JP6901263B2 - Cell observation system and cell observation method - Google Patents

Description

The present invention relates to a cell observation system.

Conventionally, in cell culture, each time a cell becomes confluent, the culture vessel is taken out from the incubator, the cell is peeled from the culture vessel, diluted, seeded in a new culture vessel and cultured, that is, passage is repeated. Is done. Generally, when cultured cells are passaged many times, deterioration such as a decrease in proliferative ability occurs, and such deteriorated cells may affect the test results.

Therefore, it is desirable that the cells used in the test have stable quality, and that there is some index for evaluating the quality. For example, the cultured cell evaluation device described in Patent Document 1 classifies cells using the morphological characteristics of the cells as an index, and evaluates the deterioration of the cells.

International Publication No. 2011/021391

However, the evaluation by the cultured cell evaluation device described in Patent Document 1 requires a complicated algorithm, and has a problem that the work is complicated and time-consuming.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a cell observation system capable of easily evaluating the quality of cells.

In order to achieve the above object, the present invention provides the following means.
One aspect of the present invention is an image acquisition unit that acquires an image in a culture vessel for culturing cells over time, and is cultured in the culture vessel based on each of the images acquired by the image acquisition unit. An image analysis unit that quantitatively analyzes the culture status of the cells, a statistical analysis unit that statistically analyzes the data analyzed by the image analysis unit, and the above-mentioned within a plurality of passage cycles obtained by the statistical analysis unit. A cell observation system including a display unit that displays the statistical analysis results in the culture vessel in a contrastable manner, and the display unit displays the growth curve of the cells in each passage cycle as the statistical analysis results. ..

According to this aspect, the image acquisition unit acquires the image in the culture vessel over time, and the image analysis unit quantitatively analyzes the culture state of the cells in the culture vessel based on each image. By statistically analyzing the analyzed data by the statistical analysis unit, it is possible to know the change in the number of cells cultured in the culture vessel. Here, when the passage is repeated, the cells deteriorate, and the deteriorated cells have a reduced proliferative ability.

In this case, by displaying the statistical results in the culture vessel in the culture vessel in the plurality of passage cycles obtained by the statistical analysis unit in a contrastable manner by the display unit, it is possible to display between each passage cycle without requiring a complicated algorithm. Changes in the proliferative capacity of the cells can be visually recognized. This makes it possible to evaluate the quality of cells with a simple operation without any trouble.

In the above aspect, the display unit displays the growth curve of the cell within each passage cycle as the result of the statistical analysis .
When cells deteriorate and proliferative capacity declines due to repeated passages, the rising curve of the proliferative curve becomes gentle. With this configuration, changes in the proliferative capacity of cells in each passage cycle can be grasped at a glance from the slope of the growth curve displayed on the display unit.

In the above aspect, the display unit may display the growth curves of a plurality of the passage cycles in an overlapping manner.
With this configuration, it is possible to grasp at a glance the difference in the proliferative ability of cells in a plurality of passage cycles of interest.

In the above aspect, a comparison unit may be provided which compares the time change of the number of the cells in each passage cycle and outputs the change of the proliferation rate of the cells.
With such a configuration, the comparison unit can easily grasp the change in the proliferative ability of the cells in each passage cycle, and the user can save the trouble of comparing.

In the above aspect, a quality evaluation unit for evaluating the quality of the cells may be provided based on the proliferation rate of the cells in the passage cycle.
With this configuration, the quality evaluation unit can easily divide the cells into passage cycle cells having the desired quality and passage cycle cells not having the desired quality.
In the above aspect, the display unit may display the growth curves of the plurality of passage cycles in series along the time axis.

According to the cell observation system according to the present invention, there is an effect that the quality of cells can be easily evaluated.

