JP2010279321A - Cell reaction observation apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell reaction observation apparatus which can sufficiently reproduce the cell environment in an organism to allow accurate observation of the reactivity of cells to a chemical. <P>SOLUTION: The cell reaction observation apparatus 1 includes at least a main passage 5, branch passages communicating with the main passage 5 (a first branch passage 11, a second branch passage 12, a third branch passage 13, and a fourth branch passage 14), and pumps provided in the branch passages (a first pump 31, a second pump 32, a third pump 33, and a fourth pump 34). A culture medium 7 is made to flow out from the main passage 5 to the branch passages or made to flow from the branch passages into the main passage 5 by operating the pumps. A rotating flow is formed in the culture medium 7 in a state filled in the main passage 5 by controlling the operation timing of the pumps to hold a cell piece 6, filled in the main passage 5, in the center of the rotating flow. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cell reaction observation apparatus. Specifically, the present invention relates to a cell reaction observation apparatus capable of holding cells at a desired position in a culture solution.

  Conventionally, a cell reaction observation device has been used in a test process in drug discovery. A number of tests can be performed under the same conditions by performing drug reactivity tests on cells cultured in the cell reaction observation apparatus. In addition, the efficacy and toxicity of the new drug can be examined without conducting animal experiments.

  As a medium used for culturing cells, a gel-like medium having a high viscosity is usually used. For example, in the apparatus described in Patent Document 1, agar or gelatin is used as the medium. In addition, a fluid passage part is provided so as to be in contact with the culture medium, and gas or the like is supplied to the fluid passage part to exchange gas in the culture medium.

JP 2002-101868 A

  In order to accurately observe the reactivity or the like of a cell to a drug, it is necessary to make the cell environment in the apparatus and the cell environment in the living body match well. However, when agar or gelatin is used as a medium as in the apparatus described in Patent Document 1, it is difficult to accurately position cells. This is because the medium is solidified after the cells are mixed in a fluid state, so that the position of the cells deviates from the desired position until the medium is solidified. Moreover, the surrounding environment of the cells in the solidified medium cannot be freely controlled. Therefore, the environment reproduced in the apparatus is greatly different from that in the living body, and there is a problem that it is impossible to accurately observe the reactivity of the cells to the drug.

  The present invention has been made to solve the above-described problems, and a cell reaction observation apparatus capable of accurately observing the reactivity of a cell to a drug by well reproducing the cell environment in a living body. The purpose is to provide.

  In order to solve the above-mentioned problems, the cell reaction observation device according to the first aspect of the present invention includes a main channel that is a channel filled with a culture solution and a tributary that is a plurality of channels communicating with the main channel. A pump that is provided at the end of the branch channel opposite to the side that communicates with the main channel, and that allows the culture medium to flow into and out of the main channel via the branch channel, A pump that causes a swirl flow to be formed in the culture solution filled in the main flow path by inflow and outflow of the culture liquid, and that holds a cell piece injected into the main flow path at the center of the swirl flow.

  In addition to the configuration of the invention according to claim 1, in the cell reaction observation device according to the invention according to claim 2, the branch channel has two surface portions arranged opposite to each other among the inner wall surfaces constituting the main channel. A first branch channel and a second branch channel disposed adjacent to one surface portion, and a third branch channel and a fourth branch channel disposed on the other surface portion. And a third branch channel disposed at a position facing the first branch channel and a fourth branch channel disposed at a position facing the second branch channel.

  In addition to the configuration of the invention according to claim 2, the cell reaction observation device according to claim 3 is in communication with the main channel and for injecting a drug solution into the cell piece in the main channel. A first injection channel disposed between the first branch channel and the second branch channel in the one surface portion, and the third branch channel in the other surface portion. And a second injection channel disposed between the fourth branch channel and the fourth branch channel.

  In addition to the configuration of the invention according to any one of claims 1 to 3, the cell reaction observation device according to the invention according to claim 4 is characterized in that the swirl flow is formed in the culture solution in the main channel. In order to hold the piece at the center of the swirling flow, an operating means for performing switching control of the operation of the pump according to the position of the cell piece is provided.

  In addition to the configuration of the invention according to claim 4, in the cell reaction observation device according to the invention according to claim 5, the operating means first causes the culture solution to flow out from the main channel to the first branch channel. Then, the culture solution is caused to flow from the third branch channel to the main channel, and then the culture solution is caused to flow out from the main channel to the fourth branch channel, and then the culture medium is transferred from the second branch channel to the main channel. A liquid is allowed to flow in.

  In addition to the configuration of the invention according to claim 4 or 5, the cell reaction observation device according to the invention of claim 6 is a photographing means for photographing the cell piece, and photographing of the result photographed by the photographing means. Position specifying means for specifying the position of the cell piece based on the image, and the operating means controls the operation of the pump based on the position of the cell piece specified by the position specifying means. It is characterized by.

  The cell reaction observation apparatus according to the first aspect of the present invention includes a main channel, a plurality of branch channels communicating with the main channel, and a pump provided in the branch channel. The culture medium is flowed into and out of the main channel by the pump, and a swirling flow is formed in the culture solution filled in the main channel. This keeps the cell debris at the center of the swirl flow. Since the cell piece can be held at a desired position, the cell piece can be cultured in a state close to a biological system. Since the cell debris can be held in the medium, the reactivity of the cell debris can be accurately observed by quickly changing the environment around the cell debris.

