JP2011050344A - Apparatus for culturing cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for culturing cells which is capable of improving the proliferation efficiency of cells cultured in a liquid in a culturing container. <P>SOLUTION: The apparatus for culturing cells includes detecting means 30 for detecting the density distribution of a cell group cultured in the liquid in the culturing container, agitating means 28 for agitating the cells in the culturing container by oscillating the culturing container, environment-regulating means 12 for regulating the environment of the culturing container during a detecting time and an agitating time to a previously set environment, and action-regulating means 81 for setting the patterns of the oscillation by the agitating means based on the density distribution detected by the detecting means. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cell culture apparatus capable of observing cells cultured in a liquid in a culture container.

  When performing subculture of cells, it is necessary to replace the culture solution in the culture vessel or to inoculate the cells into a new culture solution. Recently, subculture for stem cells for regenerative medicine has also been performed, and a cell culture apparatus that improves workability of subculture has also been proposed (see Patent Document 1).

JP 2004-016194 A

  In such subculture, depending on the relationship between the adhesion strength of the cells to the bottom surface of the culture vessel (hereinafter referred to as “adhesion surface”) and the adhesion strength between the cells, some of the cells grow in the process of proliferation. A group of cells may aggregate and non-uniform density (hereinafter referred to as “aggregation unevenness”) may occur in the culture vessel. If culturing is continued in this state, the cell growth efficiency in the culture vessel may be significantly reduced because more cells in the densely aggregated cells cannot absorb sufficient nutrients and die. .

  An object of this invention is to provide the cell culture apparatus which can improve the proliferation efficiency of the cell cultured in the liquid in a culture container.

  The cell culture device of the present invention includes a detection means for detecting a density distribution of a group of cells cultured in a liquid in a culture container, and agitation for stirring the cells in the culture container by swinging the culture container. Means for controlling the environment of the culture vessel at the time of detection and at the time of stirring to a preset environment, and based on the density distribution detected by the detection means, And an operation control means for setting a pattern.

  An object of the present invention is to provide a cell culture device capable of improving the proliferation efficiency of cells cultured in a liquid in a culture vessel.

The figure which shows the outline of a cell culture apparatus. The figure which shows the outline of the cell culture apparatus which opened the outer door. Schematic which shows the structure inside a temperature-controlled room. Schematic which shows the structure of a stirring apparatus. The functional block diagram which shows the electric constitution of a cell culture apparatus. Schematic which shows the structure of an observation unit. An example of a culture vessel. The operation | movement flowchart of CPU at the time of registration. Example of whole image acquired at the time of registration. Outline of agitation pattern for eliminating sowing unevenness. An example of a stirring pattern for eliminating sowing unevenness. The operation | movement flowchart of CPU at the time of observation. The example of the whole image acquired at the time of observation. The example of the area | region extracted by step S25. An example of an agglomerated region. The figure explaining step S25. The figure explaining area division in Step S28. The figure explaining the information calculated in step S28. Outline of agitation pattern for eliminating aggregation unevenness. An example of a stirring pattern for eliminating aggregation unevenness. The operation | movement flowchart of CPU at the time of stirring processing. The figure explaining a tilt stage.

  Hereinafter, the structure of the cell culture apparatus of this embodiment is shown in detail. As shown in FIGS. 1 to 3, the cell culture device 10 includes a first housing 11 that cultures a sample such as a microorganism or a cell, and a second housing 12 that houses a control unit 13. In the assembled state of the cell culture device 10, the first housing 11 is disposed on the upper portion of the second housing 12.

  The first housing 11 includes a temperature-controlled room 15 whose interior is covered with a heat insulating material. The temperature-controlled room 15 communicates with the outside through a front opening 16 formed on the front surface of the first housing 11. The front opening 16 of the first housing 11 is closed by an inner door 17 and two outer doors 18a and 18b. Packing etc. are provided in the peripheral part of the back side of this inner door 17 and outer doors 18a and 18b, and the inside of temperature-controlled room 15 is kept airtight when each door is closed. That is, when only the inner door 17 is closed, or when not only the inner door 17 but also the two outer doors 18a and 18b are closed, the cell culture device 10 can be moved from the outside to the inside of the temperature-controlled room 15. Inflow of heat and outflow of heat from the inside of the temperature-controlled room 15 to the outside of the cell culture device 10 are prevented. Note that the inside of the temperature-controlled room 15 is managed so as to have predetermined environmental conditions (temperature, humidity, carbon dioxide concentration, etc.) by a temperature adjustment device, a spray device, etc. (not shown).

  Of the outer doors 18a and 18b described above, the outer door 18b is provided with a loading door 22. The carry-in door 22 is opened when the carry 25 containing the culture vessel 31 is carried into the carry installation table 26 or when the carry 25 installed on the carry installation table 26 is carried out. When the loading door 22 is opened, the small door 24 provided on the inner door 17 is exposed through the opening 23. The inner door 17 and the small door 24 are made of glass or transparent synthetic resin, and even when the outer doors 18a and 18b are opened, the internal environmental conditions do not change abruptly. . By opening the small door 24 provided on the inner door 17, the carry 25 in which the culture vessel 31 is stored can be carried into the carry installation base 26, or the carry 25 installed on the carry installation base 26 can be carried out. It becomes possible. In addition, the carry 25 can store three culture containers 31 up and down at a predetermined interval, for example.

  Inside the temperature-controlled room 15, a carry installation table 26, a stocker 27, a container transport mechanism 28, a stirring device 29, an observation unit 30 and the like are arranged. In addition, although illustration is abbreviate | omitted, the inside of this temperature-controlled room 15 is provided with the culture medium exchange mechanism which performs culture medium exchange, the seeding mechanism which seeds | inoculates a cell, etc.

  As described above, the carry 25 is placed on the carry installation base 26. Although not shown, the carry installation base 26 is provided with a guide rail on the side surface. This guide rail regulates the moving direction of the carry 25 carried in or out by entering a guide groove provided at an interval in which the guide rail enters the lower surface of the carry 25. The extending direction of the guide groove is orthogonal to the direction in which the culture vessel 31 is carried into the carry 25 or the direction in which the culture vessel 31 is carried out from the carry.

  The stocker 27 is disposed on the left side of the temperature-controlled room 15 when viewed from the front of the first housing 11. The stocker 27 has a plurality of shelves, and a culture vessel 31 is stored in each shelf. In the stocker 27, a position for storing the culture vessel 31 (hereinafter referred to as storage position) is set in advance. Each storage position is associated with an identification number for identifying it from other storage positions, and the observation schedule, image data, and the like of the culture container 31 are managed for each culture container 31 by this identification number.

