Patent Publication Number: US-2022213424-A1

Title: Culture apparatus for drug discovery research

Description:
TECHNICAL FIELD 
     The present invention relates to an apparatus for culturing a three-dimensional cell structure, which is specialized in drug discovery research. 
     BACKGROUND ART 
     Conventionally, a technique of producing a three-dimensional cell structure by temporarily fixing a cellular aggregate (spheroid) in needles arranged in a pinholder shape has been known (Patent Literature 1). This technique is characterized in that a three-dimensional structure can be formed only with cells. 
     It has previously been known that spheroid culture provides higher cell activity than ordinary plate culture. In the three-dimensional cell structure form by the above-described technique, the cells are each transformed into three-dimensional forms, and thus, the cells have high metabolic activity. In addition, since the cells are directly contacted with a culture solution, the cells have high nutrient gas exchange efficiency. 
     In view of the foregoing, attempts have been made to use such a three-dimensional cell structure in a drug toxicity test and the like, and as a result, the human-specific hepatotoxicity of a drug, which could not have been detected by other human hepatocyte culture methods, could be detected (Non Patent Literature 1). Moreover, it is also likely that a three-dimensional structure constructed with myocardial cells can be utilized as a pulsation analysis tool. 
     In order to stick a spheroid into a pinholder, a special device (bio 3D printer) is required. However, since this device is expensive, it is difficult to acquire the device. 
     In drug discovery research, the number of spheroids, which is needed to be able to sufficiently analyze pharmacological activity, is 9. Thus, it is not necessary to use a bio 3D printer that treats a large number of spheroids (too high-spec device). 
     On the other hand, in order to carry out drug discovery, it is needed to treat a large amount of specimen at once. Hence, it has been desired to develop a screening system capable of performing a large-scale analysis with a small amount of cells. 
     PRIOR ART DOCUMENTS 
     Patent Literature 
     Patent Literature 1: Japanese Patent No. 4517125 
     Non Patent Literature 
     Non Patent Literature 1: Kizawa et, al., BBR 2017 
     SUMMARY OF INVENTION 
     Technical Problem 
     In order to carry out drug discovery, a screening system capable of performing a large-scale analysis with a small amount of cells has been desired. In addition, it has also been desired to develop a method for producing a simple and inexpensive cell structure that is for use in drug discovery tests. 
     Solution to Problem 
     The present inventor has conducted intensive studies directed towards achieving the aforementioned objects. As a result, the present inventor has successfully achieved the aforementioned objects by using a cell-holding container, in which the center on the bottom surface is formed as a protrusion and a recessed part is formed between the center and a side wall, thereby completing the present invention. 
     Specifically, the present invention is as follows.
     (1) A cell culture apparatus having a cell-holding container and a pinholder-shaped member comprising needle-shaped bodies arranged on a substrate, wherein   

     a protruding part is formed in the center of the bottom surface of the cell-holding container, a recessed part is formed between the center and a side wall, and through-holes through which the needle-shaped bodies penetrate are established on the bottom surface of the recessed part, 
     the needle-shaped bodies are arranged in correspondence with the positions of the through-holes, and 
     the pinholder-shaped member is arranged, such that a tip-side portion of each of the needle-shaped bodies penetrates through the corresponding through-hole from the bottom surface side or upper surface side of the cell-holding container.
     (2) A cell culture apparatus having a cell-holding container and a pinholder-shaped member comprising needle-shaped bodies arranged on a substrate, wherein   

     a protruding part is formed in the center of the bottom surface of the cell-holding container, and a recessed part is formed between the center and a side wall, 
     the needle-shaped bodies are arranged in correspondence with the positions of the bottom surface of the recessed part, and 
     the pinholder-shaped member is arranged, such that a tip of each of the needle-shaped bodies is directed from the upper surface side of the cell-holding container towards the bottom surface of the recessed part.
     (3) The cell culture apparatus according to the above (1) or (2), wherein a plurality of the cell-holding containers and a plurality of the pinholder-shaped members are arranged in the form of an array.   (4) The cell culture apparatus according to any one of the above (1) to (3), wherein the cell-holding container is subjected to a cell non-adhesive coating treatment.   (5) A method for producing a cell structure, comprising pouring a cell suspension into a cell-holding container of the cell culture apparatus according to any one of the above (1) to (4), and agglutinating the cells so that the cells cover the needle-shaped bodies.   (6) The method according to the above (5), wherein the cells are hepatocytes or myocardial cells.   (7) A cell testing method, comprising contacting a test substance with a cell structure produced by the method according to the above (5) or (6), and testing the toxicity of the test substance to the cells or the metabolic activity of the cells in the cell structure.   (8) The method according to the above (7), wherein the cells are hepatocytes or myocardial cells.   (9) A cell testing device, including the cell culture apparatus according to any one of the above (1) to (4).   (10) The device according to the above (9), wherein the cells are hepatocytes or myocardial cells.   

