JP4468661B2 - Cell culture observation apparatus and cell culture observation method - Google Patents

Description

  The present invention relates to a cell culture observation apparatus and a cell culture observation method for observing information from a cell culture reaction while culturing cells.

With recent advances in gene analysis technology, gene sequences in many organisms including humans have been clarified, and the causal relationship between diseases and gene products such as analyzed proteins has begun to be gradually elucidated. Further, in the future, various examination methods and apparatuses using cells have been considered for comprehensive and statistical analysis of various proteins and genes.
Usually, the cells are seeded in a plastic or glass dish or flask and cultured in an incubator. The inside of this incubator is, for example, set at a carbon dioxide concentration of 5%, a temperature of 37 ° C., and a humidity of 100%, and is maintained in an environment suitable for cell growth. Further, in the incubator, the culture solution is changed every 2-3 days in order to feed the cells with nutrients and maintain a pH suitable for the culture.

  Several methods are known for observing cells in culture, and one of them is the above-mentioned dish or flask taken out of an incubator and using an inverted microscope such as a phase contrast microscope. A method of performing observation is known. In this method, it is necessary to observe the cells as quickly as possible and return the cells to the incubator after the observation. This is to prevent the cell activity from being impaired by the cell being placed in a normal environment for a long time. That is, if the cell activity is unstable, it is difficult to perform accurate evaluation. In addition, when removing cells from the incubator, care is taken to prevent contamination and the like.

As another cell observation method, a method using a transparent constant temperature culture vessel for microscopic observation in which various cell culture conditions can be set is also known (see, for example, Patent Document 1).
This transparent thermostat container for microscopic observation is sealed with a pair of transparent heating plates that can be controlled to a predetermined temperature by a temperature controller, a carbon dioxide supply port and a discharge port for adjusting the carbon dioxide concentration inside the container, and a seal packing. An evaporating dish for maintaining humidity is provided in the sealed container.
In observation using this transparent constant temperature culture vessel for microscopic observation, the temperature, carbon dioxide concentration and humidity inside the vessel can be controlled, so that observation can be performed while culturing cells. That is, for example, by observing from below the transparent heat generating plate with an objective lens, it is possible to continuously and easily observe and record changes with time in the culture state of the cells.

As another cell observation method, an observation method for evaluating different dish cells for each measurement is also known. That is, when detecting changes in cells over time, a large number of dishes or the like seeded with cells under the same conditions are prepared, and each dish is taken out of the incubator at predetermined measurement times for evaluation.
In this method, one or several dishes are used in one observation, and the activity of the cells may be impaired by various observation operations. Therefore, one dish can be used for only one measurement. It is a common method not to use.
Japanese Patent Laid-Open No. 10-28576 (paragraph numbers 0004-0007, FIGS. 1 to 4)

However, in the observation using the inverted microscope described above, the cells need to be taken in and out of the incubator for each observation, so the observation position of the cells is different, and it is difficult to observe the same cell group every time. Met.
Moreover, in the observation using the transparent constant temperature culture container for microscopic observation described in Patent Document 1, it is possible to observe while culturing cells. However, when the culture medium in the container is replaced, the cells Therefore, it is difficult to observe the same cell group every time. That is, in this method, it is possible to observe the cell culture for about 2 to 3 days without exchanging the culture solution. However, if the cell culture is performed for a longer period of time, the same cell is chased. Had difficulty.

  In addition, in the method for evaluating cells of different dishes for each measurement described above, each dish does not necessarily have the same conditions. It was difficult. In particular, since the expression of proteins and the like varies depending on the cell cycle, the cell needs to be measured after the cell cycle of the cell is adjusted depending on the evaluation target. However, since the cell cycles coincide with each other only about 2 cycles, there has been a disadvantage that the experimental protocol has to be limited due to the gradually increasing deviation.

As described above, since environmental factors act strongly on cell activity, it is difficult to always observe the same cell group under a microscope. For this reason, for example, the method of using the microscope etc. of the type which covers the whole microscope with a box etc., and maintains temperature and humidity environment is also considered. However, in this case, in an environment where carbon dioxide does not exist, the pH optimum for culture cannot be maintained, and the cell activity can be maintained only for several hours.
In addition, it is conceivable to introduce carbon dioxide under a high humidity environment, but in this case, the life of a very expensive microscope may be impaired.

  The present invention has been made in consideration of such circumstances, and its purpose is to continuously observe accurate changes over time of cells in a simple and short time while growing cells for a long time. An object of the present invention is to provide a cell culture observation apparatus and a cell culture observation method.