  Examples of the culture container 31 placed on the stocker 27 include a dish, a well plate, and a flask. Each culture vessel 31 holds a sample to be cultured such as a cell together with a culture solution. These culture vessels 31 are handled in a state where they are positioned and fixed on a transparent tray-like holder 32 when performing cell culture and cell observation using this cell culture apparatus.

  The container transport mechanism 28 is disposed substantially at the center of the temperature-controlled room 15 when viewed from the front of the first housing 11. The container transport mechanism 28 transports the culture container 31 stored in the carry 25 to the stocker 27, or transports the culture container 31 stored in the stocker 27 to the carry 25, the observation unit, the stirring device 29, or the like. . The position of each part of the container transport mechanism 28 is monitored by the control unit 13 via an encoder or the like.

  The stirrer 29 is provided to eliminate seeding unevenness that occurs immediately after seeding cells in the culture solution of the culture vessel 31 and cell aggregation unevenness that occurs during the culture process.

  Next, a detailed configuration of the stirring device 29 will be described with reference to FIG. As shown in FIG. 4, the stirring device 29 includes an X stage 35, a Y stage 36, a rotary stage 37, and a base 38. The X stage 35 is disposed at the uppermost part of the stirring device, and a plate 39 for positioning the culture vessel 31 is attached to the upper surface of the X stage 35. The holder 32 to which the culture vessel 31 is fixed is positioned and fixed on the upper surface of the plate 39. The X stage 35 is driven in the X-axis direction by the X-axis drive unit 40 and moves in the X-axis direction.

  The Y stage 36 is disposed between the X stage 35 and the rotary stage 37. The Y stage 36 is driven by the Y-axis drive unit 41 and moves in the Y-axis direction. When the Y stage 36 moves in the Y axis direction, the X stage 35 also moves in the Y axis direction together with the Y stage 36.

  The rotary stage 37 is disposed between the Y stage 36 and the base 38. The rotation stage 37 is driven by the rotation drive unit 42 and rotates around the rotation axis L (θ direction). Along with the rotation of the rotary stage 37, the X stage 35 and the Y stage 36 also rotate.

  In FIG. 4, in order to eliminate the complexity of the drawing, the X-axis drive unit 40, the Y-axis drive unit 41, and the rotation drive unit 42 are replaced with an X stage 35, a Y stage 36, a rotation stage 37, and a base 38. However, in reality, it is assumed that it is arranged inside the stirring device 29.

  In the stirring device 29 having such a configuration, the culture vessel 31 is swung by at least one of the movement of the X stage 35, the movement of the Y stage 36, the rotation of the rotation stage 37, or a combination thereof, and the culture vessel The cells in the culture medium in 31 are agitated. Note that the X-axis drive unit 40, the Y-axis drive unit 41, and the rotation drive unit 42 of the stirrer 29 drive and control the X stage 35, the Y stage 36, and the rotation stage 37, respectively, according to instructions from the control unit 13 described later.

  The entire stirring device 29 is housed inside the scattering prevention case 45, and can be removed together with the scattering prevention case 45 and taken out of the temperature-controlled room 15. This scattering prevention case 45 is used when a culture solution (including cells in the culture solution) is scattered from a gap (for example, a gap formed between the vessel and the lid) of the culture vessel 31 during the stirring process. Has the function of preventing the problem of scattering to other mechanisms of the temperature-controlled room 15.

  In addition, an opening 46 is provided in the upper part of the side surface of the scattering prevention case 45 that faces the container transport mechanism 28. By providing this opening 46, the culture container 31 transported by the container transport mechanism 28 is carried into the stirring device 29 disposed inside the scattering prevention case 45, and the culture container positioned at the stirring device 29 is provided. 31 can be carried out of the scattering prevention case 45. An introduction door 47 is attached to the opening 46.

  The introduction door 47 is rotatable between a position where the opening 46 is shielded (shielding position) and a position where the opening 46 is exposed (exposing position). The introduction door 47 is normally held at the shielding position. The introduction door 47 is used for carrying out the culture vessel 31 stirred by the stirring device 29 to the outside of the scattering prevention case 45, or for placing the culture vessel 31 subjected to the stirring treatment on the stirring device 29. When it is carried into the inside of 45, the opening / closing mechanism 48 is rotated from the blocking position to the exposing position. Note that when the introduction door 47 is in the shielding position, the airtightness of the diffusion preventing case 45 is maintained.

  A weight sensor (not shown) is provided between the plate 39 of the stirring device 29 and the X stage 35. If this weight sensor detects a change in the weight of the culture vessel 31 before and after the stirring process, it is possible to detect the presence or absence of scattering of the culture solution during the stirring process (details will be described later).

  Further, in the stirring device 29, a connector for electrical wiring (not shown) protrudes from the mounting surface side of the base 28, and the connector is inserted into the bottom surface portion of the scattering prevention case 45. The opening (not shown) is formed. When the stirring device 29 and the scattering prevention case 45 are attached to the temperature-controlled room 15, the connector of the stirring device 29 is for electric wiring provided on the side of the temperature-controlled room 15 through the opening of the scattering prevention case 45. It is inserted into a connector (not shown). Accordingly, each of the X-axis drive unit 40, the Y-axis drive unit 41, the rotation drive unit 42, the opening / closing mechanism 48, and the weight sensor (not shown) is electrically connected to the control unit 13.

  Further, a gas exchange connector 49 is provided on the side surface opposite to the side surface on which the opening 46 of the scattering prevention case 45 is provided. A connector 52 provided at one end of the tube 51 of the compressor 50 (see FIG. 2) is fitted into the connector 49. When the compressor 50 is operated, the air pressure inside the scattering prevention case 45 becomes lower than the air pressure inside the temperature-controlled room 15. The compressor 50 is driven in response to an instruction from the control unit 13.

  The main body of the observation unit 30 is installed inside the second housing 12, and the upper part of the observation unit 30 is arranged so as to enter the inside from the lower side of the first housing 11. The observation unit 30 is arranged on the right side of the temperature-controlled room 15 when viewed from the front of the first housing 11. The observation unit 30 is disposed by being fitted into the opening on the bottom surface of the first housing 11. The observation unit 30 acquires a partial image of the culture vessel 31, an entire image, or the like (details of the observation unit 30 will be described later).

  Next, the electrical configuration of the cell culture device 10 will be described with reference to FIG. As shown in FIG. 5, the main body portion 58 of the observation unit 30 and the control unit 13 are stored in the second housing 12. An operation panel 65 including a monitor 65a and an input unit 65b is disposed on the front surface of the second housing 12. The input unit 65b includes a keyboard and a mouse. A computer 67 can also be connected to the control unit 13 via a communication line 66.