  In addition, in the cell reaction observation device of the invention according to claim 2, in addition to the effect of the invention according to claim 1, the main flow passage is surrounded by the communication portion of the first branch passage to the fourth branch passage. It becomes possible to form a swirl flow in the portion.

  In addition, in the cell reaction observation device according to the invention of claim 3, in addition to the effect of the invention of claim 2, the chemical solution can be injected into the main channel via the first injection channel and the second injection channel. it can. This makes it possible to cause the drug solution to react with the cell pieces.

  In addition, in the cell reaction observation device according to the invention according to claim 4, in addition to the effect of the invention according to any one of claims 1 to 3, by performing switching control of the operation of the pump based on the position of the cell piece. It becomes possible to form a swirl flow in the culture solution filled in the main channel.

  In addition, in the cell reaction observation device according to the invention according to claim 5, in addition to the effect of the invention according to claim 4, first, the culture solution is allowed to flow out from the main channel to the first branch channel, and then from the third branch channel. The culture solution is caused to flow into the channel, then the culture solution is caused to flow from the main channel to the fourth branch channel, and then the culture solution is caused to flow from the second branch channel to the main channel. Thereby, a swirl flow can be easily formed in the culture solution filled in the main channel. Moreover, a cell piece can be induced | guided | derived to the part enclosed by the communication part of a 1st branch channel-a 4th branch channel.

  Further, in the cell reaction observation device according to the invention of claim 6, in addition to the effect of the invention of claim 4 or 5, the operation of the pump is controlled according to the position of the cell piece, and the main flow path is filled. A swirl flow can be formed in the culture solution. Cell debris can be easily guided to the portion surrounded by the communication portion of the first branch channel to the fourth branch channel.

It is a schematic diagram which shows the outline | summary of a physical structure of the cell reaction observation apparatus 1, and an operation principle. 4 is a right side view of the main body 2. FIG. 3 is a schematic diagram showing a flow of a culture solution 7 generated in the main channel 5. FIG. 3 is a schematic diagram showing a flow of a culture solution 7 generated in the main channel 5. FIG. 3 is a schematic diagram showing a flow of a culture solution 7 generated in the main channel 5. FIG. 3 is a schematic diagram showing a flow of a culture solution 7 generated in the main channel 5. FIG. 3 is a schematic diagram showing a flow of a culture solution 7 generated in the main channel 5. FIG. 2 is a block diagram showing an electrical configuration of the cell reaction observation device 1. FIG. It is a flowchart which shows the process in the case of forming a swirl flow. It is a schematic diagram which shows the mode of the flow of the culture solution 7 which generate | occur | produces in the main flow path 5 in a modification. It is a schematic diagram which shows a simulation result. It is a schematic diagram which shows the simulation result in a modification. It is a photograph which shows the cell reaction observation apparatus 1 made as an experiment. It is a figure which shows the main-body part 2 of the cell reaction observation apparatus 1 made as an experiment.

  Hereinafter, a cell reaction observation device 1 according to an embodiment of the present invention will be described with reference to the drawings. These drawings are used for explaining the technical features that can be adopted by the present invention, and the configuration of the apparatus described, the flowcharts of various processes, and the like are not intended to be limited thereto. It is just an illustrative example.

  First, with reference to FIG.1 and FIG.2, the outline | summary of the physical structure of the cell reaction observation apparatus 1 and an operation principle are demonstrated. The cell reaction observation device 1 includes a main body 2 and pumps (a first pump 31, a second pump 32, a third pump 33, and a fourth pump 34) connected to the main body 2. Although not shown, a camera 66 (see FIG. 8) capable of photographing the main body 2 and a PC 60 (see FIG. 8) for controlling the operation of the pump and the camera 66 are provided. The main body 2 is placed on a table with the front side in FIG.

  As shown in FIG. 1, a main channel 5 that is a channel filled with cell pieces 6 and a culture solution 7 is formed in the main body 2. Further, branch channels (first branch channel 11, second branch channel 12, third branch channel 13, and fourth branch channel 14) that are channels communicating with the main channel 5 are formed. In addition, an injection path (injection path 15, injection path 16) that is a flow path communicating with the main flow path 5 is formed. The main body 2 includes a main channel 5, a branch channel (first branch channel 11, second branch channel 12, third branch channel 13, fourth branch channel 14), and injection path (injection path 15, injection path 16). A cover 3 (see FIG. 2) covering from above is provided. As a material constituting the main body 2, a general material used as a cell reaction observation device such as plastic, resin, glass, rubber, or the like can be used. In addition, by using a transparent material as the cover 3 of the main body 2, the cells filled in the apparatus can be easily observed. The branch channels (first branch channel 11 to fourth branch channel 14) and pumps (first pump 31 to fourth pump 34) are tubes (first tube 21, second tube 22, third tube 23, fourth). The tubes 24) are connected to each other. Details will be described below.