     Effect of the Invention 
     According to the present invention, it has become possible to produce a simple and inexpensive cell structure, and thereby, it has become possible to carry out drug discovery tests such as cytotoxicity. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a view showing one aspect of the cell culture apparatus of the present invention. 
         FIG. 2  includes central cross-sectional views of the cell culture apparatus of the present invention. 
         FIG. 3  is a view showing an aspect in which cells form spheroids, while covering needle-shaped bodies. 
         FIG. 4  is a view showing an aspect of producing a cell structure using the cell culture apparatus of the present invention. 
         FIG. 5  is a view showing an aspect in which a cell-holding container is slided in the longitudinal direction of needle-shaped bodies. 
         FIG. 6  is a view showing an aspect in which a cell structure is retained in a ring form at the tip portions of needle-shaped bodies. 
         FIG. 7  is a view showing an aspect in which the tips of needle-shaped bodies on a pinholder-shaped member are arranged from the upper surface of a cell-holding container towards a recessed part. 
         FIG. 8  is a view showing an aspect in which a plurality of the cell culture apparatuses of the present invention are arranged in the form of an array. 
         FIG. 9  is a view showing a cell testing device including the cell culture apparatus of the present invention. 
         FIG. 10  is a view showing an example of producing a cell structure according to the Example. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     The present invention provides a cell culture apparatus having a cell-holding container and a pinholder-shaped member comprising needle-shaped bodies arranged on a substrate, wherein 
     a protruding part is formed in the center of the bottom surface of the cell-holding container, a recessed part is formed between the center and a side wall, and through-holes through which the needle-shaped bodies penetrate are established on the bottom surface of the recessed part, 
     the needle-shaped bodies are arranged in correspondence with the positions of the through-holes, and 
     the pinholder-shaped member is arranged, such that a tip-side portion of each of the needle-shaped bodies penetrates through the corresponding through-hole from the bottom surface side or upper surface side of the cell-holding container. 
     An aspect of the cell culture apparatus of the present invention is shown in  FIG. 1 . 
       FIG. 1A  is a perspective view of the cell culture apparatus of the present invention. A cell culture apparatus  1  comprises a cell-holding container  10 , and a pinholder-shaped member  2  comprising needle-shaped bodies  21  arranged on a substrate  20 . 
     A protruding part  11  is formed in the center of the bottom surface of the cell-holding container  10 , and a recessed part  13  is formed between the center of the bottom surface of the cell-holding container  10  and a side wall  12 . A shape, in which the center of the bottom surface becomes a protruding part and forms a recessed part between the center and the side wall, is similar to, what is called, a Mexican hat. Accordingly, in the present description, the cell-holding container  10  is also referred to as a “Mexican hat type culture vessel” or is simply referred to as a “hat type culture vessel.” 
       FIG. 1B  is a plan view obtained by seeing a cell culture apparatus  10  from above. In this aspect, ten needle-shaped bodies  21  are arranged on a substrate  20 , and the needle-shaped bodies  21  are allowed to penetrate through through-holes  14  (as described later) of the cell-holding container  10  (the needle-shaped bodies  21  are arranged in correspondence with the positions of the through-holes  14 ). 
       FIG. 2  is a central cross-sectional view of a cell culture apparatus  1 . In  FIG. 2A , through-holes  14 , through which needle-shaped bodies  21  on a pinholder-shaped member  2  penetrate, are established on the bottom surface  13  of a cell-holding container  10 . Accordingly, the needle-shaped bodies  21  are arranged on a substrate  20 , so that the needle-shaped bodies can be in correspondence with the positions of the through-holes  14  that are, for example, in the normal directions of the substrate  20 .  FIG. 2B  shows an aspect, in which parts of needle-shaped bodies  21  on a pinholder-shaped member  2  are allowed to penetrate through the corresponding through-holes  14  from the bottom surface portions of a cell-holding container  10 . 
     In the present invention, the recessed part  13  of the cell-holding container  10  forms a cell-holding part that holds cells, and when a cell suspension is poured into the recessed part  13 , the cells simultaneously aggregate and form a spheroid, while covering a needle-shaped body. 
       FIG. 3  is a schematic view showing an aspect in which cells form a spheroid, while covering a needle-shaped body. 