It is a schematic block diagram which shows the cell observation system which concerns on 1st Embodiment of this invention. It is a vertical cross-sectional view which shows the culture observation apparatus of FIG. It is a block diagram of the PC main body of FIG. It is a figure which shows an example of the growth curve of each passage cycle displayed on the monitor in chronological order. It is a figure which shows an example of the growth curve of the number of cells in one passage cycle. It is a vertical cross-sectional view which shows the modification of the culture observation apparatus of FIG. It is a vertical cross-sectional view which shows the culture observation apparatus of the cell observation system which concerns on 2nd Embodiment of this invention. It is a vertical cross-sectional view which shows the culture observation apparatus of the cell observation system which concerns on 3rd Embodiment of this invention. It is a top view which shows the internal structure of the culture observation apparatus of FIG. It is a partial vertical sectional view which shows the culture observation apparatus of the cell observation system which concerns on 4th Embodiment of this invention. It is a top view which shows an example of the arrangement of the LED light source in the light source part of the culture observation apparatus of FIG. It is a modification of the culture observation apparatus of FIG. 10, and is a partial vertical sectional view which shows the case where the illumination light is limited by a light-shielding member. In the example of the light-shielding member of FIG. 12, when (a) has a single circular opening, (b) the radial position of the opening is different from (a), (c) the opening is 2 It is a top view which shows each case. FIG. 12 is a plan view showing another example of the light-shielding member of FIG. 12, in which (a) a fan-shaped opening is provided and (b) an annular opening is provided. It is a partial vertical sectional view which shows the other modification of the culture observation apparatus of FIG. It is a partial vertical sectional view which shows the other modification of the culture observation apparatus of FIG. It is a partial vertical sectional view which shows the other modification of the culture observation apparatus of FIG.

[First Embodiment]
The cell observation system according to the first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the cell observation system 1 according to the present embodiment is acquired by a culture observation device (image acquisition unit) 3 capable of culturing cells A and acquiring an image over time, and a culture observation device 3. A PC (Personal Computer) main body 5 for processing the image and a monitor (display unit) 7 for displaying the image acquired by the culture observation device 3 and the processing result obtained by processing the image by the PC main body 5. I have. Reference numeral 9 indicates an incubator.

As shown in FIG. 2, the culture observation device 3 includes a base 11 on which a culture container C containing cells A to be cultured is housed together with a culture solution B, a light source unit 13 provided on the base 11, and an imaging unit 15. It includes a transmission unit 17 and a control unit 19.

The culture vessel C is, for example, a flask for cell culture and is made of an optically transparent material. Reference numeral C1 indicates the bottom surface of the culture vessel C.
As shown in FIG. 2, the base 11 has a mounting surface 11a made of an optically transparent material that adheres the lower surface of the culture container C, and a culture container that is provided upright from the mounting surface 11a and mounted on the mounting surface 11a. It is provided with an abutting surface 11b that brings one side surface of C into close contact. Since the inside of the incubator 9 is in a humid state, the base 11 has a waterproof structure.

The light source unit 13 is arranged on the abutting surface 11b, and includes a plurality of LED light sources 13a arranged at a predetermined distance from the mounting surface 11a and in a direction parallel to the mounting surface 11a. The optical axis 13b of the illumination light emitted from each LED light source 13a is set so as to be substantially parallel to the mounting surface 11a.

The image pickup unit 15 includes a condenser lens 15a arranged below the mounting surface 11a inside the base 11, and an image pickup element 15b that captures the light focused by the condenser lens 15a and acquires an image. .. The image sensor 15b is also arranged on the side opposite to the mounting surface 11a with the condensing lens 15a interposed therebetween, and is also arranged on the optical axis 15c of the condensing lens 15a.

The transmission unit 17 wirelessly transmits the image acquired by the image sensor 15b to the outside.
The control unit 19 includes, for example, a timer (not shown), and periodically operates the light source unit 13, the image pickup unit 15, and the transmission unit 17.

As shown in FIG. 3, the PC main body 5 is a receiving unit 21 that receives an image transmitted from the transmitting unit 17, and cells cultured in the culture vessel C based on the image received by the receiving unit 21. It is provided with a cell analysis unit (image analysis unit, statistical analysis unit) 23 that quantitatively analyzes the culture state of A and statistically analyzes the obtained data.