  The main flow path 5 will be described. The main channel 5 is a hollow channel formed in the main body 2. The main channel 5 is filled with cell pieces 6 and a culture solution 7. The shape of the flow path is not particularly limited. The diameter of the main flow path 5 is not particularly limited as long as the cell pieces 6 can be filled.

  The branch channel (the first branch channel 11 to the fourth branch channel 14) will be described. The branch flow path is arranged in communication with two face portions arranged opposite to each other among the inner wall surfaces constituting the main flow path 5. The first branch channel 11 and the second branch channel 12 are arranged on one surface portion of the inner wall surface. The third branch channel 13 and the fourth branch channel 14 are disposed on the other surface portion. The third branch channel 13 is arranged at a position facing the first branch channel 11. The fourth branch channel 14 is disposed at a position facing the second branch channel 12. In the example shown in FIG. 1, the first branch channel 11 is disposed on the right side of the inner wall surface of the main channel 5 on the lower side of the drawing. The second branch channel 12 is disposed on the left side of the inner wall surface below the paper surface. A third branch channel 13 is arranged on the right side of the inner wall surface on the upper side of the drawing. The fourth branch channel 14 is disposed on the left side of the inner wall surface on the upper side of the drawing.

  A pump (first pump 31 to fourth pump 34) is connected to an end portion of each branch flow channel on the side opposite to the side communicating with the main flow channel 5. The pump and the branch flow path are connected by tubes (first tube 21 to fourth tube 24). In the example shown in FIG. 1, a first pump 31 is connected to the first branch channel 11, a second pump 32 is connected to the second branch channel 12, a third pump 33 is connected to the third branch channel 13, A fourth pump 34 is connected to the four branch flow paths 14. The first branch channel 11 and the first pump 31 are connected by a first tube 21. The second branch flow path 12 and the second pump 32 are connected by a second tube 22. The third branch flow path 13 and the third pump 33 are connected by a third tube 23. The fourth branch flow path 14 and the fourth pump 34 are connected by a fourth tube 24.

  By operating the pump, the culture solution 7 is allowed to flow out from the main channel 5 to the branch channel (first branch channel 11 to fourth branch channel 14) or from the branch channel (first branch channel 11 to fourth branch channel 14). The culture solution 7 can flow into the channel 5. In the present embodiment, a swirling flow is formed in the culture solution 7 filled in the main channel 5 by operating the pump. And the cell piece 6 with which the main flow path 5 was filled is hold | maintained in the center of the formed swirl | flow. Details will be described later.

  The injection path (injection paths 15 and 16) will be described. The injection path is disposed so as to communicate with two face portions disposed opposite to each other among the inner wall surfaces constituting the main flow path 5. An injection path 15 is disposed on a surface portion of the inner wall surface between the third branch channel 13 and the fourth branch channel 14. The injection path 16 is disposed at a position on the inner wall surface between the first branch path 11 and the second branch path 12 and facing the injection path 15. When the predetermined drug is reacted with the cell piece 6 in the main channel 5, the drug to be reacted is injected into the injection path 16. The drug injected from the injection path 16 comes into contact with the cell debris 6 in the main flow path 5 and is then discharged from the injection path 15. Thereby, the reactivity test of the drug with respect to the cell piece 6 can be easily performed.

  The state of the flow of the culture solution 7 when a swirling flow is formed in the culture solution 7 in the main channel 5 will be described with reference to FIGS. Note that a line segment 55 extending in the left-right direction is defined at the upper and lower central portions of the main channel 5 shown in FIG. A line segment 56 extending in the vertical direction on the paper passes through the midpoint between the first branch channel 11 and the second branch channel 12 and the midpoint between the third branch channel 13 and the fourth branch channel 14. Define And among each area | region of the main flow path 5 divided | segmented by the line segment 55 and the line segment 56, the upper right part is the 1st quadrant 51, the lower right part is the 2nd quadrant 52, the lower left part is the 3rd quadrant 53, The upper left part is defined as the fourth quadrant 54. The first quadrant 51 corresponds to the vicinity of the communication portion of the third branch channel 13 in the main channel 5. Similarly, the second quadrant 52 is near the communication portion of the first branch flow path 11, the third quadrant 53 is near the communication portion of the second branch flow path 12, and the fourth quadrant 54 is near the communication portion of the fourth branch flow path 14. Equivalent to each other. Of the flow paths communicating with the main flow path 5, the injection paths (injection paths 15 and 16) are omitted in the following.

  First, as shown in FIG. 3, the first pump 31 (see FIG. 1) is operated to inhale the culture solution 7. As a result, the culture solution 7 in the main channel 5 flows out from the main channel 5 to the first branch channel 11 (arrow 25 in FIG. 3). With the outflow of the culture solution 7 from the main channel 5, a flow in the direction toward the first branch channel 11 (line 35 in FIG. 3) is generated in the first quadrant 51 and the second quadrant 52.

  In this state, it is assumed that the cell piece 6 is injected into the fourth quadrant 54 of the main channel 5. In this case, a force (right direction in FIG. 3) toward the first quadrant 51 is applied to the cell piece 6 located in the fourth quadrant 54 by the flow generated in the culture solution 7. As a result, the cell piece 6 located in the fourth quadrant 54 is guided to the first quadrant 51. Further, the cell piece 6 that has moved to the first quadrant 51 is subjected to a force toward the second quadrant 52 (downwardly to the right in FIG. 3) by the flow generated in the culture solution 7. Thereby, the cell piece 6 located in the first quadrant 51 is guided to the second quadrant 52.