In order to achieve the above object, the present invention provides the following means.
The invention according to claim 1 is a cell culture observation apparatus for continuously observing a change with time of one or a plurality of cells present on a carrier or in a solution, and stores the cells and maintains the cell activity. A culture vessel that holds the culture vessel, an imaging unit that divides the cells in the culture vessel into regions corresponding to the cells, and images the regions captured by the imaging unit. An analysis unit that extracts and analyzes at least one of the geometric feature amount or the optical feature amount of the cells in the region based on the captured image of the culture container, and the culture vessel is on one end side of the culture vessel And a culture solution supply pipe for supplying a culture solution to the inside of the culture vessel and a culture solution supply pipe that is connected to the other end side and the upper side of the culture vessel and is unnecessary from the inside of the culture vessel. The culture medium drainage port for draining the liquid And a flow of the culture solution supplied from the culture solution supply pipe to the inside of the culture vessel. And a commutator member that disperses the flow of the culture solution discharged from the inside of the culture vessel to the culture solution discharge pipe, and the commutator member is a plate-like member and has a pore diameter over the entire surface. Two different types of through-holes are formed, and one of the two types of through-holes is arranged in a grid pattern at regular intervals, and the other through-hole having a smaller diameter than one through-hole is equally spaced A cell culture observation device arranged in a grid is provided.

In the cell culture observation apparatus according to the present invention, cells can be cultured in a culture container without being put into and out of an incubator or the like with a culture container capable of maintaining cell activity. In addition, the culture vessel can be moved by a movable stage. Thereby, the imaging part can image all the positions of the culture vessel even in a temporarily fixed state. At this time, since the imaging unit divides the image into regions corresponding to the respective cells, it is possible to focus only on a region desired to be observed, for example, a region of a specific cell group after imaging. Furthermore, the analysis unit can extract and analyze the cells in each region by reliably distinguishing cells one by one based on the geometric feature value or the optical feature value.
In other words, the analysis unit can reliably recognize and follow each cell without making a mistake while culturing the cells in the culture container, and pay attention to a region corresponding to each cell, for example, a region of a cell group. You can also chase. Therefore, based on the analysis result of the analysis unit, while cultivating the cells for a long time, that is, culturing, time-dependent changes such as the behavior that occurs during the culturing process are observed accurately and continuously for each cell or each region, for example. can do. Moreover, since each cell and each region desired to be observed can be easily recognized, the observation is easy and the time required for the observation can be shortened.
Furthermore, when culturing cells for a long time, it is equipped with a culture solution supply pipe, a culture solution discharge pipe, and a commutator member, so that a concentrated culture solution flow can be converted into a dispersed flow, and the cross-sectional area of the culture vessel is almost the same. The culture solution can be exchanged while flowing the culture solution at a constant flow rate and flow rate in the entire region.

Furthermore, the staying portion generated on the downstream side of the commutator member by the discharge flow of the culture solution from the through-hole having a large diameter can be agitated by the discharge flow from the through-hole having a small diameter. Therefore, it is possible to exchange the culture solution while circulating the culture solution without stagnation.

The invention according to claim 2 provides the cell culture observation apparatus according to claim 1 , wherein the geometric feature amount is at least one of a gravity center position and an area.
In the cell culture observation apparatus according to the present invention, the analysis unit can distinguish and recognize each cell clearly and accurately based on the difference in the center of gravity and the area of the cell. In addition, changes in cells over time can be easily observed from changes in the center of gravity and area of the cells.

The invention according to claim 3 provides the cell culture observation apparatus according to claim 1 or 2 , wherein the optical characteristic amount is luminance.
In the cell culture observation apparatus according to the present invention, the analysis unit can distinguish and recognize each cell clearly and accurately due to the difference in luminance of the cells, and more easily changes over time of the cells than changes in luminance. Can be observed.

The invention according to claim 4 provides the cell culture observation apparatus according to claim 3 , wherein the luminance is a luminance for each wavelength.
In the cell culture observation apparatus according to the present invention, the analysis unit can observe various types of proteins corresponding to different wavelengths in one observation, and the time and effort required for the observation are omitted, and the efficiency of the observation Can be improved.

The invention according to claim 5 uses the cell culture observation device according to any one of claims 1 to 4 to continuously change the cells over time while culturing the cells in the culture vessel. A cell culture observation method for observing automatically, an imaging step in which the cells in the culture container are segmented into regions corresponding to the cells and imaged, and cells in each region imaged in the imaging step There is provided an analysis step of extracting and analyzing at least one of a geometric feature value and an optical feature value, and a cell culture observation method.

In the cell culture observation method according to the present invention, it is possible to take an image in a state in which the cells being cultured are stored in the culture container by the imaging step, and therefore, during imaging, the possibility of contamination or the like is imposed on the cells. There is nothing. In addition, since the image is divided into regions corresponding to the respective cells, it is possible to pay attention only to the region desired to be observed, for example, the region of the cell group. Further, the analysis step can reliably extract and analyze cells in each region one by one based on geometric feature values or optical feature values. Therefore, each cell and region can be traced reliably while culturing the cell, and the change with time in the culturing process can be observed accurately and continuously. In addition, when the culture conditions are changed, the reaction of cells to be observed can be measured in real time.