The cell analysis unit 23 counts the number of cells A cultured in the culture vessel C by a conventional technique. In addition, the cell analysis unit 23 plots the counted number of cells on the time axis to create a growth curve showing the time change of the number of cells A in the culture vessel C within each passage cycle. ing. The period from the passage work to the next passage work is defined as one passage cycle. The growth curve of the number of cells in each passage cycle generated by the cell analysis unit 23 is sent to the monitor 7 as information on the number of cells A.

The monitor 7 can compare and display the time change (statistical analysis result) of the number of cells in the culture vessel C in the plurality of passage cycles based on the information on the number of cells A sent from the cell analysis unit 23. It is designed to do.

For example, as shown in FIG. 4, the monitor 7 displays the growth curve of the number of cells in each passage cycle sent from the cell analysis unit 23 in chronological order. In the example shown in FIG. 4, one passage cycle is set to 3 days, and the growth curves of each passage cycle obtained by repeating the passage from the 1st day to the 21st day are arranged in series along the time axis. ing.

Further, the monitor 7 can display the growth curves of a plurality of passage cycles designated by the operator (user) by superimposing the growth curves of each passage cycle displayed in chronological order. There is. In the example shown in FIG. 4, the growth curve of the passage cycle a from the 10th day to the 12th day and the growth curve of the passage cycle b from the 19th day to the 21st day are superimposed and displayed. ..

The operation of the cell observation system 1 according to the present embodiment configured in this way will be described below.
In order to observe the culture state of the cells A using the cell observation system 1 according to the present embodiment, the culture container C containing the cells A to be cultured and the culture solution B is placed on the mounting surface 11a of the base 11 on the lower surface thereof. It is mounted on the base 11 so as to be in close contact with each other and one side surface abuts on the abutting surface 11b of the base 11.

In this state, the culture observation device 3 equipped with the culture vessel C is housed in the incubator 9 and arranged so that the mounting surface 11a is horizontal, so that the temperature and humidity in the incubator 9 are controlled. The culture of cell A in the culture vessel C is started. Further, at this point, the timer in the control unit 19 is activated to start the time measurement.

When the culture is started, the control unit 19 operates the light source unit 13 to turn on the LED light source 13a and take a picture with the image sensor 15b according to a schedule set in advance according to the time measurement result of the timer.

The LED light source 13a is provided on the abutting surface 11b that abuts the side surface of the culture container C, and illuminates light from the side surface of the culture container C into the culture container C in the direction of the optical axis 13b along the bottom surface C1 of the culture container C. .. As a result, the cells A adhering to the bottom surface C1 of the culture vessel C and growing are illuminated from the side in the same manner as the oblique illumination or the dark field illumination, and the shadow of the cells A is formed.

A part of the scattered light scattered in the cell A passes through the bottom surface C1 of the culture vessel C and the mounting surface 11a of the base 11, is collected by the condensing lens 15a in the base 11, and is photographed by the image pickup element 15b. .. In the image pickup unit 15, for example, the light from the cell A is photographed by the image pickup device 15b every few hours, and an image is acquired.

After each image is acquired, the LED light source 13a is turned off. By intermittently operating the light source unit 13 and the like in this way, it is possible to suppress the temperature rise of the apparatus and reduce the influence of heat on the cells A.

Then, the images acquired by the image sensor 15b over time are sent from the control unit 19 to the transmission unit 17, and are sequentially transmitted to the outside by the transmission unit 17. Outside the incubator 9, the images transmitted from the transmitting unit 17 are sequentially received by the receiving unit 21 of the PC main body 5 and displayed on the monitor 7, so that the culture vessel C is taken out from the incubator 9 and observed. Furthermore, the culture state of the cells A in the culture vessel C can be confirmed outside the incubator 9 without opening the door of the incubator 9. That is, there is an advantage that the troublesomeness of the confirmation work at the time of cell culture can be significantly reduced. Further, since it is not necessary to take the culture vessel C out of the incubator 9, it is possible to eliminate changes in the environment (changes in temperature, pH, etc.) with respect to the cells A.