  Next, the operation of the first pump 31 is stopped. Next, as shown in FIG. 4, the third pump 33 (see FIG. 1) is operated, and the culture solution 7 is discharged. As a result, the culture solution 7 flows from the third branch channel 13 into the main channel 5 (arrow 26 in FIG. 4). Along with the inflow of the culture solution 7 into the main flow path 5, a flow in the direction from the second quadrant 52 to the third quadrant 53 is generated in the second quadrant 52 and the third quadrant 53 (line 36 in FIG. 4). .

  In this case, a force toward the third quadrant 53 (in the diagonally lower left direction in FIG. 4) is applied to the cell piece 6 located in the second quadrant 52. Thereby, the cell piece 6 located in the second quadrant 52 is guided to the third quadrant 53.

  Next, the operation of the third pump 33 is stopped. Next, as shown in FIG. 5, the fourth pump 34 (see FIG. 1) is operated to inhale the culture solution 7. As a result, the culture solution 7 in the main channel 5 flows out from the main channel 5 to the fourth branch channel 14 (arrow 27 in FIG. 5). Along with the outflow of the culture solution 7 from the main flow path 5, a flow in the direction toward the fourth branch flow path 14 (line 37 in FIG. 5) is generated in the third quadrant 53 and the fourth quadrant 54.

  In this case, a force toward the fourth quadrant 54 is applied to the cell piece 6 located in the third quadrant 53 (upwardly obliquely in the left direction in FIG. 5). As a result, the cell piece 6 located in the third quadrant 53 is guided to the fourth quadrant 54.

  Next, the operation of the fourth pump 34 is stopped. Next, as shown in FIG. 6, the second pump 32 (see FIG. 1) is operated, and the culture solution 7 is discharged. As a result, the culture solution 7 flows from the second branch channel 12 into the main channel 5 (arrow 28 in FIG. 6). As the culture solution 7 flows into the main flow path 5, a flow in the direction from the fourth quadrant 54 to the first quadrant 51 is generated in the fourth quadrant 54 and the first quadrant 51 (line 38 in FIG. 6). .

  In this case, a force toward the first quadrant 51 (upward and downward in FIG. 6) is applied to the cell piece 6 located in the fourth quadrant 54. Thereby, the cell pieces 6 arranged in the fourth quadrant 54 are guided to the first quadrant 51.

  Next, the operation of the second pump 32 is stopped. Next, as shown in FIG. 3, the first pump 31 (see FIG. 1) is operated to inhale the culture solution 7. Then, the above process is repeatedly executed. As a result, as shown in FIG. 7, a swirling flow is formed in the culture solution 7 in the main channel 5 (line 39 in FIG. 7). The cell piece 6 is held at the center of the swirl flow (the origin portion of the first quadrant 51 to the fourth quadrant 54).

  With reference to FIG. 8, the electrical configuration of the cell reaction observation apparatus 1 will be described. As shown in FIG. 8, the cell reaction observation device 1 includes a PC 60 that performs operation control of a pump and a camera 66. The PC 60 includes a CPU 61 that performs overall control, a ROM 62 that stores a BIOS, a RAM 63 that temporarily stores various data, and a hard disk drive 64 (hereinafter referred to as “HDD 64”) that stores various programs. The CPU 61, the ROM 62, the RAM 63, and the HDD 64 are electrically connected. The CPU 61 can access storage areas of the ROM 62, RAM 63, and HDD 64.

  The cell reaction observation device 1 includes a CD-ROM drive 65. A CD-ROM 651 inserted into the CD-ROM drive 65 stores a program executed by the CPU 61 and the like. When the cell reaction observation apparatus 1 is introduced, these various programs are set up and stored in the HDD 64 from the CD-ROM 651.

  The cell reaction observation device 1 includes a camera 66. The CPU 61 and the camera 66 are electrically connected. The CPU 61 can recognize an image photographed by the camera 66. In the present embodiment, the camera 66 is disposed so as to face the main body 2 (see FIG. 1). The camera 66 images the main channel 5, the branch channel, and the cell pieces 6 (see FIG. 1) filled in the main channel 5.

  The cell reaction observation apparatus 1 includes the first pump 31, the second pump 32, the third pump 33, the fourth pump 34 described above, and the pump driver 67 that can operate the first pump 31 to the fourth pump 34. ing. The CPU 61 and the pump driver 67 are electrically connected so that the CPU 61 can control the operation of the first pump 31 to the fourth pump 34. The pump driver 67 and the first pump 31 to the fourth pump 34 are electrically connected to each other.

  With reference to the flowchart of FIG. 9, the process performed in CPU61 of PC60 with which the cell reaction observation apparatus 1 is provided is demonstrated. In this processing, after the main flow path 5 is filled with cell pieces 6 (see FIG. 1), the keyboard or mouse (not shown) of the PC 60 is operated, and an instruction to start the cell reaction observation apparatus 1 is input by the user. In this case, it is activated and executed by the CPU 61.