  The control unit 13 is connected to the container transport mechanism 28, the stirring device 29, the opening / closing mechanism 48, the observation unit 30, the monitor 65a of the operation panel 65, and the input unit 65b. Then, the control unit 13 comprehensively controls each part of the cell culture device 10 according to a predetermined program. As an example, the control unit 13 automatically determines the observation schedule of the culture vessel 31 according to the type of the culture vessel 31 or the type of cells input by the operation of the input unit 65b of the operation panel 65 or the operation of the computer 67. To do. Then, the control unit 13 automatically executes the observation sequence of the culture vessel 31 based on the determined observation schedule.

  Here, the control unit 13 includes a communication unit 71, a database unit 72, a memory 73, and a CPU 74. The communication unit 71, the database unit 72, and the memory 73 are each connected to the CPU 74. The communication unit 71 performs data transmission / reception with the computer 67 outside the cell culture device 10 via a wireless or wired communication line.

  The memory 73 stores a program and calculation data related to the above observation sequence. The calculation data includes information (such as a look-up table described later) necessary for the CPU 74 to determine a stirring pattern described later (stirring pattern for eliminating seeding unevenness, stirring pattern for eliminating aggregation unevenness).

  In the database unit 72, a management file 75 is prepared for each culture vessel 31, and the management file 75 of each culture vessel 31 includes an observation schedule of the culture vessel 31 and environmental conditions (temperature, humidity, History information of carbon dioxide concentration, stirring pattern, etc.), image data of the culture vessel 31 and the like are appropriately written. A common identification number is assigned to each culture vessel 31 and the management file 75 corresponding thereto.

  The CPU 74 is a processor that executes various arithmetic processes of the control unit 13. The CPU 74 has functions of an image analysis unit 81 and a stirring pattern setting unit 82. The image analysis unit 81 has a function of performing image analysis processing on the image acquired by the observation unit 30, and the stirring pattern setting unit 82 has a function of setting and adjusting the stirring pattern of the stirring device 29 (theses Details of the function will be described later.)

  Next, the configuration of the observation unit 30 will be described with reference to FIG. As shown in FIG. 6, the observation unit 30 includes a sample stage 347, a first illumination unit 351, a second illumination unit 352, a microscopic observation unit 353, and a container observation unit 354.

  Among these, the microscopic observation unit 353 is built in the main body portion 349, and the illumination unit 351 is disposed at a position of the arm 348 facing the microscopic observation unit 353. The microscopic observation unit 353 and the first illumination unit 351 constitute an observation system for phase difference observation.

  The container observation unit 354 is housed in the arm 348, and the second illumination unit 352 is disposed at a position facing the container observation unit 354 in the main body portion 349. These container observation part 354 and the 2nd illumination part 352 comprise the observation system for whole observation.

  The sample stage 347 is made of a transparent member, and the culture vessel 31 is placed thereon. The sample stage 347 is composed of a high-precision stage, and moves the culture vessel 31 in the horizontal direction (XY direction), thereby observing the culture vessel 31 with a phase difference observation microscope (microscopic observation unit 353 and first illumination unit 351). ) Or the optical path of the observation system for overall observation (the container observation unit 354 and the second illumination unit 352).

  The sample stage 347 can adjust the focus of the microscope for phase difference observation by changing the positional relationship in the Z direction between the culture vessel 31 and the microscope for phase difference observation. Moreover, the sample stage 347 can change the observation point of the microscope for phase difference observation by changing the positional relationship in the XY direction between the culture vessel 31 and the microscope for phase difference observation.

  The observation unit 30 can capture the entire image of the culture vessel 31 in a state where the culture vessel 31 is inserted into the optical path of the observation system for overall observation (the container observation unit 354 and the second illumination unit 352).

  In addition, the observation unit 30 captures a partial enlarged phase difference image of the culture vessel 31 in a state in which the culture vessel 31 is inserted into the optical path of a phase difference observation microscope (microscopic observation unit 353 and first illumination unit 351). can do.

Note that the combination of the objective lens 361 and the phase filter 362 of the microscopic observation unit 353 can be switched between a plurality of combinations having different observation magnifications. For example, the combination of the objective lens 361 and the phase filter 362 includes a combination for double observation (objective lens 361 _low and phase filter 362 _low ) and a combination for quadruple observation (objective lens 361 _high and phase filter 362 _high ). Switch between.

The observation unit 30 can acquire a phase difference image (low-magnification phase difference image) with a magnification of 2 in a state where the objective lens 361 _low and the phase filter 362 _low are set in the optical path, and the objective lens 361 _high and the phase filter 362. A phase difference image (high magnification phase difference image) with a magnification of 4 can be acquired in a state where _high is set in the optical path.

  Next, the culture container 31 of this embodiment is demonstrated based on FIG. In the present embodiment, the culture vessel 31 is assumed to be a transparent dish with a lid as shown in FIGS. The culture vessel 31 is filled with the culture solution 31b, and cells are seeded over almost the entire area of the culture solution 31b during subculture. The cells immediately after seeding are floating without adhering to the bottom surface (adhesion surface) of the culture vessel 31 and other cells as shown in FIG. 7A, and the aforementioned seeding unevenness is the density of cells in this state. It is unevenness.

  Thereafter, as the culture proceeds, the cells adhere to the bottom surface (adhesion surface) of the culture vessel 31 and proliferate as shown in FIG. In the state where the proliferation has progressed, many cells adhere to the bottom surface (adhesion surface) of the culture vessel 31 or other cells. The above-mentioned aggregation unevenness is the density unevenness of cells in this state.

  Next, the operation of the control unit 13 (the operation of the CPU 74) when registering the culture vessel 31 will be described with reference to FIG. Hereafter, each step of FIG. 8 is demonstrated in order. At the time of disclosure of this flow, it is assumed that the culture vessel 31 immediately after the passage is stored in the carry 25 and set on the carry installation base 26. This culture vessel 31 is a culture vessel immediately after being subjected to passage processing by the above-described medium exchange mechanism and seeding mechanism, or a culture vessel immediately after being subjected to passage processing by the user's hand.

  Step S11: The CPU 74 gives an instruction to the container transport mechanism 28 to move the culture container 31 set on the carry installation base 26 to the observation unit 30. The container transport mechanism 28 transports the culture container 31 to a predetermined position on the sample stage 347 of the observation unit 30. In addition, the mounting position of the culture vessel 31 on the sample stage 347 is positioned as determined in advance by a positioning pin or the like.

  When the user inputs the type (container type) of the culture container 31 and the type (cell type) of the cells seeded in the culture container 31 via the input unit 65 or the like, the CPU 74 determines the input container type and The observation schedule of the culture vessel 31 is determined according to the combination with the cell type. The observation schedule includes an observation period of the culture vessel 31 and observation timing (observation frequency) within the observation period.

  Then, when the management file 75 dedicated to the culture vessel 31 is created in the database unit 72, the CPU 74 writes the inputted vessel type and cell type and the determined observation schedule into the management file 75.