  As described above, according to the cell culture observation apparatus and the cell culture observation method according to the present invention, there is no possibility of contamination or the like and no burden on the cells at the time of imaging. In addition, each cell in culture and the region corresponding to each cell can be reliably recognized without making a mistake. Therefore, it is possible to accurately and continuously observe changes with time in regions such as cells and cell groups generated in the culture process. Furthermore, since the cells and regions to be observed can be easily recognized, the observation is easy and the time required for the observation can be shortened.

Hereinafter, an embodiment of a cell culture observation apparatus 1 according to the present invention will be described with reference to FIGS.
The cell culture observation apparatus 1 according to the present embodiment is an apparatus that continuously observes changes over time of a plurality of cells A present on a slide glass (carrier) 2 shown in FIG. That is, as shown in FIG. 1 and FIG. 2, the cell culture observation apparatus 1 accommodates the slide glass 2 inside and maintains a culture vessel 10 capable of maintaining the cell activity of the cells A on the slide glass 2, and the culture An inverted microscope 20 capable of holding the container 10 is provided.
Note that the glass slide 2 of the present embodiment is used after a base material such as a polymer material for fixing the cells A is formed by treatment.

The inverted microscope 20 includes an electric stage (movable stage) 30 that holds the culture vessel 10 and an imaging mechanism (imaging unit) 40 that divides the cells A in the culture vessel 10 into regions corresponding to the cells A and images them. have.
In addition, the cell culture observation apparatus 1 extracts and analyzes at least one of the geometric feature amount or the optical feature amount of the cell A based on the image captured by the imaging mechanism 40 (hereinafter referred to as a PC). (Analysis unit) 50 is provided.

  As shown in FIGS. 1 and 2, the electric stage 30 is driven by a motor (not shown) and is supported by the main gantry 21 so as to be movable in the XY directions (horizontal directions). Further, a culture vessel fixing portion 31 for fixing the culture vessel 10 is provided on the electric stage 30 so that the horizontal angle (tilt) can be adjusted. That is, the culture vessel fixing part 31 is formed in a flat plate shape as shown in FIGS. 3 and 4, and is fixed on the electric stage 30 by surrounding mounting screws. At this time, tilt adjustment with respect to the horizontal plane of the electric stage 30 can be performed by adjusting the tightening amount of each mounting screw. The culture vessel 10 is fixed by being fitted into a locking port 31a formed at the center of the culture vessel fixing portion 31. Therefore, the culture vessel 10 can adjust the horizontal plane via the culture vessel fixing portion 31 and can move in the XY directions via the electric stage 30.

  Further, an objective lens 41 for observing the cells A on the slide glass 2 accommodated in the culture vessel 10 is disposed below the electric stage 30, as shown in FIG. The formed image is output to a CCD camera 42 disposed above the main mount 21. That is, the objective lens 41 and the CCD camera 42 constitute the image pickup mechanism 40. The CCD camera 42 has a function of outputting a captured image to the PC 50 via an interface (not shown). The objective lens 41 has a plurality of lenses having different magnifications, and a desired lens can be selected by rotating and changing a revolver (not shown).

  Further, as shown in FIGS. 1 and 2, the inverted microscope 20 includes a microscope control device 22, an electric shutter (not shown), and an electric fluorescent mirror unit. The microscope control device 22 includes an XY scanning control unit 22 a that controls the operation of the electric stage 30, a coordinate detection unit 22 b that detects the coordinates of the electric stage 30, and a parallel light source control unit 22 c that controls the light applied to the cell A. Have. The coordinate detection unit 22b has a function of outputting the detected detection value to the PC 50.

  The PC 50 comprehensively controls the microscope control device 22, that is, the XY scanning control unit 22a and the parallel light source control unit 22c. The PC 50 also stores an image memory unit 51 that stores the captured image sent from the imaging mechanism 40, and image processing that analyzes the captured image stored in the image memory unit 51 (described in detail later). Unit 52 and a data processing unit 53 for detecting a change with time of the cell A based on the data processed by the image processing unit 52. The image processing unit 52 and the data processing unit 53 function according to a desired cell A observation method.

As shown in FIGS. 3 and 4, the culture vessel 10 has a through-hole 11 that can accommodate the slide glass 2 and a housing 12 made of a material excellent in heat conduction, for example, stainless steel or aluminum, A pair of optically smooth glass plates 13 that close the through-hole 11 of the housing 12 from above and below are provided. At the lower end of the housing 12, a locking portion 12a that can be fitted into the locking port 31a of the culture vessel fixing portion 31 is formed, and when the locking portion 12a and the locking port 13a are fitted, the culture vessel It is fixed to the fixing part 31.
In addition, a fluorine resin (not shown) such as a packing made of ethylene tetrafluoride is disposed on the joint surface between the housing 12 and the pair of glass plates 13 to ensure watertightness inside. Thereby, since the slide glass 2 is accommodated in the culture container 10, even if a pair of glass plate 13 is removed from the housing | casing 12, the inside of the culture container 10 can be made watertight.
In addition, it is good to give the highly hydrophilic process etc. in order to prevent the bubble of the culture solution B from adhering to the inner surface of a pair of glass plate 13. FIG. Further, after the slide glass 2 is stored in the culture vessel 10, the pair of glass plates 13 may be completely sealed and fixed to the housing 12 with a silicon adhesive or the like.