  First, photographing by the camera 66 (see FIG. 8) is started (S10). Next, the first pump 31 (see FIG. 1) is controlled. Then, the culture solution 7 in the main channel 5 flows out to the first branch channel 11 (S11). By the operation of the first pump 31, the cell pieces 6 in the culture solution 7 are guided to the first quadrant 51 (see FIG. 3). Furthermore, the cell piece 6 is induced | guided | derived to the 2nd quadrant 52 (refer FIG. 3).

  An image photographed by the camera 66 is acquired while the first pump 31 is in operation. The acquired image is analyzed, and the position of the image portion corresponding to the cell piece 6 is specified. It is determined whether the specified position of the cell piece 6 is included in the second quadrant 52 (see FIG. 3) (S13). When not included in the second quadrant 52 (S13: NO), the process returns to S13, and the position of the cell piece 6 is continuously specified.

  When the position of the cell piece 6 is specified as described above, a part of the image portion of the cell piece 6 specified by the image analysis is included in the second quadrant 52. It is determined that the position is included in the second quadrant 52. Similar determinations are made below.

  When the specified position of the cell piece 6 is included in the second quadrant 52 (S13: YES), the operation of the first pump 31 is stopped (S15). Next, the third pump 33 (see FIG. 1) is controlled. Then, the culture solution 7 flows from the third branch channel 13 into the main channel 5 (S17). By the operation of the third pump 33, the cell pieces 6 in the culture solution 7 are guided to the third quadrant 53 (see FIG. 4).

  An image photographed by the camera 66 is acquired while the third pump 33 is in operation. The acquired image is analyzed, and the position of the image portion corresponding to the cell piece 6 is specified. It is determined whether the image position of the identified cell piece 6 is included in the third quadrant 53 (see FIG. 3) (S19). If it is not included in the third quadrant 53 (S19: NO), the process returns to S19, and the position of the cell piece 6 is continuously specified.

  When the specified position of the cell piece 6 is included in the third quadrant 53 (S19: YES), the operation of the third pump 33 is stopped (S21). Next, the fourth pump 34 (see FIG. 1) is controlled. Then, the culture solution 7 in the main channel 5 flows out to the fourth branch channel 14 (S23). By the operation of the third pump 33, the cell pieces 6 in the culture solution 7 are guided to the fourth quadrant 54 (see FIG. 3) (see FIG. 5).

  An image photographed by the camera 66 is acquired in a state where the fourth pump 34 is operated. The acquired image is analyzed, and the position of the image portion corresponding to the cell piece 6 is specified. It is determined whether the image position of the specified cell piece 6 is included in the fourth quadrant 54 (see FIG. 3) (S25). If not included in the fourth quadrant 54 (S25: NO), the process returns to S25, and the position of the cell piece 6 is continuously specified.

  When the identified position of the cell piece 6 is included in the fourth quadrant 54 (S25: YES), the operation of the fourth pump 34 is stopped (S27). Next, the second pump 32 (see FIG. 1) is controlled. Then, the culture solution 7 flows from the second branch channel 12 into the main channel 5 (S29). By the operation of the second pump 32, the cell pieces 6 in the culture solution 7 are guided to the first quadrant 51 (see FIG. 6).

  An image photographed by the camera 66 is acquired in a state where the second pump 32 is operated. The acquired image is analyzed, and the position of the image portion corresponding to the cell piece 6 is specified. It is determined whether the image position of the specified cell piece 6 is included in the first quadrant 51 (see FIG. 3) (S31). When it is not included in the first quadrant 51 (S31: NO), the process returns to S31 and the position of the cell piece 6 is continuously specified.

  When the position of the specified cell piece 6 is included in the first quadrant 51 (S31: YES), the operation of the second pump 32 is stopped (S33). And it returns to S11 and the above-mentioned process is repeatedly performed.

  As described above, the culture solution 7 is caused to flow into and out of the main channel 5 by the pump, and a swirling flow is formed in the culture solution 7 filled in the main channel 5. Thereby, the cell piece 6 can be held at the center of the swirling flow. Since the cell piece 6 can be held at a desired position, the cell piece 6 can be cultured in a state close to a biological system. Since a stable flow can be formed, a highly reproducible test can be performed. Since the cell piece 6 can be held in the medium, the reactivity of the cell piece 6 can be accurately observed by quickly changing the environment around the cell piece 6. The cells can be held in a desired position and the cells can be made independent and the reactivity can be observed.

  Also, the first pump 31 is activated (the culture fluid 7 flows out from the main flow path 5 to the first branch flow path 11) → (the first pump 31 is stopped) → the third pump 33 is activated (the culture is performed from the third branch flow path 13 to the main flow path 5) Liquid 7 flows in) → (Third pump 33 stopped) → Fourth pump 34 operates (the culture fluid 7 flows out from the main channel 5 to the fourth branch channel 14) → (Fourth pump 34 stops) → Second pump 32 operates By repeating (the culture solution 7 flows into the main channel 5 from the second branch channel 12), a swirl flow can be easily formed in the culture solution 7 filled in the main channel 5. Moreover, the cell piece 6 can be induced | guided | derived to the part enclosed by the communication part of the 1st branch flow path 11-the 4th branch flow path 14. FIG.