  Step S12: The CPU 74 instructs the observation unit 30 to acquire the entire image of the culture vessel 31. However, the whole image here is an entire image that can visualize the density distribution of cells in the culture vessel 31. Therefore, it is assumed here that the entire image is an entire image obtained by phase difference observation. The observation unit 30 moves the sample stage 347 in the X and Y directions, thereby inserting the culture vessel 31 into the optical path of the microscopic observation unit 353 and changing the combination of the objective lens 361 and the phase filter 362 to a combination for quadruple observation, for example. Set. However, in this state, the phase difference can be observed not only in the whole culture vessel 31 but only in a part. Therefore, the observation unit 30 repeatedly acquires phase difference images while moving the sample stage 347 stepwise, thereby acquiring a plurality of partial images in which different regions of the culture vessel 31 are copied, and these partial images. Is sent to the CPU 74. The image analysis unit 81 of the CPU 74 creates a whole image showing the whole culture vessel 31 by appropriately joining the plurality of partial images, and displays the whole image on the monitor 65a (see FIG. 9).

  In this whole image, an almost whole image of the culture vessel 31 is accommodated, and the center of the culture vessel 31 coincides with the center of the whole image. Since the whole image is a phase difference observation image, not only the outline of the culture vessel 31 but also the seeded cells are shown. The brightness of the area where the cells are present at a high density on the whole image is relatively high, and the brightness of the area where the cells are present at a low density is relatively low. Here, it is assumed that the brightness of the outline of the culture vessel 31 is higher than the brightness of the region where the cells are floating at the highest density. In FIG. 9, a region with high luminance is represented with a high density, and a region with low luminance is represented with a low concentration. The relationship between brightness and density is the same in the following figures.

  Step S13: The image analysis unit 81 of the CPU 74 performs binarization processing on the entire image acquired in step S12, and extracts an area where the luminance is within the threshold range from the entire image. However, the lower limit of the threshold range is a value between the brightness of the area where cells are present at high density and the brightness of the area where cells are present at low density in the expected seeding unevenness. The upper end value of the threshold range is set to a value between the brightness of the contour of the culture vessel 31 and the lower end value. Therefore, the image analysis unit 81 finds a region having the largest size among the extracted regions, and calculates the size of the region as an index of the degree of sowing unevenness.

  Step S14: The CPU 74 determines whether or not the size calculated in step S13 exceeds a threshold value. If it exceeds, the CPU 74 regards that sowing unevenness to be eliminated has occurred, and proceeds to step S15. If not exceeded, it is considered that there is no seeding unevenness to be eliminated, and the process proceeds to step S19.

Step S15: The stirring pattern setting unit 82 of the CPU 74 refers to the container type information stored in the management file 75, and sets the stirring pattern of the stirring device 29 to a stirring pattern suitable for the container type. However, where agitation pattern set is stirred pattern for sowing unevenness eliminated, for example, as shown in FIG. 10, the linear oscillating in the X-axis direction D _X, and a linear oscillating in the Y-axis direction D _X It is a combination.

Specifically, the stirring pattern setting unit 82 in this step writes, for example, a driving waveform as shown in the upper part of FIG. 11 to the memory of the X-axis driving unit 40 of the stirring device 29 and writes it to the memory of the Y-axis driving unit 41. For example, a drive waveform as shown in the middle part of FIG. 11 is written, and a drive waveform (flat drive waveform) as shown in the lower part of FIG. In FIG. 11, the horizontal axis t is time, the vertical axis V _X is the speed of the X stage 35, the vertical axis V _Y is the speed of the Y stage 36, and the vertical axis Vθ is the angular speed of the rotary stage 37. (The same applies to FIG. 20 described later.)

  These driving waveforms include a linear oscillation wa that applies an external force for agitation in the + X axis direction to the culture vessel 31, a linear oscillation wb that applies an agitation force in the −X axis direction to the culture vessel 31, and the culture. It is a waveform for performing in order a linear rocking wc that applies a stirring force in the + Y-axis direction to the vessel 31 and a linear rocking wd that applies a stirring force in the -Y-axis direction to the culture vessel 31.

  The shape of the drive waveform is common between these linear swings wa to wd, and the individual drive waveforms suddenly increase the speed of the stage immediately after the start of the swing, and then slowly decrease, and the stage stops. It reverses the moving direction of the stage at the time and slowly returns the stage to the state of the start of rocking, and applies stirring force to the cells in the culture vessel 31 during the rapid acceleration period at the beginning of rocking Can do.

  It should be noted that the rocking strength of each of the linear rocking wa to wd (here, the magnitude of acceleration immediately after the rocking starts) is set to a predetermined value (a value for eliminating seeding unevenness). Further, the number of swings of each of the linear swings wa to wd is also set to a predetermined value (a value for eliminating seeding unevenness). In addition, the swing amplitude of each of the linear swings wa to wd (here, the length of the acceleration period immediately after the start of swing) is also set to a predetermined value (a value for eliminating seeding unevenness).

  Step S16: The CPU 74 gives an instruction to the opening / closing mechanism 48 to open the introduction door 47 of the stirring device 29, and gives an instruction to the container transport mechanism 28 to move the culture vessel 31 to the stirring device 29. The opening / closing mechanism 48 sets the rotation position of the introduction door 47 to the aforementioned exposure position, and the container transport mechanism 28 transports the culture container 31 from the opening 46 opened in that state to the inside of the stirring device 29, Mount on the holder 32. In addition, the mounting position of the culture vessel 31 with respect to the plate 39 is positioned as determined in advance by the holder 32. Hereinafter, when the culture vessel 31 is positioned with respect to the plate 39 and each of the X stage 35, the Y stage 36, and the rotation stage 37 is in the reference state (non-driven state), the rotation axis L is positioned at the center of the culture vessel 31. Assume that

  Thereafter, when the CPU 74 issues an instruction to the opening / closing mechanism 48 to close the introduction door 47, the opening / closing mechanism 48 returns the introduction door 47 to the shielding position so that the stirring process can be started.

  Step S17: The CPU 74 executes a stirring process according to the stirring pattern being set (FIG. 11). Details of the stirring process will be described later. According to the stirring pattern during setting (FIG. 11), the culture solution in the culture vessel 31 is stirred uniformly over the X-axis direction and the Y-axis direction.