The images received by the receiving unit 21 are sequentially sent to the cell analysis unit 23, and the cell analysis unit 23 sequentially counts the number of cells A cultured in the culture vessel C based on each image. Then, the cell analysis unit 23 plots the counted number of cells on the time axis, and creates a growth curve of the number of cells in the culture vessel C within one passage cycle. The growth curve of the number of cells generated by the cell analysis unit 23 is displayed on the monitor 7.

When the proliferated cells A reach a state (confluent) spread over one surface of the culture vessel C, the operator once removes the culture observation device 3 from the incubator 9, dilutes the number of cells, and sprinkles the cells into a new culture vessel C. Do substitute work.

The new culture vessel C after the subculture work is mounted on the base 11, and the culture observation device 3 is housed in the incubator 9 again. Then, similarly to the cell A in the culture vessel C above, the cell A is cultured in an environment in which the temperature and humidity in the incubator 9 are controlled, and the image is acquired and displayed on the monitor 7. Further, the cell analysis unit 23 counts the number of cells using the image, creates a growth curve of the number of cells in the culture vessel C within one passage cycle, and displays it on the monitor 7. This is repeated for multiple passage cycles.

On the monitor 7, as shown in FIG. 4, the growth curves of the number of cells in the culture vessel C in the plurality of passage cycles are displayed in series along the time axis. By displaying in this way, it is possible to easily compare the time change of the number of cells in the culture vessel C in each passage cycle. In addition, when cell A deteriorates and the proliferative capacity decreases due to repeated passage, the rising curve of the growth curve becomes gentle, and the slope of the growth curve of each passage cycle causes the growth curve of each passage cycle. The change in the proliferative capacity of cell A can be grasped at a glance.

Further, when the operator specifies the growth curves of any one or more of the passage cycles, the growth curves of those designated passage cycles are superimposed and displayed on the monitor 7. This makes it possible to grasp at a glance the difference in the proliferative ability of cells A in a plurality of passage cycles of interest.

In addition, the operator can evaluate the degree of deterioration of cell A by using the change in the growth curve of each passage cycle as an index. For example, the slope of each growth curve in each passage cycle may be calculated and used as an index for quantitative comparison. In this case, for example, as shown in FIG. 5, the number of cells at _{the beginning of the passage cycle (time t 0 ) is N 0,} and the number of cells at the end of the passage cycle (time t) is N _t . Then, the slope a of the growth curve can be calculated by _{a = (N t} −N ₀ ) / (t −t _0).

Alternatively, the slopes of any two points in the passage cycle may be calculated and used as an index for quantitative comparison. The slope a of the growth curve is a = (N _2- N _{), where N 1} is the number of cells at an arbitrary time point (time t _{1 ) and N 2} is the number of cells at another time point (time t _2). It can be calculated by ₁ ) / (t _2- t _1).

Alternatively, the growth rate may be calculated and used as an index for quantitative comparison. In this case, the growth rate α can be calculated by _{(N t} −N ₀ ) / N _0. The proliferation rate α is (N _2- N ₁ ) / N, where N ₁ is the number of cells at an arbitrary time point (time t _{1 ) and N 2} is the number of cells at another time point (time t _2). It can be calculated by _1.

In addition, the time (doubling time) required for the number of cells to double for the growth curve of each passage cycle may be calculated and quantitatively compared using this as an index. In this case, the doubling time T can be calculated by _{(t-t 0} ) log ₁₀ 2 / log ₁₀ (N _t / N ₀ ) or (t-t ₀ ) / log ₂ (N _t / N _0). ..

Alternatively, the doubling time of any two points in the passage cycle may be calculated and used as an index for quantitative comparison. The number of cells at an arbitrary time point (time t _{1 ) is N 1} , the number of cells at another time point (time t ₂ ) is N ₂ , and the doubling time T is (t _2- t ₁ ) log _10. It can be calculated by 2 / log ₁₀ (N ₂ / N ₁ ) or (t _2- t ₁ ) / log ₂ (N ₂ / N _1). Here, t ₁ and t ₂ are preferably any two points in the logarithmic growth phase.