  The pump control described above is executed based on the position of the cell piece 6 specified based on the image taken by the camera 66. Accordingly, the cell piece 6 can be reliably held at a desired position in the main flow path 5 without depending on the size of the cell piece 6 or the viscosity of the culture solution 7.

  The injection path 16 in FIG. 1 corresponds to the “first injection path” of the present invention, and the injection path 15 corresponds to the “second injection path” of the present invention. The CPU 61 that performs the processes of S11, S15, S17, S21, S23, S27, S29, and S33 in FIG. 9 corresponds to the “operation means” of the present invention. The camera 66 in FIG. 8 corresponds to the “photographing means” of the present invention. The CPU 61 that performs the processes of S13, S19, S25, and S31 in FIG. 9 corresponds to the “position specifying means” of the present invention.

  In addition, this invention is not limited to the said embodiment, A various change is possible. In the above-described embodiment, the front side of the sheet in FIG. 1 is defined as the upper side of the main body 2 of the cell reaction observation device 1, and is assumed to be placed on the table in this direction. However, the present invention is not limited to this arrangement. For example, the upper side of the paper surface in FIG. 1 may be defined as the upper side of the main body 2 of the cell reaction observation device 1 and placed on the table in this direction.

  In the above-described embodiment, the cell reaction observation device 1 has a configuration in which the first branch channel 11 to the fourth branch channel 14 communicate with the main channel 5. However, the present invention is not limited to this configuration. Therefore, for example, a configuration in which the set of the first branch channel 11 to the fourth branch channel 14 is repeatedly arranged in the main channel 5 may be employed. By adopting such a configuration, a plurality of cell pieces can be arranged at different locations, and thus it becomes possible to perform a reactivity test between a plurality of cell pieces arranged at different locations.

  In the above-described embodiment, when the position of the cell piece 6 is specified, if a part of the image portion of the cell piece 6 specified by the image analysis is included in the specific quadrant, the cell piece It was determined that position 6 was included in a specific quadrant. However, the present invention is not limited to this method. Therefore, for example, when all the image portions of the specified cell piece 6 are in a specific quadrant, it may be determined that the image portion is included in the specific quadrant.

  In the above-described embodiment, once the swirl flow is formed and the cell pieces 6 are held at the origin of the first quadrant 51 to the fourth quadrant 54, the operation of the first pump 31 to the fourth pump 34 is stopped. You may let them. In addition, after that, when the cell piece 6 held at the origin part moves and enters a state included in any of the first quadrant 51 to the fourth quadrant 54, the first pump 31 to the fourth pump 34. Either of them may be operated. Specifically, when the state is included in the first quadrant 51, the first pump 31 is operated (inhaled), and when the state is included in the second quadrant 52, the third pump When the pump 33 is operated (discharged) and is included in the third quadrant 53, the fourth pump 34 is operated (suctioned) and is included in the fourth quadrant 54. May operate (discharge) the second pump 32.

  In the above-described embodiment, the first quadrant 51 to the fourth quadrant 54 are defined for the main flow path 5, and the operation of the pump is controlled by determining whether or not the cell pieces 6 are included in these regions. Is held at the origin of the first quadrant 51 to the fourth quadrant 54. However, the present invention is not limited to this method. For example, the pump is operated and controlled by defining a different area and determining whether the cell piece 6 is included in this area. Thereby, the cell piece 6 can be held in a predetermined region. Hereinafter, modifications of the present invention will be described.

  A modification of the present invention will be described with reference to FIG. In the modification, as shown in FIG. 10, a square 81 whose center of gravity is the origin in the first quadrant 51 to the fourth quadrant 54 (see FIG. 3) of the main flow path 5 is defined. A line segment 82 extending from the upper right corner of the square 81 in the right direction on the paper surface is defined. Similarly, a line segment 83 extending from the lower right corner to the lower side of the page, a line segment 84 extending from the lower left corner to the left side of the page, and a line segment 85 extending from the upper left corner to the upper side of the page are defined. In the main flow path 5, the area within the square 81 is the A area 86, the area divided by the line segment 82 and the line segment 85 is the area divided by the B quadrant 87, the line segment 82, and the line segment 83. Are defined as the C quadrant 88, the area delimited by the line segment 83 and the line segment 84 is defined as the D quadrant 89, and the area delimited by the line segment 84 and the line segment 85 is defined as the E quadrant 90. In the modification, the cell pieces 6 filled in the main flow path 5 are guided and held in the A area 86.

  An outline of the operation control method of the pump when the cell piece 6 is held in the area A 86 will be described. First, imaging of the main channel 5 by the camera 66 is started. The first pump 31 (see FIG. 1) is operated, and the culture solution 7 is caused to flow from the main channel 5 to the first branch channel 11. As a result, a flow (line 91) in the direction from the Bth quadrant 87 to the Cth quadrant 88 is generated. In this state, when the cell piece 6 is injected into the main channel 5, the cell piece 6 is guided to the C quadrant 88 via the B quadrant 87.