In addition, the culture vessel 10 includes a culture solution supply pipe 14 that supplies the culture solution B into the housing 12, a culture solution discharge pipe 15 that discharges the culture solution B that is no longer needed from the inside of the housing 12, and the flow of the culture solution B. Is provided with a pair of commutator members 16.
The culture solution supply pipe 14 is provided on one end side and the lower side of the casing 12, and the culture solution discharge pipe 15 is provided on the other end side and the upper side of the casing 12. That is, the culture solution B supplied from the culture solution supply pipe 14 fills the inside of the culture vessel 10 and is then discharged from the culture solution discharge pipe 15. Moreover, the culture solution supply pipe 14 is connected to a culture solution bottle 61 whose culture solution temperature is controlled by the culture solution temperature control unit 60 as shown in FIG. In addition, a culture solution pump 62 is interposed between the culture solution supply pipe 14 and the culture solution bottle 61, and the temperature of the culture solution is controlled from the culture solution bottle 61 by driving the solution culture solution pump 62. B is supplied into the culture vessel 10. The culture solution pump 62 is, for example, a circulation pump such as a verista pump, and the intermittent operation timing and flow rate thereof are controlled by the culture solution pump control unit 63.
In addition, the carbon dioxide concentration of the culture solution B stored in the culture solution bottle 61 is controlled by the carbon dioxide concentration control unit 64 such that the flow rate of carbon dioxide and the intermittent supply timing are maintained so as to maintain a predetermined pH value. Has been.

  As shown in FIGS. 3 and 4, the pair of commutator members 16 is disposed inside the housing 12 and between the culture solution supply pipe 14 and the culture solution discharge pipe 15 and the slide glass 2. . The pair of commutator members 16 is formed of a plate-like porous member having a plurality of through holes in the thickness direction. That is, as shown in FIG. 5, in the commutator member 16, through holes 16a having a diameter of about 0.1 mm are arranged in a lattice pattern at intervals of 0.3 mm in the thickness direction, and a diameter of about 0.03 mm. Through-holes 16b are formed in the center at intervals of 0.3 mm. As described above, the commutator member 16 has two types of through holes 16a and 16b having different diameters.

Accordingly, as shown in FIGS. 3 and 4, the commutator member 16 on the side of the culture solution supply pipe 14 allows the culture solution B supplied from the culture solution supply pipe 14 to the inside of the culture vessel 10 to have a plurality of through holes 16 a, It is possible to distribute and distribute in 16b. Further, the commutator member 16 on the culture medium discharge pipe 15 side distributes the culture medium B discharged from the inside of the culture vessel 10 through the culture medium discharge pipe 15 to the plurality of through holes 16a and 16b and distributes it. It is possible. Therefore, the concentrated flow of the culture solution B can be converted into a dispersed flow, and in the vicinity of the slide glass 2 where the cells A are arranged, the culture solution B is maintained at a constant flow rate and flow rate in almost the entire area of the culture vessel 10. It is possible to flow.
In particular, the commutator member 16 has through-holes 16a and 16b having different diameters. Therefore, the stay generated on the downstream side of the commutator member 16 by the discharge flow of the culture medium B from the through-hole 16a having a large diameter. The part can be agitated by the discharge flow from the through-hole 16b having a small diameter. Therefore, it is possible to exchange the culture solution B by circulating the culture solution B without stagnation and reliably releasing it from the inside of the culture vessel 10.
For example, a minute through hole having a diameter of about 0.2 μm may be adopted as the through hole of the commutator member 16. In this case, it is possible to prevent the occurrence of contamination through the culture broth B.

  A temperature control unit 17 is attached to the culture vessel 10. The temperature control unit 17 forms therein a hot water circuit 18 for circulating the hot water W around the culture vessel 10. The hot water W is supplied from the hot water supply pipe 17 a and the hot water circuit 18 for supplying the hot water W to the hot water circuit 18. A hot water liquid discharge pipe 17b for discharging is provided. Thereby, the hot water W can be circulated in the hot water circuit 18, and the heat of the hot water W can be transmitted to the culture solution B inside the culture vessel 10 through the casing 12 of the culture vessel 10.

