from which isolated cells are to be obtained is loaded into the container and the blade member is rotated, the tissue in the state where the blade member bites into the tissue is rotated together, and collides with the baffle. The tissue is cut by the blade member because of the resistance provided by the baffle. When the rotation of the blade member is continued for a predetermined time period, the cutting is repeated, so that the tissue is minced. An enzyme treatment solution is previously loaded into the container, and therefore the enzyme process is performed in parallel. 
         [0005]    In the configuration disclosed in JP-T-2007-505631, the mincing process and the enzyme process can be automatized. In order to recover isolated cells, however, a cell suspension which is obtained as a result of the mincing process and the enzyme process must be taken out to the outside of the container, and then subjected to a filtering process. In realization of an automated system which stably recovers a large amount of isolated cells, the burden on the user must be mitigated, and influences of human procedures on the number and quality of isolated cells must be reduced. Therefore, it is necessary to reduce processes to be performed by the user, as far as possible. In the configuration disclosed in JP-T-2007-505631, however, the reduction of processes to be performed by the user is insufficient. Consequently, the configuration is inadequate for realizing an automated system. 
       SUMMARY 
       [0006]    It is therefore an object of the invention to provide a cell isolation instrument in which processes to be performed by the user can be reduced as far as possible, and cells isolated from a tissue (body tissue) can be efficiently recovered. 
         [0007]    In order to achieve the object, according to the invention, there is provided a cell isolation instrument comprising: a first container which has an opening in an upper portion; a filtration member which defines at least a part of a lower portion of the first container; an isolation member which is movably housed in the first container to collide with a tissue, thereby isolating cells; and a second container which houses the first container in a manner that the first container can be taken out. 
         [0008]    The isolation member may include: a blade member; and a shaft member which rotatably supports the blade member in the first container. An upper end portion of the shaft member may be connectable to a cell isolation apparatus which rotates the shaft member. 
         [0009]    The cell isolation instrument may further comprise a lid member which is fitted to the first container to close the opening. A through hole through which the shaft member is passed may be formed in the lid member, and at least a part of a portion, which is located below the lid member, of the shaft member may be larger in diameter than the through hole. 
         [0010]    The lid member may include: a first lid member in which a first through hole through which the shaft member is passed, and at least one opening are formed; and a second lid member in which a second through hole through which the shaft member is passed, and which covers the at least one opening of the first lid member above the first lid member. 
         [0011]    A groove may be formed in at least one of an outer wall of the first container and an inner wall of the second container. 
         [0012]    In a state where the first container is housed in the second container, an upper end portion of the second container may be positioned above an upper end portion of the first container. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0013]      FIG. 1  is an exploded perspective view showing a cell isolation instrument of an embodiment of the invention. 
           [0014]      FIG. 2  is a longitudinal sectional view showing a state where the cell isolation instrument of  FIG. 1  is in the course of assembling. 
           [0015]      FIG. 3  is a longitudinal sectional view showing a state where the cell isolation instrument of  FIG. 1  is assembled. 
           [0016]      FIG. 4  is a longitudinal sectional view showing a state where the cell isolation instrument of  FIG. 1  is connected to a cell isolation apparatus. 
           [0017]      FIG. 5  is a longitudinal sectional view showing a state where a filtered cell suspension is recovered by using the cell isolation instrument of  FIG. 1 . 
       
    
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
       [0018]    Hereinafter, an embodiment of the invention will be described in detail with reference to the accompanying drawings. 
         [0019]      FIG. 1  shows a state where a cell isolation instrument  1  of an embodiment of the invention is disassembled. The cell isolation instrument  1  includes an isolation member  10 , an inner container (first container)  20 , an outer container (second container)  30 , an inner lid (lid member, first lid member)  40 , and an outer lid (lid member, second lid member)  50 . These components are formed by a material (such as a resin) which has resistance to ethylene oxide gas sterilization. 
         [0020]    The isolation member  10  includes a shaft member  11  and a plurality of blade members  12 . The shaft member  11  includes a large-diameter portion  11   a,  a small-diameter portion  11   b,  and a connecting portion  11   c.  The blade members  12  are supported by the lower end of the large-diameter portion  11   a.  The diameter of the small-diameter portion  11   b  is smaller than that of the large-diameter portion  11   a,  and a step  11   d  is formed in the interface between the large-diameter portion  11   a  and the small-diameter portion  11   b.  The connecting portion  11   c  is disposed in the upper end of the small-diameter portion  11   b,  and configured as a portion which is to be connected to a cell isolation apparatus  60  that will be described later. 