As described above, according to the cell observation system 1 according to the present embodiment, it is complicated by displaying the time change of the number of cells in the culture vessel C in the culture vessel C in a plurality of passage cycles in a contrastable manner by the monitor 7. It is possible to visually recognize changes in the proliferative capacity of cell A during each passage cycle without requiring a complicated algorithm. As a result, the quality of the cell A can be evaluated by a simple operation without any trouble.

Further, since the imaging unit 15 photographs the scattered light transmitted through the bottom surface C1 of the culture container C among the scattered light in the cultured cells A, the culture container C is used when the cells are cultured in a high temperature and high humidity environment. A clear image can be obtained without being affected by water droplets formed by dew on the upper surface of the surface.

Further, in the present embodiment, since the LED light source 13a is used as the light source unit 13, there is an advantage that heat generation can be suppressed, the influence on the cells A can be reduced, and power consumption can be suppressed.

In the present embodiment, a cell proliferation curve may be created by calculating the cell density (the number of cells per unit area) instead of the number of cells and plotting it on the time axis. Alternatively, a cell proliferation curve may be created by calculating the area occupied by cells A in the culture vessel C instead of the number of cells and plotting it on the time axis.

Further, in the present embodiment, for example, the cell analysis unit 23 may function as a comparison unit that compares the time change of the number of cells in each passage cycle and outputs the change in the proliferation rate of the cell A. .. By doing so, the operator can easily grasp the change in the proliferative ability of the cell A for each passage cycle, and the operator himself can save the trouble of comparing.

Further, in the present embodiment, for example, the cell analysis unit 23 may function as a quality evaluation unit that evaluates the quality of the cell A based on the proliferation rate of the cell A in each passage cycle. By doing so, the cell analysis unit 23 can easily evaluate the cell A having a desired quality.

In this case, for example, when the proliferation rate of cell A is larger than a predetermined threshold value at the final stage of the culture step, it may be determined that the cell A in the culture vessel C is of good quality. Further, when the cell A deteriorates and the proliferative ability decreases, the doubling time of the number of cells becomes long. Therefore, when the doubling time until reaching the predetermined number of cells is shorter than the predetermined threshold value, the doubling time in the culture vessel C becomes longer. Cell A may be judged to be of good quality.

Further, in the present embodiment, the illumination light from the light source unit 13 is incident on the culture vessel C in the horizontal direction along the optical axis 13b parallel to the bottom surface C1 of the culture vessel C. The illumination light from the light source unit 13 may be incident on the culture vessel C at an angle of ± 30 ° or less with respect to the horizontal direction without limitation. Even if such an angle is adopted, a shadow similar to dark field illumination or oblique illumination can be formed on the cell A, and a three-dimensional image can be taken.

Further, in the present embodiment, as shown in FIG. 6, the optical axis 15c of the condenser lens 15a is orthogonal to the bottom surface C1 of the culture vessel C, and a partial image of the bottom surface C1 is acquired. Good. In this case, for example, the optical axis 15c of the condenser lens 15a may be bent by one or more mirrors 15d. Although the certainty of determining the culture state is lower than when the entire bottom surface C1 of the culture container C or a relatively wide part of the bottom surface C1 is photographed, the culture state may be estimated from the image of this partial region. ..

Further, in the present embodiment, the control unit 19 is provided with a timer to periodically operate the light source unit 13 and the like, but instead of this, a receiving unit (not shown) is connected to the control unit 19. Therefore, a command signal from the outside of the incubator 9 may be received, and the control unit 19 may drive the light source unit 13 and the like in response to the command signal. The light source unit 13 may be turned on / off and photographed by remote control according to an instruction input in advance by the operator, or the light source unit 13 may be turned on / off and photographed by remote control at an arbitrary timing.
The transmission and reception of the image signal and the command signal may be performed wirelessly or by wire.

Further, in the present embodiment, the aspect of displaying the cell proliferation curve is shown, but it may be displayed as a straight line by plotting on the logarithmic axis. By doing so, the portion corresponding to the logarithmic proliferation phase in the passage cycle is displayed as a straight line, and the cell proliferation ability can be grasped and compared more intuitively.
Further, instead of the cell proliferation curve, the change in the number of cells may be displayed as a bar graph or may be displayed as discontinuous dots.