Further, the hot water supply pipe 17a is connected to a hot water bottle 66 whose temperature is controlled by a hot water temperature control unit 65 as shown in FIG. In addition, a hot water pump 67 is interposed between the hot water supply pipe 17 a and the hot water bottle 66, and the hot water W whose temperature is controlled from the hot water bottle 65 is driven by the temperature control unit 17 by driving the hot water pump 67. It is designed to be supplied inside. The hot water pump 67 is a circulation pump such as a verista pump, for example, and its hot operation control unit 68 controls its intermittent operation timing and flow rate.
Further, the hot water pump control unit 68 uses a temperature sensor (not shown) such as a thermocouple, thermistor, side temperature resistor, etc. to maintain the temperature of the culture vessel 10 in the range of 37 ± 0.5 ° C. And a function of controlling the circulation flow rate. Therefore, the culture vessel 10 is heated like a water bath, and the temperature of the culture solution B can be kept constant without causing a rapid temperature change.

  The culture medium pump control unit 63, the carbon dioxide concentration control unit 64, and the hot water pump control unit 68 described above are connected to the PC 50 and controlled comprehensively. Further, in order to establish sterility to the cell A, it relates to the flow path of the culture solution B such as the inside of the culture vessel 10, the culture solution bottle 61, the culture solution pump 62, the culture solution supply pipe 14, and the culture solution discharge pipe 15. The portion to be configured is configured to be sterilizable.

A case where cell culture is observed with the cell culture observation apparatus 1 configured as described above will be described with reference to FIGS. 2 and 6 to 12.
First, the cell culture observation apparatus 1 is set to an initial state. That is, as shown in FIG. 2, the culture solution pump control unit 63, the carbon dioxide concentration control unit 64, and the hot water pump control unit 68 are operated to supply the culture solution B into the culture vessel 10, and for example, the temperature is It is set to a predetermined value of 37 ± 0.5 ° C. and a carbon dioxide concentration of 5%. Further, the culture vessel fixing part 31 is adjusted so that the entire surface of the slide glass 2 in the culture vessel 10 is within the focal depth of the objective lens 41, and the slide glass 2 is orthogonal to the optical axis (not shown). The slide container 2 is positioned by adjusting the culture vessel fixing part 31 in a tilted manner.

  After the initial state described above, the operator selects the measurement target range 100 of the slide glass 2 that he wishes to observe as shown in FIG. 6 (S1). That is, the operator numerically inputs the coordinate positions of the measurement start position 101 and the measurement end position 102 with the corner on the slide glass 2 (for example, the lower left corner with respect to the paper surface) as the origin. Thereby, the range surrounded by both the positions 101 and 102 is recognized as the measurement target range 100.

  Next, the operator operates the electric stage 30 by pressing a stage movement switch (not shown) on the PC 50 (S2), and confirms whether or not the input measurement target range 100 is a desired range. That is, in the case of YES when the stage movement switch is pressed (S3), the PC 50 reads the input measurement start position 101 (S10), and the measurement start position 101 is positioned within the photographing field of the objective lens 41. The electric stage 30 is moved (S11). When the electric stage 30 moves, a preview screen of the measurement start position 101 is displayed on a monitor (not shown) of the PC 50 (S12). The operator confirms that the slide glass 2 is located near the measurement start position 101 and confirms that the image on the slide glass 2 is clearly obtained on the preview screen.

After confirming the measurement start position 101, the operator presses a confirmation switch (not shown) of the PC 50 (S13). When the confirmation switch is pressed (in the case of YES), the PC 50 reads the input measurement end position 102 (S14) and moves the electric stage 30 so that the measurement end position 102 is located within the imaging field of view of the objective lens 41. (S15). When the electric stage 30 moves, a preview screen of the measurement end position 102 is displayed on a monitor (not shown) of the PC 50 (S16). The operator presses the confirmation switch after determining that the measurement end position 102 is appropriate as described above (S17).
Thereby, for example, even if the operator erroneously inputs the coordinate positions of the measurement start position 101 and the measurement end position 102, it can be determined before the measurement is started. In this case, that is, in the case of NO in which the confirmation switch is not pressed, the operator can obtain the desired measurement target range 100 by inputting the coordinate positions of the measurement start position 101 and the measurement end position 102 again.

  After completing the setting of the measurement target range 100, the operator selects a fluorescent protein to be used, for example, GFP, HC-Red, or the like from a list stored in advance in the PC 50 (S4). The PC 50 automatically selects an optimal cube (optical filter) according to the selected fluorescent protein. This makes it possible to detect desired fluorescence from the cell A. This setting is not limited to one, and a plurality of settings can be set. Multi-color measurement can be performed by setting a plurality. In particular, it is effective in detecting many kinds of proteins from the cell A by one observation. The cube change during measurement is automatically performed in synchronization with the driving of the electric stage 30.