  Step S18: The CPU 74 gives an instruction to the opening / closing mechanism 48 to open the introduction door 47 of the stirring device 29, and gives an instruction to the container transport mechanism 28 to move the culture vessel 31 to the sample stage 347 of the observation unit 30. The opening / closing mechanism 48 sets the rotation position of the introduction door 47 to the aforementioned exposure position, and the container transport mechanism 28 takes out the culture container 31 from the opening 46 and transports it to a predetermined position on the sample stage 347. In addition, the mounting position of the culture vessel 31 on the sample stage 347 is positioned as determined in advance by a positioning pin or the like. Thereafter, the CPU 74 instructs the opening / closing mechanism 48 to close the introduction door 47 of the transport mechanism 29. The opening / closing mechanism 48 returns the introduction door 47 to the shielding position again, and returns to step S12.

  Step S19: The CPU 74 writes the entire image data acquired in the immediately preceding step S12 into the management file 75 of the culture vessel 31 after associating it with the current date and time.

  Step S20: The CPU 74 gives an instruction to the container transport mechanism 28 to store the culture container 31 in the stocker 27. The container transport mechanism 28 stores the culture container 31 placed on the sample table 347 in the stocker 27. This completes the registration flow.

  Next, the operation of the control unit 13 during the observation of the culture vessel 31 (the operation of the CPU 74) will be described with reference to FIG. Hereafter, each step of FIG. 12 is demonstrated in order. Here, the first observation is performed after the elapse of the fixing time (about 2 hours) necessary for the cells to adhere to the adhesive surface after the end of the flow in FIG. It is assumed that it is performed every predetermined time (for example, every 24 hours) sufficiently longer than the fixing time.

  Step S21: The CPU 74 compares the observation schedule written in the management file 75 with the current date and time to determine whether or not the observation time has come. When the observation time has come, the process proceeds to step S22, and when the observation time has not come, the process waits.

  Step S22: The CPU 74 turns on the initial flag. This initial flag is a flag for indicating whether or not the stirring to be performed is re-stirring.

  Step S23: The CPU 74 gives an instruction to the container transport mechanism 28 to transport the culture container 31 to the observation unit 30. The container transport mechanism 28 transports the culture container 31 stored in the stocker 27 to a predetermined position on the sample stage 347 of the observation unit 30. In addition, the mounting position of the culture vessel 31 on the sample stage 347 is positioned as determined in advance by a positioning pin or the like.

  Step S24: The CPU 74 acquires the entire image of the culture vessel 31 in the same procedure as in Step S12 described above, and displays the entire image on the monitor 65a (FIG. 13). In this whole image, cells aggregated inside the culture vessel 31 and cells floating inside the culture vessel 31 are shown. The brightness of the area where the cells are present at a high density on the whole image is relatively high, and the brightness of the area where the cells are present at a low density is relatively low. Here, it is assumed that the brightness of the outline of the culture vessel 31 is lower than the brightness of the region where the cells are aggregated at the highest density.

  Step S25: The image analysis unit 81 of the CPU 74 performs a binarization process on the entire image acquired in Step S24, and extracts an area where the luminance exceeds the threshold value from the entire image (FIG. 14). . However, the threshold value is set to a value between the brightness of the outline of the culture vessel 31 and the brightness of the region where the cells are aggregated at the highest density. Therefore, the image analysis unit 81 regards the area having the largest size among the extracted areas as an area (aggregation area) Aa in which cells are aggregated at a high density and in a wide range (FIG. 15). The size is calculated as an indicator of the degree of aggregation unevenness.

  Step S26: The CPU 74 determines whether or not the size calculated in step S25 exceeds a threshold value. If the size is exceeded, the CPU 74 regards that aggregation unevenness to be eliminated has occurred, and proceeds to step S27. If not, it is considered that there is no aggregation unevenness to be eliminated, and the process proceeds to step S36.

  Step S28: The stirring pattern setting unit 82 of the CPU 74 sets the stirring pattern of the stirring device 29 by the following procedures (a) to (e).

(A) stirring pattern setting unit 82, as shown in FIG. 16, and calculates the center coordinates of aggregation region Aa in the entire image _{(X 1, Y} 1), as shown in FIG. 17, the center coordinates _(X 1, Y ₁ ) Assuming a plurality of line segments (here, four line segments shifted by 90 °) radially extending in different directions at equal angles from ₁ ), the entire image is divided into a plurality of partial regions by these line segments. (Here, it is divided into _four partial areas A ₁ , A ₂ , A ₃ , A ₄ ).

(B) agitating the pattern setting unit 82, a luminance average value of the partial area A ₁ in the entire image, the average luminance value of the partial area A _2, the brightness average value of the partial area A _3, the average brightness of the partial region A ₄ Each of the partial areas A ₁ , A ₂ , A ₃ , and A ₄ is regarded as a non-aggregated area An. In the calculation of the average brightness value in this procedure (b), it is desirable that the pixels in the aggregated area Aa are excluded from the averaging target.

(C) The stirring pattern setting unit 82 calculates the center coordinates (X ₁ ′, Y ₁ ′) of the non-aggregation area An, and the center of the non-aggregation area An from the center coordinates (X ₁ , Y ₁ ) of the aggregation area Aa. coordinates _{(X 1 ', Y 1'} ) obtains a unit vector _{D 1} toward obtains a vector _{D 1X} an X component of the unit vector _{D 1,} and a vector _{D 1Y} is Y-component of the unit vector _{D 1} ( FIG. 18).

(D) The stirring pattern setting unit 82 places the rotation axis L of the stirring device 29 at the center of the aggregation region Aa by applying the center coordinates (X ₁ , Y ₁ ) of the aggregation region Aa to a predetermined conversion formula. It calculates the amount of movement _{L Y} moving amount _{L X} and Y stage 36 of the X stage 35 necessary. As a result, all the information on the aggregation unevenness to be eliminated is collected.

(E) The stirring pattern setting unit 82 refers to the container type and cell type information stored in the management file 75, determines the stirring pattern of the stirring device 29, the combination of the container type and the cell type, and the aggregation unevenness described above. Set a stirring pattern suitable for However, agitation pattern set here is stirred pattern for eliminating agglomeration unevenness, for example, as shown in FIG. 19, combined with rotation swinging around the center of the aggregate area Aa, and a linear oscillating the D ₁ direction It is a thing.

  Specifically, the stirring pattern setting unit 82 in the procedure (e) writes a drive waveform as shown in the upper part of FIG. For example, a drive waveform as shown in the middle part of FIG. 20 is written, and a drive waveform as shown in the lower part of FIG.

These drive waveforms are applied to the culture vessel 31 and the linear movement wa for shifting the culture vessel 31 so that the rotation axis L coincides with the center of the aggregation region Aa while keeping the external force applied to the culture vessel 31 to a minimum. On the other hand, by applying an external force in the + θ direction, a rotational swing wb that imparts a stirring force in the + θ direction to the cells, and by applying an external force in the -θ direction to the culture vessel 31, the stirring force in the -θ direction is applied to the cells. The rotation swing wc applied to the cell and the linear swing wd that applies the stirring force in the −D ₁ direction to the cell by applying an external force in the −D ₁ direction to the culture vessel 31 are sequentially performed.