[Second Embodiment]
Next, the cell observation system according to the second embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 7, the cell observation system 31 according to the present embodiment is different from the cell observation system 1 according to the first embodiment in the imaging unit 33.
In the description of the present embodiment, the same reference numerals are given to the parts having the same configuration as the cell observation system 1 according to the first embodiment described above, and the description thereof will be omitted.

The image pickup unit 33 is arranged below the mounting surface 11a of the base 11, a microlens array 35 having a plurality of microlenses 35a arranged on a plane substantially parallel to the mounting surface 11a, and further below the microlens array 35. It is provided with the image sensor 35b. The microlens 35a of the microlens array 35 is arranged corresponding to each pixel of the image sensor 35b.

The focal length of each microlens 35a is set to be larger than the thickness dimension of the transparent member constituting the mounting surface 11a and the thickness dimension of the bottom surface C1 of the culture vessel C mounted on the mounting surface 11a. Has been done. Each microlens 35a is focused on the cell A adhering to the bottom surface C1 of the culture vessel C and is arranged below the mounting surface 11a, so that the image of the cell A is projected on the imaging surface of the image sensor 35b. You can do it.

It is not always necessary for the image sensor 35b to take an image of the entire bottom surface C1 of the culture vessel C, and an image is acquired for an arbitrary partial region such as the central portion where the cell A is likely to be present, and the acquired image is used. Based on this, the culture state may be estimated.

[Third Embodiment]
Next, the cell observation system according to the third embodiment of the present invention will be described below with reference to the drawings.
As shown in FIGS. 8 and 9, the cell observation system 41 according to the present embodiment includes a focus adjustment mechanism 43 for moving the condenser lens 15a of the imaging unit 15 in a direction along the optical axis 15c, and an imaging unit 15. It differs from the first embodiment in that it includes a moving mechanism 45 that moves two-dimensionally in a direction along the mounting surface 11a.
In the description of the present embodiment, the same reference numerals are given to the parts having the same configuration as the cell observation system 1 according to the first embodiment described above, and the description thereof will be omitted.

The focus adjusting mechanism 43 is, for example, a linear motion mechanism including a motor 43a and a ball screw 43b. The focus adjusting mechanism 43 is fixed to the nut 43c by rotating the ball screw 43b by the operation of the motor 43a and linearly moving the nut 43c meshing with the ball screw 43b along the optical axis 15c of the condenser lens 15a. The condenser lens 15a can be moved along the optical axis 15c.

As shown in FIG. 9, the moving mechanism 45 is composed of two linear motion mechanisms 47 and 49 arranged orthogonally. The first linear motion mechanism 47 includes a guide rail 47a fixed to the base 11, a slider 47b movably supported in the first horizontal direction X along the guide rail 47a, and a drive mechanism 47c for moving the slider 47b. It has. The drive mechanism 47c includes a motor 47d and a ball screw 47e.

Further, the second linear motion mechanism 49 includes a guide rail 49a fixed to the slider 47b of the first linear motion mechanism 47, a slider 49b movably supported in the second horizontal direction Y along the guide rail 49a, and the like. It is provided with a drive mechanism 49c for moving the slider 49b. The drive mechanism 49c includes a motor 49d and a ball screw 49e.

The operator confirms whether or not the focus of the condenser lens 15a is properly aligned with the cell A by the image acquired by the image sensor 15b, and if not, the focus adjusting mechanism 43 is used. A command signal for operating in that direction is input, and the focus adjusting mechanism 43 is operated by the control unit 19.

When the ball screw 43b is rotated by the rotation of the motor 43a, the nut 43c moves in any of the horizontal directions according to the rotation direction of the ball screw 43b, and the condenser lens 15a fixed to the nut 43c moves in the horizontal direction. As a result of being moved, the focal position of the condenser lens 15a is moved in the vertical direction.

That is, when the condenser lens 15a is moved in a direction close to the mirror 15d, the focal position is raised, and when the condenser lens 15a is moved in a direction away from the mirror 15d, the focal position is lowered. .. As a result, the focal position can be adjusted to an appropriate position and a clear image can be obtained.