  From the result of photographing by the camera 66, the position of the cell piece 6 is determined. When it is determined that the cell piece 6 is included in the C quadrant 88, only the third pump 33 (see FIG. 1) is operated, and the culture solution 7 is caused to flow from the third branch channel 13 into the main channel 5. Stop other pumps. As a result, a flow (line 92) in the direction from the C quadrant 88 to the D quadrant 89 is generated. In this state, the cell piece 6 is guided from the C quadrant 88 to the D quadrant 89.

  On the other hand, when it is determined that the cell piece 6 is included in the D quadrant 89, only the fourth pump 34 (see FIG. 1) is operated, and the culture solution 7 is caused to flow out from the main channel 5 to the fourth branch channel 14. Stop other pumps. As a result, a flow (line 93) in the direction from the D quadrant 89 to the E quadrant 90 is generated. In this state, the cell piece 6 is guided from the D quadrant 89 to the E quadrant 90.

  On the other hand, when it is determined that the cell piece 6 is included in the E quadrant 90, only the second pump 32 (see FIG. 1) is operated, and the culture solution 7 is caused to flow into the main channel 5 from the second branch channel 12. Stop other pumps. As a result, a flow (line 94) in the direction from the E quadrant 90 to the B quadrant 87 is generated. In this state, the cell piece 6 is guided from the E quadrant 90 to the B quadrant 87.

  On the other hand, when it is determined that the cell piece 6 is included in the A region 86, the operation of all the pumps is stopped. As a result, the cell piece 6 is held in the A region 86. When the cell piece 6 moves and deviates from the A area 86 and is included in any of the B quadrant 87 to the E quadrant 90, the above-described processing is executed according to the area including the cell piece 6. Is done.

  By executing the above processing, the cell pieces 6 injected into the main flow path 5 are gradually guided to the A area 86 and held. Thus, in a modification, the cell piece 6 can be hold | maintained in a predetermined area | region (A area | region 86).

A first embodiment of the present invention will be described with reference to the drawings. In the first example, the flow formed in the culture solution 7 when the culture solution 7 is flowed into and out of the main flow channel 5 based on the method described in the above-described embodiment (particularly FIG. 9). The situation was simulated by a simulator. Then, it was confirmed that a swirling flow was formed in the main flow path 5. Hereinafter, (1) simulation method and principle, (2) simulation environment, and (3) simulation results will be described.
(1) Method and principle

As an equation governing the flow of the culture solution 7, an incompressible viscous flow equation of motion (Navier-Stokes equation) was adopted. The Navier-Stokes equation is shown in Equation 1. In Equation 1, u represents the flow rate of the culture solution 7 flowing in and out of the main channel 5. ρ indicates the fluid density of the culture solution 7. v represents the kinematic viscosity coefficient of the culture solution 7. b shows an external force. By substituting each parameter and numerically integrating Equation 1, a simulation for confirming the operation of the cell reaction observation device 1 can be performed.

A formula for calculating the Reynolds number Re indicating the nature of the flow in the fluid is shown in Formula 2. In Equation 2, L represents a typical diameter of the main flow path 5. U represents a typical flow rate of the culture solution 7. In this simulation, we considered the Reynolds number when the swirl flow is well formed. Details will be described later.
(2) Simulation environment

  A self-made fluid simulator was used as a simulator. The diameter of the main flow path 5 was set to 100 μm, and the diameter of the branch flow paths (the first branch flow path 11 to the fourth branch flow path 14) was set to 50 μm. Further, the distance between adjacent branch channels (between the first branch channel 11 and the second branch channel 12 and between the third branch channel 13 and the fourth branch channel 14) was set to 100 μm. The first pump 31 to the fourth pump 34 were set to be controlled based on the flowchart shown in FIG.

For the physical properties of the culture solution 7, the physical properties of “Leibovitz ′S 1-15 Medium” manufactured by Introgen Co., Ltd. were referred to. The kinematic viscosity coefficient was set to 15.4 × 10 ⁻⁶ m ² / s. As the cell piece 6, a hepatocyte was assumed, and the size was set to 20 μm.

In view of the diameter of the main flow path 5 and the kinematic viscosity coefficient of the culture solution 7, among the parameters based on the Reynolds number Re, U is 1 mm / s, L is 100 μm, and v is 10 ⁻⁶ m ^2. / S. As a result, the Reynolds number Re was set to 0.1.
(3) Simulation results

  The simulation results are shown in FIG. A line 47 in FIG. 11 indicates the swirl flow formed in the main flow path 5 by the operation control of the pumps (the first pump 31 to the fourth pump 34). This result shows that the swirl flow of the culture solution 7 is well formed in the main channel 5. In FIG. 11, a line 48 indicates a moving path of the cell pieces 6 filled in the main flow path 5. This result shows that the cell pieces 6 filled in the main flow path 5 are guided to the center of rotation and are held well.

  Among the above parameters, when the Reynolds number was set to be greater than 0.1, a phenomenon was observed in which turbulent flow was generated in the flow of the culture solution 7 and the cell pieces 6 were not induced in the central portion of the swirling flow. From this result, it has been clarified that the cell piece 6 (see FIG. 3 and the like) can be favorably guided to the turning center in the main flow path 5 by setting the Reynolds number to at least 0.1 or less. . That is, by reducing the Reynolds number and making it a macroscale phenomenon, the generated flow velocity pattern becomes a laminar flow, so that the cell debris 6 can be well guided to the turning center in the main flow path 5.