The drive waveform (the drive waveform in the X-axis direction and the Y-axis direction) relating to the linear movement wa is calculated from the movement amounts L _X and L _Y calculated in the procedure (d), and the drive waveform (Y-axis relating to the linear movement wd). Drive waveform in the direction and the Y-axis direction) is calculated from the vectors D _1X and D _1Y calculated in step (c).

  In addition, the drive waveforms of the rotation swing wb and the rotation swing wc increase the angular velocity of the stage abruptly immediately after the start of the swing, and then slowly decrease it. When the stage stops, the rotation direction of the stage is changed. The stage is slowly reversed and returned to the posture at the time of starting rocking, and stirring force can be applied to the cells in the culture vessel 31 during the rapid acceleration period at the beginning of rocking.

  Further, the drive waveform of the linear swing wd is rapidly increased immediately after the swing is started and then slowly decreased. When the stage is stopped, the moving direction of the stage is reversed, and the drive waveform at the start of the swing is reversed. The stage is slowly returned to the position.

  In addition, each of the rotational swing wb, the rotational swing wc, and the linear swing wd (here, the magnitude of acceleration immediately after the start of the swing) is set to a value suitable for the combination of the container type and the cell type. Is done. Information about what value is suitable for which combination is stored in advance in the memory 73 in the form of, for example, a lookup table, and the stirring pattern setting unit 82 in this step performs the lookup. It is assumed that the swing strength can be set immediately by referring to the table.

  In addition, the number of rotations wb, rotation wc, and linear swing wd is set to a value suitable for the combination of container type and cell type. However, FIG. 20 shows an example in which the number of times of each swing is 1. Information about what value is suitable for which combination is stored in advance in the memory 73 in the form of, for example, a lookup table, and the stirring pattern setting unit 82 in this step performs the lookup. It is assumed that the number of swings can be set immediately by referring to the table.

  In addition, the magnitude of the oscillation amplitude (ie, the length of the initial acceleration period) of each of the rotation oscillation wb, the rotation oscillation wc, and the linear oscillation wd is set to a value suitable for the combination of the container type and the cell type. The Information about what value is suitable for which combination is stored in advance in the memory 73 in the form of, for example, a lookup table, and the stirring pattern setting unit 82 in this step performs the lookup. It is assumed that the swing amplitude can be set immediately by referring to the table.

  Step S28: The CPU 74 determines whether or not the initial flag is turned on. If it is turned on, the CPU 74 proceeds to step S29 to immediately start the stirring process, and if it is turned off, adjusts the stirring pattern being set. Therefore, the process proceeds to step S34.

  Step S29: The CPU 74 stores the culture vessel 31 in the stirring device 29 in the same procedure as in Step S16 described above.

Step S30: The CPU 74 executes a stirring process with the stirring pattern being set (FIG. 20) (details of the stirring process will be described later). According to the stirring pattern being set (FIG. 20), the center of the rotation and swing is set to the center of the aggregation region Aa, and therefore, it adheres to other cells without adhering to the adhesion surface in the aggregation region Aa. The cells can be separated and suspended toward the outside of the aggregation region Aa. Further, according to this stirred pattern (FIG. 20), since the direction of the stirring force with a linear oscillating is set to -D ₁ direction, adhere to other cells not adhere to the adhesive surface in the aggregation region Aa Cells can be separated and suspended towards the non-aggregated region An.

  Step S31: The CPU 74 turns off the initial flag.

  Step S32: The CPU 74 gives an instruction to the opening / closing mechanism 48 to open the introduction door 47 of the stirring device 29, and gives an instruction to the container transport mechanism 28 to move the culture vessel 31 to the stocker 27. The opening / closing mechanism 48 sets the rotation position of the introduction door 47 to the aforementioned exposure position, and the container transport mechanism 28 transports the culture container 31 from the opening 46 opened in that state to the outside of the stirring device 29, Store in the stocker 27. Thereafter, when the CPU 74 instructs the opening / closing mechanism 48 to close the introduction door 47, the opening / closing mechanism 48 returns the introduction door 47 to the shielding position.

  Step S33: The CPU 74 waits for the above-described fixing time (about 2 hours), returns to step S23, and re-executes steps S23 to S26. In step S26, if there is still an aggregation non-uniformity that should be eliminated (YES in step S26), the CPU 74 performs the processing subsequent to step S27 again. When steps S23 to S26 are re-executed, the initial flag is turned off, so that the result of determination in subsequent step S28 is NO, and step S34 is executed.

  Step S34: The CPU 74 compares the size of the aggregation region Aa with the previous value, and when the size reduction is not sufficient, the CPU 74 determines that the stirring pattern being set has no stirring effect and proceeds to step S35. If sufficient size reduction is observed, it is determined that the stirring pattern being set has a stirring effect, and the process proceeds to step S29.

  Step S35: The stirring pattern setting unit 82 of the CPU 74 rewrites the contents of the memory of the rotation driving unit 42, the memory of the X-axis driving unit 40, and the memory of the Y-axis driving unit 41, thereby setting the stirring pattern being set (see FIG. 20). ) And then the process proceeds to step S29.

  Specifically, the agitation pattern setting unit 82 in this step sets the number of rotations / oscillations wb and the number of rotations / oscillations wc in a predetermined number of times (for example, 1) among the agitation patterns being set (see FIG. 20). Increase the stirring effect by increasing the number of times).

  Here, the increase target is only the number of rotations wb and wc, but only the amplitude of rotations wb and wc, or both the number and amplitude. Further, here, the object to be adjusted is only the rotational swing wb, wc, but only the linear swing wd, or both the rotational swing wb, wc and the linear swing wd may be used.

  Step S36: The CPU 74 writes the entire image acquired in the immediately preceding step S24 in the management file 75 in association with the current date and time.

  Step S37: The CPU 74 gives an instruction to the container transport mechanism 28 to store the culture container 31 in the stocker 27. The container transport mechanism 28 stores the culture container 31 placed on the sample table 347 in the stocker 27.

  Step S38: The CPU 74 compares the observation schedule written in the management file 75 with the current date and time to determine whether or not the observation period has ended. If the observation period has not ended, the process returns to step S21. If the observation period has ended, the observation flow ends.

  Next, the operation of the control unit 13 during the stirring process (the operation of the CPU 74) will be described with reference to FIG. Hereafter, each step of FIG. 21 is demonstrated in order.

  Step S42: The CPU 74 refers to the output signal of the weight sensor provided in the stirring device 29 and detects the weight of the culture vessel 31. Note that the weight detected here may include the weight of the holder 32 or the plate 39.