Further, when the operator wants to observe different positions, he / she inputs a command signal for moving the moving mechanism 45 in any direction, and the control unit 19 operates the moving mechanism 45. By moving the imaging unit 15 along the second linear motion mechanism 49, the observation position is moved in the horizontal direction Y, and the second linear motion mechanism 49 and the imaging unit 15 are moved along the first linear motion mechanism 47. This moves the observation position in the horizontal direction X. Thereby, the observation position can be adjusted two-dimensionally.

The light source unit 13 of the present embodiment may be arranged at a position to be illuminated from above the culture container C. Further, the light source unit 13 may be arranged at a position where it is illuminated from the side surface of the culture vessel C. Further, the light source unit 13 may be arranged at a position to be illuminated from the bottom surface of the culture vessel C, or the light source unit 13 may be moved together with the image pickup unit 15 by the moving mechanism 45.

[Fourth Embodiment]
The first embodiment and the second embodiment show the mode of irradiating the cell A with the illumination light from the side, but the mode of irradiating the cell A with the illumination light from below will be described below.
In the cell observation system 51 according to the present embodiment, as shown in FIG. 10, the culture observation device 3 is arranged in a stage 53 on which the culture vessel C containing the cells A is placed and a stage below the stage 53. The objective lens 55 that collects the light transmitted through the 53 from above, the photographing optical system 57 that captures the light transmitted through the cell A, and the objective lens 55 are arranged on the outer side in the radial direction and pass through the stage 53. It is provided with a light source unit 59 that emits illumination light upward.

An optically transparent material such as a glass plate 53a is arranged on the stage 53 so as to cover the upper part of the objective lens 55 and the light source portion 59, and the culture vessel C is placed on the upper surface of the glass plate 53a. It has become.
The bottom surface of the culture vessel C is indicated by reference numeral C1, and the top plate of the culture vessel C is indicated by reference numeral C2.

As shown in FIGS. 10 and 11, the light source unit 59 is provided on a plurality of LED light sources (light sources) 61 arranged around the objective lens 55 at intervals in the circumferential direction and the radial direction, and each LED light source 61. A plurality of collimating lenses 63 which are arranged correspondingly and make the illumination light generated in each LED light source 61 substantially parallel light, and a diffuser plate 65 which diffuses the illumination light made substantially parallel by the collimating lens 63 are provided. There is.

The light source unit 59 can independently light the specific LED light source 61 (FIGS. 10 and 11 show the lit LED light source 61 by hatching).
That is, by lighting only the LED light sources 61 at different positions in the radial direction of the objective lens 55, the glass plate 53a and the bottom surface C1 of the culture vessel C are transmitted from the bottom to the top as shown by the solid line in FIG. After that, the angle of the illumination light that is reflected on the inner surface of the top plate C2 of the culture vessel C, passes through the cell A, the bottom surface C1 of the culture vessel C and the glass plate 53a from diagonally above, and is incident on the objective lens 55 is broken. It is possible to switch as shown by.

Further, by lighting only the LED light source 61 at a specific position in the circumferential direction of the objective lens 55, the cell A can be illuminated only from a specific direction in the circumferential direction. Further, as shown in FIG. 11, by lighting the LED light source 61 arranged in two or more directions in the circumferential direction of the objective lens 55, particularly in a direction axially symmetric with respect to the optical axis S of the objective lens 55. It is possible to irradiate the cell A with illumination light with reduced illumination unevenness.

The observation method using the cell observation system 51 according to the present embodiment configured in this way will be described below.
In order to observe transparent cells A using the cell observation system 51 according to the present embodiment, as shown in FIG. 10, the culture vessel C is in a state where the cells A are adhered to the bottom surface C1 of the culture vessel C. Is placed on the glass plate 53a of the stage 53 so that the bottom surface C1 is on the lower side.