  A second embodiment of the present invention will be described with reference to the drawings. In the second embodiment, the state of the movement of the cell pieces 6 injected into the main channel 5 when the culture solution 7 is flowed into and out of the main channel 5 based on the method shown in the above-described modification. Simulated by simulator. Since the simulation method, the principle, and the simulation environment are the same as those in the first embodiment described above, description thereof will be omitted.

  The simulation results are shown in FIG. A line 95 in FIG. 12 indicates the swirl flow formed in the main flow path 5 by the operation control of the pumps (the first pump 31 to the fourth pump 34, see FIG. 1). This result shows that the swirl flow of the culture solution 7 is well formed in the main channel 5. In FIG. 12, a line 96 indicates a movement path of the cell piece 6 (see FIG. 3 and the like) filled in the main channel 5. This result shows that the cell pieces 6 filled in the main flow path 5 are satisfactorily guided to the A region 86 at the turning center.

  A third embodiment of the present invention will be described with reference to the drawings. In the third example, the cell reaction observation device 1 was actually created. The created cell reaction observation apparatus 1 is shown in FIG. In FIG. 13, “DSZT-70IFL” manufactured by Carton was used as the camera 66. A pump was created by combining a motor “graphite brush 20w” made by Maxon, a slider “LM guide actuator KR20” made by THK, and a micro syringe “1701LT” made by Hamilton, and the first pump 31 to the fourth pump 34 were made.

  FIG. 14 shows a schematic diagram of the main body 2 in the cell reaction observation apparatus 1 that has been created. In FIG. 14, the diameter of the main flow path 5 was set to 100 μm. The diameters of the first branch channel 11 to the fourth branch channel 14 were 50 μm. The distance between the first branch channel 11 to the second branch channel 12 and the distance between the third branch channel 13 to the fourth branch channel 14 were set to 100 μm. The diameter of the tubes (first tube 21 to fourth tube 24) is 1.5 mm, and each of the branch channels (first branch channel 11 to fourth branch channel 14) is connected to the branch channel. The end portion was circular (71 to 74 in FIG. 14). The diameter of the circular portion was 1.5 mm, the same as the diameter of the tube. Moreover, the both ends of the main flow path 5 were made circular so that the cell pieces 6 could be easily filled into the main flow path 5 of the tube. The diameter of the circular portion was 1.5 mm.

  It was confirmed that a swirl flow can be formed in the culture solution 7 by controlling the operation of the pump using the cell reaction observation device 1 created as described above. It was also confirmed that the cell piece 6 can be held at the center of the swirling flow.

DESCRIPTION OF SYMBOLS 1 Cell reaction observation apparatus 5 Main flow path 6 Cell piece 7 Culture solution 11 First branch flow path 12 Second branch flow path 13 Third branch flow path 14 Fourth branch flow path 15 and 16 Injection path 31 First pump 32 Second pump 33 Third Pump 34 Fourth pump

Claims (6)

A main channel that is a channel filled with a culture solution;
A branch channel which is a plurality of channels communicating with the main channel;
A pump that is provided at an end of the branch channel opposite to the side communicating with the main channel, and that causes the culture solution to flow into and out of the main channel via the branch channel, A cell reaction observation apparatus comprising: a pump that forms a swirling flow in the culture solution filled in the main flow path by inflow and outflow and holds a cell piece injected into the main flow path at the center of the swirling flow. The branch channel is
The first branch channel and the second branch channel that are disposed adjacent to one of the surface portions, and are arranged in communication with two surface portions of the inner wall surface constituting the main channel. A third branch channel and a fourth branch channel disposed on the surface portion, the third branch channel disposed at a position facing the first branch channel, and a position facing the second branch channel; The cell reaction observation apparatus according to claim 1, further comprising at least a fourth branch channel.   A flow path that communicates with the main flow path and for injecting a drug solution into the cell debris in the main flow path, and is between the first branch flow path and the second branch flow path of the one surface portion. The first injection path disposed and a second injection path disposed between the third branch channel and the fourth branch channel among the other surface portions are provided. The cell reaction observation device according to 1.   An operation means for performing switching control of the operation of the pump according to the position of the cell debris in order to form the swirl flow in the culture solution in the main channel and hold the cell debris at the center of the swirl flow; The cell reaction observation apparatus according to any one of claims 1 to 3, further comprising a cell reaction observation apparatus. The operating means is
First, the culture solution is allowed to flow from the main channel to the first branch channel, then the culture solution is allowed to flow from the third branch channel to the main channel, and then the culture solution is transferred from the main channel to the fourth branch channel. The cell reaction observation apparatus according to claim 4, wherein the culture solution is allowed to flow out from the second branch channel and then into the main channel. Photographing means for photographing the cell piece;
A position specifying means for specifying the position of the cell piece based on a photographed image obtained by the photographing means;
The operating means is
The cell reaction observation device according to claim 4 or 5, wherein operation of the pump is controlled based on a position of the cell piece specified by the position specifying means.