After the setting of the fluorescent protein is completed, the operator sets the measurement magnification and the measurement time interval (S5). After completion of all the settings described above, the operator presses a measurement start switch (not shown) of the PC 50 (S6) to start the imaging step. If it is desired to change the setting described above, that is, if the measurement start switch is not pressed, various settings can be reset from the setting of the measurement target range 100.
When the measurement start switch is pressed (in the case of YES) (S7), the PC 50 reads the input measurement start position 101 (S20), and the measurement start position 101 is positioned within the photographing field of the objective lens 41. The electric stage 30 is moved (S21). Then, the PC 50 changes the revolver according to the set measurement magnification (S22) and selects the objective lens 41 having a desired magnification (S23). Next, the PC 50 controls the parallel light source control unit 22c to select an optimal cube according to the set fluorescent protein (S24) (S25).

  Next, after opening the shutter (S26), the imaging mechanism 40 captures the fluorescence amount of the cell A corresponding to the wavelength of the selected fluorescent protein and outputs the captured image to the PC 50 (S27). When the captured image is captured, the XY scanning control unit 22a slightly moves the electric stage 30 in the X direction. That is, the XY scanning control unit 22a takes the measurement visual field 103 determined by the objective lens 41 and the CCD camera 42 as one step, and slightly moves the electric stage 30 to the next step (S28). When moving one step, the imaging mechanism 40 images the measurement visual field 103. In this way, images are continuously acquired in the X direction while repeating imaging and one-step movement. When the scanning in the X direction in the measurement target range 100 is completed, the XY scanning control unit 22a receives the end flag and scans the electric stage 30 in the Y direction for one step and scans in the X direction again. Do. The above is repeated and measurement is performed up to the measurement end position 102 (S29). Thus, in the case of YES where imaging has been completed in the entire measurement target range 100 (S30), the shutter is closed and imaging by the imaging mechanism 40 and movement of the electric stage 10 are stopped.

On the other hand, the captured image that is captured for each step, that is, for each measurement visual field 103 that is a region corresponding to each cell, is sent to the PC 50 and stored in the image memory unit 51. The image processing unit 52 recognizes the measurement visual field 103 and the coordinate position of each cell A in the measurement visual field 103 based on the captured image stored in the image memory unit 51. At this time, the image processing unit 52 calculates and extracts a geometric feature amount such as the center of gravity and area of each cell A and an optical feature amount such as fluorescence luminance in the analysis step. As a result, the image processing unit 52 accurately extracts the characteristics of each cell A, so that each cell A is accurately associated with the position information and analyzed.
Therefore, the image processing unit 52 can image the fluorescence amount distribution of the cell A at each position on the entire surface of the slide glass 2. Further, the fluorescence amount distribution or the like can be imaged by paying attention to each measurement visual field 103. Further, since the image processing unit 52 can accurately track each cell A, for example, paying attention to only an arbitrary number of cells A, the fluorescence distribution inside the cells A is locally observed while culturing. It is also possible to measure for a long time. Furthermore, while culturing the cells A, for example, the entire surface of the slide glass 2 can be measured at regular intervals, and the fluorescence amount of each cell A over time can be automatically measured.

When a plurality of fluorescent proteins and magnifications are designated, the PC 50 measures the measurement target range 100, then automatically changes the objective lens 41 and the cube, and performs the same operation as described above for measurement. In this case, since the image processing unit 52 extracts the luminance for each wavelength of the cell A according to the plurality of fluorescent proteins, it is possible to observe many kinds of proteins and the like by one observation.
In addition, when a focus shift occurs when the objective lens 41 is changed, it may be dealt with by an inter-objective confocal correction or an autofocus mechanism.

Moreover, the cell culture observation apparatus 1 of this embodiment can also culture the cell A for a long period of time and detect a change with time of one cell A.
In this case, first, geometric features such as the center of gravity and area of each cell A and optical features such as luminance are extracted with higher accuracy. That is, the image processing unit 52 extracts a background image from the captured image stored in the image memory unit 51 (S40) and removes the background from the original captured image (S41). Then, the image is emphasized so that each cell A can be easily recognized in a granular form from the image with the background removed. That is, after reading the maximum luminance range of the image that can be enhanced (S42), the image is enhanced by multiplying a predetermined numerical value accordingly (S43). Then, by extracting, for example, those having a threshold value or more from the emphasized image, the luminance of each cell A is recognized as a clear granularity (S44). As a result, the geometric feature quantity and optical feature quantity of each cell A are recognized more accurately and extracted in association with the position information of the cell A (S45). After the extraction, the emphasis work performed for recognizing the cell A is corrected. That is, the characteristic amount of the cell A is corrected by correcting the numerical change at the time of emphasis (S46). Then, the corrected feature amount is output to, for example, a file and stored in the file (S47).