         [0021]    The inner container  20  includes a body unit  21  and a filter (filtration member)  22 . The body unit  21  is a cylindrical member which is opened in upper and lower portions. The filter  22  is formed by a material which is noncytotoxic. In the embodiment, nylon mesh having openings of 50 μm is used as the filter  22 . The filter  22  is adhered or welded to the body unit  21  so as to cover the whole lower opening of the body unit  21 . 
         [0022]    The outer container  30  includes a body unit  31  and a pedestal portion  32 . The body unit  31  is a cylindrical member which is opened in an upper portion. The inner side of the body unit  31  includes a first inner wall  33   a  and a second inner wall  33   b.  In the second inner wall  33   b,  a plurality of grooves  34  are formed at regular intervals in the circumferential direction of the body unit  31 . 
         [0023]    In an upper end portion of the body unit  31 , the first inner wall  33   a  has a first inner diameter. The second inner wall  33   b  has a second inner diameter which is smaller than the first inner diameter, and which is larger than the outer diameter of the body unit  21  of the inner container  20 . An inner step  33   c  is defined in the interface between the first inner wall  33   a  and the second inner wall  33   b.  The second inner wall  33   b  is continuous from the inner step  33   c  to a lower end portion of the body unit  31 . 
         [0024]    The inner lid  40  is a disk-like member which has an upper surface  40   a  and a lower surface  40   b  (see  FIG. 2 ). A through hole (trough hole, first through hole)  41  through which the upper surface  40   a  and the lower surface  40   b  communicate with each other is formed in a center portion of the inner lid  40 . A plurality of openings  42  through which the upper surface  40   a  and the lower surface  40   b  communicate with each other are formed so as to surround the through hole  41 . The inner diameter of the through hole  41  is larger than the outer diameter of the small-diameter portion  11   b  of the shaft member  11  of the isolation member  10 , and smaller than that of the large-diameter portion  11   a.    
         [0025]    The outer diameter of the inner lid  40  is approximately equal to the inner diameter (first inner diameter) of the first inner wall  33   a  of the body unit  31  of the outer container  30 . An annular upper fitting wall  43  is formed in a circumferential portion of the upper surface  40   a.  An annular lower fitting wall  44  is formed slightly inside a circumferential portion of the lower surface  40   b,  and a step  45  is defined outside the wall (see  FIG. 2 ). The outer diameter of the lower fitting wall  44  is approximately equal to the inner diameter of the body unit  21  of the inner container  20 . 
         [0026]    The outer lid  50  is a disk-like member which has an upper surface  50   a  and a lower surface  50   b  (see  FIG. 3 ). A through hole (trough hole, second through hole)  51  through which the upper surface  50   a  and the lower surface  50   b  communicate with each other is formed in a center portion of the outer lid  50 . The inner diameter of the through hole  51  is larger than the outer diameter of the small-diameter portion  11   b  of the shaft member  11  of the isolation member  10 . The outer diameter of the outer lid  50  is approximately equal to the inner diameter of the upper fitting wall  43  of the inner lid  40 . An annular fitting wall  52  is disposed in a circumferential portion of the upper surface  50   a.    
         [0027]    Next, a method of assembling the cell isolation instrument of the embodiment, and a part of steps of a cell isolating process will be described with reference to  FIGS. 2 and 3 . 
         [0028]    First, the inner container  20  is housed in the outer container  30  in a posture in which the filter  22  is downward directed. At this time, the outer wall  21   a  of the inner container  20  is opposed to the second inner wall  33   b  of the outer container  30  through a small gap. The upper end surface  21   b  of the body unit  21  is flush with the inner step  33   c  of the body unit  31 . 
         [0029]    In the state where the inner container  20  is housed in the outer container  30 , the upper end surface (upper end portion)  31   a  of the outer container  30  is positioned above the upper end surface (upper end portion)  21   b  of the inner container  20 . 
         [0030]    Next, the isolation member  10  is housed in the inner container  20 . At this time, a lower end portion of the shaft member  11  is placed on the filer  22 . 
         [0031]    Then, the inner lid  40  is attached so that the small-diameter portion  11   b  of the shaft member  11  of the isolation member  10  is passed through the through hole  41 . At this time, the step  45  of the inner lid  40  is placed on the upper end surface  21   b  of the inner container  20 , and the inner step  33   c  of the outer container  30 . Furthermore, the lower fitting wall  44  of the inner lid  40  is fitted to an inner wall  21   c  of the inner container  20 , and the upper fitting wall  43  is fitted to the first inner wall  33   a  of the outer container  30 . 
         [0032]    In the state shown in  FIG. 2 , a predetermined amount of enzyme treatment solution which is previously heated to the activation temperature is poured into the inner container  20  through the openings  42 . A tissue which is extracted from a living body for the purpose of isolation of cells is loaded similarly through the openings  42  into the inner container  20 . 