  Step S43: The CPU 74 instructs the stirring device 29 to start stirring. The X-axis drive unit 40 of the stirring device 29 sends a drive signal corresponding to the drive waveform written in the memory in the X-axis drive unit 40 to the X stage 35, and the Y-axis drive unit 41 is the Y-axis drive unit 41. The drive signal corresponding to the drive waveform written in the internal memory is sent to the Y stage 37, and the rotation drive unit 42 outputs the drive signal corresponding to the drive waveform written in the memory in the rotation drive unit 42 to the rotation stage 37. To send. As a result, the rotary stage 37, the X stage 35, and the Y stage 36 are driven with the stirring pattern being set.

  Step S44: The CPU 74 refers again to the output signal of the weight sensor provided in the stirring device 29, and detects the current weight of the culture vessel 31 in the same manner as in step S42.

  Step S45: The CPU 74 calculates a difference by subtracting the weight detected in Step S44 from the weight detected in Step S42. If the difference is larger than zero (or a threshold value), the culture is performed at the time of stirring in Step S43. It is considered that the culture solution has scattered from the container 31, and the process proceeds to step S46. If the difference is smaller than zero (or a threshold value), it is considered that the culture solution has not been scattered from the culture vessel 31 during the stirring in step S43. End the flow.

  Step S46: The CPU 74 drives the compressor 50 connected to the scattering prevention case 45 so that the atmospheric pressure inside the scattering prevention case 45 is lower than the external atmospheric pressure (atmospheric pressure inside the temperature-controlled room 15). In this state, the possibility that the culture solution scattered inside the scattering prevention case 45 goes out of the scattering prevention case 45 is extremely low.

  Step S47: The CPU 74 prompts the user to take out the scattering prevention case 45 from the cell culture device 10 by displaying a warning on the monitor 65a. Note that the CPU 74 displays, on the monitor 65a, character information such as “Mixing failed”, “There is a possibility that the culture broth has scattered”, “Replace the culture device”, and the like. Warning by. At the same time, the CPU 74 may warn the user from the remote computer 67 via the communication line 66. If the remote computer 67 is used in this way, it is possible to promptly warn a user at a remote location.

  The user who has received the warning replaces the scattering prevention case 45 with a new one (or a washed one) and then returns the cell culture device 10. At the time of this exchange, since the inside of the scattering prevention case 45 is kept at a low pressure, even if the user does not pay particular attention, the possibility that the culture solution diffuses to each position of the temperature-controlled room 15 is extremely low. (The description of FIG. 21 above.)

  As described above, since the CPU 74 of this embodiment sets the stirring pattern of the stirring device 29 based on the entire image of the culture vessel 31, it is possible to efficiently eliminate cell aggregation unevenness and improve cell proliferation efficiency.

  In addition, since the CPU 74 of this embodiment sets the center of oscillation at the center of the aggregation region Aa, the cells that do not adhere to the adhesive surface but adhere to other cells in the aggregation region Aa are separated and aggregated. It can be floated toward the outside of the region Aa. Many of the cells floating in this manner may adhere to the adhesion surface after the fixing time (about 2 hours) has elapsed. Therefore, according to such agitation accompanied by rocking, aggregation unevenness is effectively eliminated.

  In addition, since the CPU 74 of this embodiment sets the acceleration direction of oscillation in the direction opposite to the direction from the aggregation region Aa to the non-aggregation region An, it does not adhere to the adhesion surface in the aggregation region Aa and adheres to other cells. Adherent cells can be separated and suspended toward the non-aggregated region An. Many of the cells floating in this manner may adhere to the adhesion surface after the fixing time (about 2 hours) has elapsed. Therefore, according to such agitation accompanied by rocking, aggregation unevenness is effectively eliminated.

  Further, the CPU 74 of this embodiment repeats the acquisition of the image (step S24), the setting of the stirring pattern (step S27), and the stirring (step S30) until the size of the aggregation region Aa becomes less than the threshold value. Residual can be prevented.

  Further, the CPU 74 of the present embodiment provides an appropriate standby time each time stirring for eliminating the aggregation unevenness is performed (step S33), so that it is possible to accurately determine whether the aggregation unevenness remains after the stirring.

  In addition, when the size of the aggregation region Aa does not become less than the previous value in the repetition process (NO in step S34), the CPU 74 of the present embodiment calculates at least one of the number of oscillations and the oscillation amplitude for stirring. Since the stirring effect is enhanced to improve (step S35), it is possible to prevent the number of repetitions from increasing (or becoming endless).

  Moreover, since the cell culture apparatus 10 of this embodiment is provided with the scattering prevention case 45 which covers the whole stirring apparatus 29, there is no possibility that parts other than the stirring apparatus 29 in the temperature-controlled room 15 may be contaminated with a culture solution.

  Moreover, since the scattering prevention case 45 of this embodiment is comprised so that removal with respect to the cell culture apparatus 10 is possible, the user can replace | exchange the stirring apparatus 29 with the scattering prevention case 45 as needed.

  In addition, since the stirring device 29 of the present embodiment includes the weight sensor, the CPU 74 can reliably detect the presence or absence of liquid scattering during stirring based on the presence or absence of a change in the weight of the culture vessel 31 before and after stirring.

  Further, when a change in the weight of the culture vessel 31 is detected, the CPU 74 of this embodiment issues a warning to the user that the liquid has scattered from the culture vessel 31, so that the culture is performed in an inappropriate state. Can be avoided.

  Note that the CPU 74 of the present embodiment determines the values of the rocking intensity, the number of rocking, and the rocking amplitude of the stirring pattern for eliminating the aggregation unevenness at the time of setting the stirring pattern (step S28). Since these values do not need to be changed unless the combination of the vessel type and the cell type is changed, these values are not determined every time the stirring pattern is set, but at the time of registration of the culture vessel 31 (for example, step You may go to S11). In that case, the CPU 74 may write the determined value in the management file 75 of the culture vessel 31 and read and set it when setting the stirring pattern (step S28).

  Further, the CPU 74 of the present embodiment performs binarization processing on the entire image in the detection of the seeding unevenness or the aggregation unevenness (steps S13 and S25), but before the binarization processing, before the low resolution conversion processing or the like. Processing may be performed.

  The CPU 74 of the present embodiment uses the same image as the image for storage (here, the entire image) as an image for detecting sowing unevenness or agglomeration unevenness, but an image for detecting sowing unevenness or agglomeration unevenness. May be different from the image for storage. For example, the CPU 74 may set the storage image as an entire image with an observation magnification of 4 and the unevenness detection image as an entire image with an observation magnification of 2 times.

  Moreover, although the cell culture apparatus 10 of this embodiment applied phase difference observation as an image acquisition method (observation method) for detecting seeding unevenness or aggregation unevenness, the cell density distribution in the culture vessel 31 can be visualized. Other observation methods may be used.