Note that the above-described enhancement of the captured image may be performed by recognizing a cell portion from a binarized image group having several levels of binarization and using it as a mask for the original captured image. Alternatively, only the cell luminance edge may be emphasized, and the cell may be recognized using the edge as a reference. Furthermore, a method of recognizing a cell portion by binarizing a sharpened image and using it as a mask of an original captured image may be used.
Further, as a method for removing the background, a method of flattening the luminance below a certain level may be used. Furthermore, the captured image may be discarded because of its large capacity, but can be used for recalculation by sequentially storing the images.

Next, the data processing unit 53 performs data processing of the feature amount of the cell A accumulated in the file (S50). First, the data processing unit 53 reads the feature values accumulated in the file (S51) and rearranges the cells A so as to be in time series (S52). After the data is rearranged, the data processing unit 53 graphs the change over time in the brightness, that is, the difference in the expression level for each cell A (S53).
At this time, if necessary, the grid shown in FIG. 7, that is, the cell A for each measurement visual field 103 can be edited (S54), and the change in luminance with time can be graphed (S55).
Further, the area 103a shown in FIG. 7, that is, the cell A for each range in which the measurement visual field 103 is further narrowed can be edited (S56), and the change in luminance with time can be graphed (S57). The area 103a is arbitrarily set by the operator. In addition, the cells A present at the boundaries of the grid and the area 103a are determined based on the barycentric coordinates of the cells A, and are distributed to the side where the barycentric coordinates exist.

  When the necessary graphing is completed, the data processing unit 53 outputs the graphed data to a file (S58). Thereby, the time-dependent change of one cell A when the cell A is cultured for a long period of time can be easily observed. Accordingly, it is possible to accurately and easily measure changes in the expression level of the cells A with time during culture.

As described above, in the cell culture observation apparatus 1 and the cell culture observation method, it is possible to perform observation while culturing the cells A in the culture vessel 10 capable of maintaining the cell activity. That is, since it is possible to take an image in a state where the cell A being cultured is stored in the culture vessel 10 by the imaging step, there is no possibility of contamination or a burden on the cell at the time of imaging. In addition, since the image is divided into the measurement visual fields 103 that are regions corresponding to the respective cells A, the analysis can be performed while paying attention to each measurement visual field 103. Further, the PC 50 recognizes each captured cell A by distinguishing the captured image based on the geometric feature amount and the optical feature amount of the cell A in the analysis step. In other words, it is possible to accurately and continuously observe changes over time that occur in the culture process while reliably recognizing and chasing each cell A in culture without making a mistake. Further, since the cell A is extracted based on the geometric feature quantity or the optical feature quantity, the cell A that is the observation target can be easily recognized, and the time required for the observation can be shortened.
In addition, the reaction of cell A to be observed can be measured in real time while changing the culture conditions, and the presence or absence of protein expression, the expression level, and the change in expression level over time can be accurately measured. can do.

  Further, the image processing unit 52 recognizes the center of gravity and area of the cell A as a geometric feature amount and recognizes the luminance of the cell A as an optical feature amount, so that each cell A is clearly distinguished with high accuracy. Can be recognized. Therefore, the change with time of the cell A can be reliably observed. In particular, since the image processing unit 52 recognizes the luminance for each wavelength as an optical feature quantity, it is possible to observe various types of proteins and the like corresponding to different wavelengths in one observation.

The technical scope of the present invention is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention.
For example, in the analysis step, the geometrical feature and the optical feature amount of the cell are extracted, but it is only necessary that at least one of them can be extracted. Thereby, an analysis step can be made to correspond according to the number of cells to observe, for example, one cell or a cell group.
Further, although the center of gravity position and area of the cell are used as the geometric feature amount, it is sufficient that at least one of them can be extracted. In addition, the present invention is not limited to this, and a geometric feature amount capable of recognizing each cell may be extracted.
Furthermore, although the brightness of the cells is used as the optical feature amount, the present invention is not limited to this, and an optical feature amount capable of recognizing each cell may be extracted.

In this embodiment, when inputting the measurement target range of the slide glass, the coordinate position of the measurement start position and the measurement end position is input and determined. However, the present invention is not limited to this, and other methods may be used. For example, a method of selecting the measurement target range by dividing the slide glass with a grid and inputting only the coordinate position of the measurement start and setting the number of measurement target grids in the X direction and the Y direction may be used.
That is, as shown in FIG. 13, a square of one side L1 designated by the operator is defined as the measurement target grid 110, and a range of the length L2 designated by the operator is defined as the non-measurement area 120. Then, the operator can alternately arrange the measurement target grid 110 and the non-measurement area 120 in the X direction and the Y direction by specifying the start position 101 of the measurement target grid 110 and the number thereof. Thereby, the measurement object range 100 can be set. For example, by setting the length L2 of the non-measurement area 120 to “0”, the entire surface of the slide glass 2 can be set as the observation target range.
This method is suitable when using a slide glass with a grid in advance.