         [0033]    Next, the outer lid  50  is attached so that the small-diameter portion  11   b  of the shaft member  11  of the isolation member  10  is passed through the through hole  51 . At this time, the lower surface  50   b  of the outer lid  50  covers the openings  42  of the inner lid  40  to prevent foreign substances from entering the inner container  20  in the subsequent steps. The fitting wall  52  of the outer lid  50  is fitted to the inner side of the upper fitting wall  43  of the inner lid  40 .  FIG. 3  shows this state. A small gap is formed between the small-diameter portion  11   b  of the shaft member  11  and the through holes  41 ,  51 . 
         [0034]    As a result of the above-described steps, the cell isolation instrument  1  which accommodates the tissue and the enzyme treatment solution is placed on a stage (not shown) disposed in the cell isolation apparatus  60 . As shown in  FIG. 4 , the cell isolation apparatus  60  includes a bearing member  61 , a rotation mechanism  62 , a controller  63 , a setting section  64 , and a height adjusting mechanism  65 . In accordance with stirring conditions (the number of rotations, the rotation method, the duration time, and the like) which are set by the user through the setting section  64 , the controller  63  controls the rotation mechanism  62  to rotate the bearing member  61 . 
         [0035]    In accordance with user instructions or in an automatic manner, the controller  63  controls the height adjusting mechanism  65  to vertically move at least one of the stage and the bearing member  61 . This operation causes the connecting portion  11   c  of the shaft member  11  of the isolation member  10  to be connected to the bearing member  61 . The isolation member  10  is fixed to a position where the lower end portion of the shaft member  11  is separated from the filter  22 , and the step  11   d  of the shaft member  11  is not contacted with the lower surface  40   b  of the inner lid  40 . The isolation member  10  is supported by the bearing member  61 , so that the blade members  12  can be rotated in the inner container  20  with the rotation of the bearing member  61 .  FIG. 4  shows this state. 
         [0036]    When the user sets the stirring conditions through the setting section  64  and inputs instructions for starting the process, the controller  63  controls the rotation mechanism  62  in accordance with the preset stirring conditions so that the blade members  12  are rotated in the inner container  20  through the bearing member  61 . As a result, the mincing process and the enzyme process are automatically applied on the tissue. 
         [0037]    There is a possibility that, when isolated cells are immersed in the enzyme treatment solution in the outer container  30  for a long term, the cells may be damaged. sometimes, therefore, the enzyme process is performed divisionally in a plurality of steps. Specifically, the inner container  20  is lifted, and the filtered cell suspension is once acquired. Thereafter, a new enzyme treatment solution is poured into the outer container  30 , and the inner container  20  is lowered, whereby the isolating process and the enzyme process are again performed. In a related art, these works must be manually performed. According to the configuration of the embodiment, when the user sets a plurality of enzyme processes through the setting section  64 , the desired enzyme processes can be executed automatically and at adequate timings. 
         [0038]    During the stirring operation, the enzyme treatment solution may enter the gap between the inner container  20  and the outer container  30 . However, the grooves  34  which are formed in the second inner wall  33   b  of the outer container  30  function as an escape route for the entering enzyme treatment solution. Therefore, a situation where the entering enzyme treatment solution is caused to rise through the gap by the capillary force, and the solution leaks to the outside of the inner container  20  can be prevented from occurring. Even when the enzyme treatment solution leaks out, the enzyme treatment solution remains on the upper surface  50   a  of the outer lid  50  because the upper end surface  31   a  of the outer container  30  is positioned above the upper end surface  21   b  of the inner container  20 , and therefore the solution does not leak to the outer side surface of the outer container  30 . 
         [0039]    When the stirring operation is ended, a solution in which minced tissue pieces and isolated cells are suspended in the enzyme treatment solution is obtained in the cell isolation instrument  1 . Next, a step of recovering the isolated cells from the solution will be described with reference to  FIG. 5 . 
         [0040]    First, the connection of the cell isolation instrument  1  and the cell isolation apparatus  60  is cancelled, and the cell isolation instrument  1  is taken out from the cell isolation apparatus  60 . While holding the outer container  30 , the shaft member  11  of the isolation member  10  is gripped and lifted upward, and then the step  11   d  abuts against the lower surface  40   b  of the inner lid  40  because the large-diameter portion  11   a  of the shaft member  11  is larger in diameter than the through hole  41  of the inner lid  40 . When the shaft member  11  is further upward lifted, the inner container  20  can be lifted upward while the inner lid  40  remains to be fitted into the inner container  20 . 