  In addition, the cell culture device 10 of the present embodiment, when stirring for eliminating seeding unevenness (steps S15 and S17), swings in the X-axis direction (wa, wb) and swings in the Y-axis direction (wc, wd) Are performed in order, but may be performed simultaneously.

  Further, in the cell culture device 10 of the present embodiment, the rocking in the θ direction and the rocking in the XY direction are sequentially performed during the stirring for eliminating the uneven aggregation (steps S27 and S30).

  Further, the CPU 74 of the present embodiment calculates the center coordinates of the aggregation region Aa in the detection of aggregation unevenness (step S25), but may calculate the center-of-gravity coordinates instead of the center coordinates. In that case, not only the contour shape of the aggregated area Aa but also the luminance distribution is reflected in the determination of the oscillation center.

  Further, although the CPU 74 of the present embodiment fixes the observation frequency (every 24 hours), it may be variable. In that case, for example, the CPU 74 counts the number of repetitions of the loop (steps S27 to S33 to S23 to S26) at the time of one observation, and frequent stirring is required when the counted number is equal to or greater than a threshold value. The frequency of observation may be changed to a higher value at that time.

  Further, the CPU 74 of the present embodiment repeats the loop (steps S27 to S33 to S23 to S26) until there is no aggregation unevenness, but counts the number of repetitions, and if the count number exceeds a threshold value, stirring is performed. The user may be regarded as having no effect, and a warning may be given to the user at that time.

  In addition, the agitation device 29 described above uses the X stage 35, the Y stage 36, and the rotation stage 37 to swing the culture vessel 31, but in addition to these stages, a tilt stage 300 as shown in FIG. May be used. The tilt stage 300 is provided between the X stage 35 and the plate 39 and includes at least three pins 300a that individually move the positions of the plate 39 up and down, and changes the balance of the protruding amount of the pins 300a. By doing so, the tilt direction and tilt amount of the plate 39 can be changed.

  When such a tilt stage 300 is used, since the tilt swing (or the linear swing in the vertical direction) can be combined with the above-described rotation swing and linear swing, the degree of freedom of the stirring pattern is increased. . For example, when the shape of the culture vessel 31 is complicated, the stirring effect may be improved by combining this tilting and swinging.

  Further, in the cell culture device of this embodiment, stirring was performed using a dedicated stirring device (stirring device 29). However, the culture vessel 31 was placed on the sample table 347 of the observation unit 30, and the sample table 347 of the observation unit 30 was placed. The agitation may be performed by driving in the same manner as the stage of the agitator 29. However, at the time of stirring, it is desirable that the sample stage 347 and the culture vessel 31 are fixed to each other via a holder or the like.

  Further, the CPU 74 of the present embodiment determines at least one of the rocking intensity, the number of rocking times, and the rocking amplitude in the stirring pattern for eliminating the uneven aggregation according to the combination of the cell type and the container type. However, some of the culture vessels 31 have a special surface treatment applied to the bonding surface, so that at least one of the rocking strength, the number of rocking times, and the rocking amplitude is determined based on the cell type, the container type, and the surface. You may determine according to the combination with the kind of process. In this case, the user is required to input the type of surface processing along with the cell tumor and the container type.

  In the cell culture device 10 of the present embodiment, a part of the function of the control unit 13 (for example, the function of the stirring pattern setting unit 82) may be mounted on the stirring device 29 side. Further, some or all of the functions of the X-axis drive unit 40, the Y-axis drive unit 41, and the rotation drive unit 42 may be mounted on the control unit 13 side.

  In addition, a part of the operation of the cell culture device 10 of the present embodiment can be executed by the computer 67. In that case, the operation program of the computer 67 may be installed in the computer 67 via a storage medium such as a memory card, an optical disk, or a magnetic disk.

  DESCRIPTION OF SYMBOLS 10 ... Cell culture apparatus, 30 ... Observation unit, 13 ... Control unit, 74 ... CPU, 31 ... Culture container, 39 ... Plate, 35 ... X stage, 36 ... Y stage, 37 ... Rotary stage, 32 ... Holder, 40 ... X axis drive unit, 41 ... Y axis drive unit, 42 ... rotation drive unit, 45 ... scattering prevention case, 47 ... introduction door, 49 ... connector, 52 ... connector, 51 ... tube, 50 ... compressor

Claims (12)

A detection means for detecting a density distribution of a cell group cultured in a liquid in a culture vessel;
Stirring means for stirring the cells in the culture container by swinging the culture container;
Environment control means for controlling the environment of the culture vessel at the time of detection and stirring to a preset environment;
Based on the density distribution detected by the detection means, an operation control means for setting the oscillation pattern by the stirring means;
A cell culture apparatus comprising: The cell culture device according to claim 1,
For shaking for the stirring,
Rotation swing by rotational movement is included,
The operation control means includes
The cell culturing apparatus, wherein the center of the rotational oscillation is set at the center of an area where the cell group exists at high density. The cell culture device according to claim 1,
For shaking for the stirring,
Includes linear swinging by linear motion,
The operation control means includes
The cell culture is characterized in that the acceleration direction of the linear oscillation is set in a direction opposite to a direction from a region where the cell group exists at a high density to a region where the cell group exists at a low density. apparatus. In the cell culture device according to any one of claims 1 to 3,
The operation control means includes
A series of processing consisting of the detection, the setting, and the agitation is repeated until the size of an area where the cell group exists at a high density is less than a threshold value. The cell culture device according to claim 4,
The operation control means includes
A cell culture device characterized in that a waiting time is provided between the previous stirring and the next detection. In the cell culture device according to claim 4 or 5,
The operation control means includes
In the case where the size of the region does not become less than the previous value in the repetition process, at least one of the number of oscillations for the stirring and the amplitude of the oscillations is improved. Culture device. In the cell culture device according to claim 4 or 5,
The operation control means includes
When the number of repetitions exceeds a threshold value, the frequency of execution of the series of processes is increased. In the cultured cell apparatus according to claim 4 or 5,
The operation control means includes
A cell culture device that warns a user when the number of repetitions exceeds a threshold value. In the cell culture device according to any one of claims 1 to 8,
A cell further comprising a scattering prevention case for preventing the liquid in the culture container from splashing into the cell culture device by covering the stirring means and the entire culture container during the stirring. Culture device. The cell culture device according to claim 9,
The scattering prevention case is
A cell culture device configured to be detachable from the cell culture device. In the cell culture device according to claim 9 or 10,
A cell culture apparatus, further comprising a measuring means for detecting the presence or absence of a change in the weight of the culture vessel before and after the stirring. The cell culture device according to claim 11,
The operation control means includes
A warning is given to a user when a change in weight is detected before and after the agitation.

Images