  Furthermore, as another method of setting the measurement target range, the shape of the slide glass or a pre-scan image is displayed on the PC monitor screen, and the measurement start position and the measurement end position are specified by surrounding them with markers, thereby specifying the measurement target range. The method of setting is also acceptable. The prescan image may be an image obtained by scanning the entire surface of the slide glass with a low-magnification objective lens and pasting the images with a short exposure time and a low resolution. Further, when reading the shape of the slide glass, it is necessary to read the information on the slide glass stored in advance in the memory and indicate the slide glass shape on the monitor screen.

  Moreover, although the slide glass was used as the carrier, a 96-well microplate, a 384-hole microplate, or the like may be employed. At this time, the cells can be cultured in each hole, and the fluorescence intensity from the cell culture can be measured on the lower side (bottom side) of the microplate. In this case, the culture can be continued for several days by adopting a configuration such as extending the scanning stroke, covering the opening of the microplate, or covering with a carbon dioxide supply box.

In the present embodiment, the background of the image can be further stabilized by covering the periphery with a black screen or the like and setting the amount of light around the apparatus to be as stable as possible.
Furthermore, the microscope may be upright. In this case, since it is necessary to accurately control the thickness of the culture solution, an interval ring that does not obstruct the flow of the culture solution is placed between the case of the culture vessel and the glass plate so that the distance between the cells and the glass plate is good. Good to manage. The spacing ring can be replaced with a commutator member.

It is a block diagram which shows one Example of the cell culture observation apparatus based on this invention. It is a schematic diagram which shows the cell culture observation apparatus shown in FIG. It is a top view which shows the state by which the culture container was fixed to the culture container fixing | fixed part. It is sectional drawing which shows the state by which the culture container was fixed to the culture container fixing | fixed part. It is a perspective view which shows the commutator member distribute | arranged in the culture container. It is a figure which shows the measurement object range and imaging step at the time of observing the cell on a slide glass. It is a figure which shows the relationship between a measuring object range and an area. It is a flowchart which shows the observation method of cell culture by the cell culture observation apparatus shown in FIG. FIG. 9 is a flowchart when performing stage movement in the flowchart shown in FIG. 8. FIG. 9 is a flowchart when starting measurement in the flowchart shown in FIG. 8. It is a flowchart which shows the process of an image process part in the case of observing a time-dependent change of a cell. It is a flowchart which shows the process of a data processing part in the case of observing a time-dependent change of a cell. It is a figure which shows the measurement object range at the time of observing the cell on a slide glass, and the modification of an imaging step.

Explanation of symbols

A cell 1 cell culture observation device 2 slide glass (carrier)
10 Culture vessel 30 Electric stage (movable stage)
40 Imaging mechanism (imaging unit)
50 Personal computer (analysis department)

Claims (5)

A cell culture observation apparatus for continuously observing a change with time of one or a plurality of cells present on a carrier or in a solution,
A culture vessel that houses the cells and maintains cell activity;
A movable stage for holding the culture vessel;
An imaging unit that divides and images the cells in the culture container for each region corresponding to each cell;
An analysis unit that extracts and analyzes at least one of a geometric feature amount or an optical feature amount of a cell in the region based on a captured image of each region imaged by the imaging unit, and
The culture vessel is
A culture solution supply pipe connected to one end side and the lower side of the culture vessel and supplying a culture solution to the inside of the culture vessel;
A culture medium discharge pipe connected to the other end side and the upper side of the culture container and discharging the culture medium which is no longer needed from the inside of the culture container;
Disposed inside the culture vessel so as to be adjacent to the culture solution supply pipe and the culture solution discharge pipe, respectively, and distributes the flow of the culture solution supplied from the culture solution supply pipe to the inside of the culture vessel. A commutator member that disperses the flow of the culture solution discharged from the inside of the culture vessel to the culture solution discharge pipe.
The commutator member is a plate-like member, and a plurality of two types of through holes having different hole diameters are formed over the entire surface,
Of the two types of through-holes, one through-hole is arranged in a grid at regular intervals, and the other through-hole having a smaller diameter than the one through-hole is arranged in a grid at equal intervals. A cell culture observation apparatus. In the cell culture observation apparatus according to claim 1 ,
The cell culture observation apparatus, wherein the geometric feature amount is at least one of a gravity center position and an area. In the cell culture observation apparatus according to claim 1 or 2 ,
The cell culture observation apparatus, wherein the optical feature amount is luminance. In the cell culture observation apparatus according to claim 3 ,
The cell culture observation apparatus, wherein the luminance is luminance for each wavelength. A cell culture observation method for continuously observing a change with time of a cell while culturing the cell in the culture vessel using the cell culture observation device according to any one of claims 1 to 4. Because
An imaging step of dividing and imaging the cells in the culture container for each region corresponding to each cell;
An analysis step of extracting and analyzing at least one of a geometric feature value or an optical feature value of a cell in each region imaged in the imaging step.

Images