       FIG. 3A  is a view showing an aspect of the formation of a spheroid in the absence of a needle-shaped body  21 , and scattered cells  32  in a cell suspension  31  simultaneously aggregate and form a spheroid  33 . In the present invention, as shown in  FIG. 3B , by using a needle-shaped body  21 , cells  32  form a spheroid  33 , while covering a needle-shaped body  21 . Since a plurality of the needle-shaped bodies  21  are arranged, for example, with equal intervals, the formed spheroids  33  are fused with one another, thereby producing a cell structure  34 . 
     Herein, the type of the cells used in the present invention is not particularly limited, and any given cells that form a spheroid can be used. Examples of the cells that form a spheroid may include undifferentiated cells such as stem cells (ES cells, cord blood-derived cells, undifferentiated mesenchymal stem cells, adult mesenchymal stem cells, etc.), and the differentiated cells thereof. Examples of tissues, from which the cells used herein are derived, may include articular cartilage, bone, adipose tissues, ligaments, tendons, teeth, auricle, nose, liver, pancreas, blood vessels, nerve, and heart. Among these, hepatocytes, myocardial cells, and the like are preferable. In addition, the spheroid does not always need to be formed as an aggregate of a single type of cell. The spheroid may also be formed from multiple types of cells (for example, a mixture of hepatocytes and vascular endothelial cells), as long as the cells are able to form the spheroid. 
     Moreover, a culture period required to form a spheroid and a cell structure is different depending on the size of the cell culture apparatus  1 . The culture period is approximately 2 days to 4 days under common culture conditions (for example, at 37° C., under 5% CO 2  atmosphere). 
     With regard to the material of the needle-shaped body  21 , the needle-shaped body made of stainless steel, polypropylene, nylon, etc. can be used. However, the material of the the needle-shaped body  21  is not limited thereto. 
     Moreover, the cell-holding container  10  has been preferably subjected to cell non-adhesive coating with fluorine, etc. (for example, a cell-holding container made of polydimethylsiloxane). However, a cell-holding container that has been subjected to fluorine processing or polyhydroxyethyl methacrylate polymer processing can also be used, and further, a cell-holding container made of an acrylic resin, an ABS resin, a polyester resin, a polycarbonate resin, polypropylene, polyethylene, polyacetal, polyether ether ketone, nylon, etc. can also be used. 
       FIG. 4  shows an aspect in which a cell structure  34  is produced using the cell culture apparatus  1  of the present invention, based on the mechanism of forming a cell structure  34 , as shown in  FIG. 3B . 
     First, a vessel  40  larger than the cell culture apparatus  1  of the present invention is filled with a culture solution, and the cell culture apparatus  1  is then placed in the vessel. The culture apparatus  1  has an aspect in which tip-side portions of needle-shaped bodies  21  on a pinholder-shaped member  2  penetrate through through-holes  14  of a cell-holding container  10 . When a cell suspension  31  is poured into a recessed part  13  of the cell-holding container  10 , the cells form spheroids, while covering the needle-shaped bodies  21 , and at the same time, the spheroids are fused with one another to form a cell structure  34 . In  FIG. 1  or  FIG. 4 , the periphery of the cell-holding container  10  has a circular shape, and the recessed part  13  circularly surrounds the circumference of a protruding part  11  in the center of the bottom surface. Thus, the produced cell structure has a ring shape. 
     In the present invention, the shape of the periphery of the cell-holding container  10  is not limited to a circular shape, but it can be a polygon such as a rectangle, a pentagon, a hexagon or an octagon. Moreover, then number of the needle-shaped bodies  21  (i.e., the number of through-holes  14 ) is not limited, either, and it can be, for example, 2 to 20. When the cell-holding container  10  has a circular shape, the diameter thereof or the diameter of the recessed part  13  is not particularly limited, either, and it can be, for example, 0.6 mm to 30.0 mm. 
       FIG. 5  shows an aspect in which a cell-holding container  10  is slided in the longitudinal direction of needle-shaped bodies  21  in the cell culture apparatus  1  of the present invention. 
     When the cells are cultured, the cell-holding container  10  is arranged, so that the tip-side portions of the needle-shaped bodies  21  slightly protrude from through-holes  14 , as shown in  FIG. 5A . After construction of a cell structure  34 , the cell-holding container  10  is slided downwards (on the side of a substrate  20 ) ( FIG. 5B ). Thereby, the cell structure  34  is retained in a ring form at the tip portions of the needle-shaped bodies  21  ( FIG. 6 ). Hence, by sliding the cell-holding container  10  downwards, the area of the cell structure  34  established on the cell-holding container  10  is reduced, so that an analysis can be promptly carried out on the cell structure  34 . For example, a test substance is placed into the cell-holding container  10  and/or the culture vessel  40 , in the form of the cell structure  34  shown in  FIG. 6 , and thereafter, the activity or movement of the cell structure  34 , the toxicity of the test substance to the cell structure  34 , etc. can be examined. 