         [0041]    At this time, unwanted tissue pieces  71  contained in the solution cannot be passed through the filter  22  because they are larger than the mesh openings of the filter  22 , and remain in the inner container  20 . By contrast, the cell suspension  70  containing isolated cells which are smaller than the mesh openings of the filter  22  are passed through the filter  22  to flow down into the outer container  30 . 
         [0042]    According to the configuration of the embodiment, namely, the cell suspension  70  containing isolated cells can be recovered easily and surely into the outer container  30 , simply by the simple operation of lifting up the inner container  20  to be taken out from the outer container  30 . Therefore, the efficiency of the step of recovering isolated cells can be remarkably improved. 
         [0043]    The embodiment has been described in order to facilitate understanding of the invention, and is not intended to limit the invention. It is a matter of course that the invention may be changed or improved without departing the spirit thereof, and includes equivalent embodiments. 
         [0044]    The filter  22  is not always required to cover the whole opening in the lower portion of the inner container  20 . It is required that the filter  22  forms at least a part of the lower portion of the inner container  20  in a range where the recovery of isolated cells by filtration is not disturbed. 
         [0045]    The isolation member  10  is not always required to be configured by the shaft member  11  which rotatably supports the blade members  12 . Various other configurations such as a configuration where a stirrer is used may be employed as far as a collision occurs on a tissue and cells can be isolated. 
         [0046]    The inner lid  40  is not always necessary. In the case where the inner lid is not disposed, the inner diameter of the through hole  51  of the outer lid  50  is made smaller than that of the large-diameter portion  11   a  of the shaft member  11  of the isolation member  10  because, when the shaft member  11  is lifted up, the step  11   d  must abut against the lower surface  50   b  of the outer lid  50 . 
         [0047]    The step of recovering isolated cells maybe automatized. For example, a configuration maybe employed where a structure for holding the outer container  30  is disposed on the stage of the cell isolation apparatus  60 , and, after the stirring operation, the height adjusting mechanism  65  is caused by the controller  63  to perform an operation of lifting the bearing member  61 . The shaft member  11  in the state where it is connected to the bearing member  61  is lifted up, and the inner container  20  can be taken out from the outer container  30 . 
         [0048]    It is not always necessary to form the plurality of openings  42  in the inner lid  40 . The number and size of at least one opening  42  can be adequately determined within a range where the workability of the operation of loading the enzyme treatment solution and the tissue is not impaired. 
         [0049]    The grooves  34  are not always required to be formed in the second inner wall  33   b  of the outer container  30 . Even when the grooves are formed in the outer wall  21   a  of the inner container  20  opposed to the second inner wall  33   b,  it is possible to prevent the enzyme treatment solution from leaking out. Alternatively, the grooves may be formed in both the second inner wall  33   b  and the outer wall  21   a.    
         [0050]    According to an aspect of the invention, the filtered cell suspension is allowed to be recovered into the second container, simply by taking out the first container from the second container. 
         [0051]    According to an aspect of the invention, the rotation of the shaft member can be controlled on the side of the cell isolation apparatus, and a cell isolation step including the process of mincing the tissue, and the enzyme process can be easily automatized. When living cells are to be recovered, fine adjustment is necessary because characteristics such as the hardness and the size are different depending on tissues. In the invention, however, it is requested only to adjust the rotation speed and time, kind, and the like of the blade member in accordance with the tissue to be recovered, and various cells can be recovered in the living state by the same cell isolation instrument. 
         [0052]    According to an aspect of the invention, the first container can be taken out simply by lifting the shaft member, and steps until the step of recovering the filtered cell suspension can be easily automatized. Furthermore, the works of lifting the first container to acquire the cell suspension, then pouring a new enzyme solution into the second container, again housing the first container in the second container, and performing the enzyme process a plurality of times can be easily automatized. Therefore, damage to the isolated cells caused by long-term immersion of the cells in the enzyme solution can be mitigated. 
         [0053]    According to an aspect of the invention, the tissue and the enzyme treatment solution are loaded into the first container through the minimum necessary opening, and thereafter the opening is covered by the second lid member. Therefore, it is possible to prevent foreign substances from entering the device during the cell isolating process and the recovering process. 
         [0054]    According to an aspect of the invention, the enzyme treatment solution and cell suspension which enter the gap between the first and second containers are guided to the groove. Therefore, it is possible to prevent liquids from leaking to the outside of the first container. 
         [0055]    According to an aspect of the invention, even when the enzyme treatment solution and the cell suspension leak from the first container, they cannot override the upper end of the second container. Therefore, leakage to the outside of the second container, i.e., the outside of the cell isolation instrument can be prevented from occurring.