       FIGS. 1 and 2 , etc. show an aspect in which the needle-shaped bodies  21  on the pinholder-shaped member  2  are allowed to penetrate from the bottom surface side of the cell-holding container  10  into the recessed part  13 . On the other hand,  FIG. 7  shows an aspect in which the tips of needle-shaped bodies  21  on a pinholder-shaped member  2  are arranged from the upper surface of a cell-holding container  10  towards a recessed part  13 . In this case, through-holes  14  do not need to be established, and the establishment of the through-holes  14  is arbitrary. In order to fix the needle-shaped bodies  21 , an aspect in which holes are established in the recessed part  13  of the cell-holding container  10  to such an extent that the holes do not penetrate (not shown in the figure) and the needle-shaped bodies  21  are inserted into the holes, may also be applied. As shown in  FIG. 7A , the pinholder-shaped member  2  is arranged in the direction opposite to the aspect shown in  FIG. 1 , and a cell suspension is then poured therein, followed by the formation of spheroids and the formation of the cell structure  34 . Thereafter, the direction of the pinholder-shaped member  2  is returned to the original direction (i.e, to face upwards), so as to achieve an aspect, in which the cell structures  34  are retained at the tips of the needle-shaped bodies  21 . Besides, in this case, the substrate  20  can be removed. 
       FIG. 8  is a view showing an aspect in which a plurality of the cell culture apparatuses  1  of the present invention are arranged in the form of an array. 
     In  FIG. 8 , the shape of a cell-holding container  10  is a square, and needle-shaped bodies  21  penetrate into 4 positions (4 corners) of a recessed part. Thus, by arranging the plurality of the cell culture apparatuses  1  of the present invention in the form of an array, a large number of cell structures  34  can be produced, and various types of tests can be carried out at once. Besides, with regard to the arrangement of the array, the array can be integrally molded. 
       FIG. 9  shows a view showing a cell testing device  9  including the cell culture apparatus  1  of the present invention. In the cell testing device  9 , a cell culture apparatus  1  is established on a substrate  90 , and a cell culture solution-supplying part  91  and a test substance-supplying part  92  are also established on the substrate  90 . In the cell testing device  9 , an electrode can be connected with needle-shaped bodies  21  (not shown in the figure). 
     Thereby, the metabolic activity of the cell structure, the pulsation of myocardial cells, or the like can be three-dimensionally examined. 
     Therefore, the present invention provides a cell testing method, which is characterized in that it comprises allowing a test substance to come into contact with a cell structure, and then testing the toxicity of the test substance to the cells or the metabolic activity of the cells. 
     Examples of the test substance may include naturally or artificially synthesized, various types of peptides, proteins (including enzymes and antibodies), nucleic acids (polynucleotides (DNA and RNA), oligonucleotides (siRNA, etc.), peptide nucleic acids (PNA), etc.), low molecular weight compounds, and polymeric organic compounds. 
     Moreover, the term “contact” means that a test substance is allowed to come into contact with the cell structure of the present invention. Examples of such contact may include: pouring a test substance into a culture vessel including a cell structure; and culturing a cell structure in a medium comprising a test substance. 
     EXAMPLES 
     Hereinafter, the present invention will be more specifically described in the following example. However, the following example is not intended to limit the scope of the present invention. 
     Example 1 
     Method 
     Four needles (0.17 mm) were raised on a hat type vessel (diameter: 4 mm) made of a polydimethylsiloxane (PDMS) resin ( FIG. 10A ), and a cell suspension (cell count: 5×10 6  cells) was then poured into the vessel ( FIG. 10B ). Human fibroblasts were used as cells herein. 
     Three days later, it was observed that the cells aggregated and covered the needles ( FIG. 10C ). 
     By sliding the hat type vessel downwards, a ring-shaped cell structure was retained at the tip of each needle ( FIGS. 10D  and E). In the present invention, the cell structure in this state can be directly used in a pharmacological test, but as shown in  FIG. 10F , it is also possible to remove the cell structure from the needle, and then, to subject it to a pharmacological test or to produce pathologic tissues therefrom. 
     DESCRIPTION OF REFERENCE NUMERALS 
     
         
           1 : Cell culture apparatus of the present invention,  2 : Pinholder-shaped member,  9 : Cell testing device,  10 : Cell-holding container,  11 : Protruding part,  12 : Side wall,  13 : Recessed part,  14 : Through-hole,  20 : Substrate,  21  Needle-shaped body,  31 : Cell suspension,  32 : Cell,  33 : Spheroid,  34 : Cell structure,  40 : Culture vessel,  90 : Substrate,  91 : Cell culture solution-supplying part,  92 : Test substance-supplying part