Then, in this state, any LED light source 61 of the light source unit 59 is operated to generate illumination light. The illumination light generated in the LED light source 61 is made substantially parallel by the collimated lens 63 arranged corresponding to the LED light source 61, and is diffused by the diffuser plate 65, and the bottom surface of the glass plate 53a and the culture vessel C is diffused. C1 is transmitted from the bottom to the top, reflected on the inner surface of the top plate C2 of the culture vessel C, and irradiated to the cell A from diagonally above.

Of the illumination light emitted to the cell A, the transmitted light of the illumination light transmitted through the cell A passes through the bottom surface C1 of the culture vessel C and the glass plate 53a from top to bottom and is incident on the objective lens 55. At this time, the illumination light is refracted and scattered depending on the shape and refractive index of the cell A, or dimmed by the transmittance of the cell A to become transmitted light carrying the information of the cell A by the objective lens 55. It is focused and photographed by an image pickup element (not shown).

As described above, according to the cell observation system 51 according to the present embodiment, since the photographing optical system 57 including the light source unit 59 and the objective lens 55 is arranged below the cell A, conventionally, both sides of the cell A are sandwiched. Compared with the transmitted light observation device in which the light source unit and the photographing optical system are arranged, the light source unit 59 and the photographing optical system 57 can be integrated on only one side of the cell A, and the device can be made thinner. There is. Further, even in such a thin cell observation system 51, there is an advantage that a subject such as a cell can be observed without being labeled by photographing the transmitted light.

Further, the illumination light from the light source unit 59 is emitted from the outside in the radial direction of the objective lens 55 and reflected on the inner surface of the top plate C2 of the culture vessel C, so that the cells A are irradiated from diagonally above the objective lens. Since the light is focused by 55, it is possible to form light and dark in the image of cell A by appropriately setting the angle of incidence on cell A, and it is possible to obtain an image that is easy to see even for a transparent subject such as a cell. There is an advantage that it can be done.

Further, in the present embodiment, since the light source units 59 are arranged in the radial direction around the objective lens 55 and include a plurality of LED light sources 61 that can be turned on independently, as shown by a broken line in FIG. In addition, the irradiation angle of the illumination light incident on the cell A can be changed by changing the radial position of the LED light source 61 to be lit. As a result, when the incident angle is smaller than the capture angle of the objective lens 55, the bright field illumination has less uneven illumination, and when the incident angle is larger than the capture angle of the objective lens 55, the dark field illumination emphasizes the fine structure. Further, when the incident angle is the same as the capture angle of the objective lens 55, the oblique illumination can be used so that the cell A can be seen three-dimensionally.

Further, in the present embodiment, since the light source units 59 are arranged in the circumferential direction around the objective lens 55 and include a plurality of LED light sources 61 that can be independently lit, the circumferential direction of the LED light source 61 to be lit is provided. By changing the position, the irradiation direction of the illumination light incident on the cell A can be changed. As a result, the direction of the shadow of the formed cell A image can be changed to change the appearance.

Further, as shown in FIG. 11, by simultaneously lighting a plurality of LED light sources 61 at different positions in the circumferential direction, in particular, by simultaneously lighting a plurality of LED light sources 61 arranged axially symmetrically, uneven illumination There is an advantage that the image of the cell A with less unevenness can be obtained.

Further, in the present embodiment, since the diffuser plate 65 is provided corresponding to each LED light source 61, the illumination light emitted from the LED light source 61 is uniformly diffused, and the illumination has a uniform intensity with less uneven illumination. The cell A can be irradiated with light.

In the present embodiment, a plurality of LED light sources 61 are arranged in an array and turned on independently to switch the irradiation angle, irradiation direction, etc. of the illumination light. As shown in FIGS. 12 to 14, the light source unit 59 includes a light source 67 arranged around the objective lens 55 and a light-shielding member 69 arranged above the light source 67 and shielding the illumination light from the light source 67. You may be prepared.

That is, the light-shielding member 69 has an opening 69a that opens in a part in the circumferential direction or a part in the radial direction, and a transmission that reflects light on the inner surface of the top plate C2 of the culture vessel C and transmits the light transmitted through the cell A. The holes 69b are provided, and by replacing the light-shielding member 69, the position of the opening 69a can be adjusted to change the irradiation angle and irradiation direction of the illumination light.

The light source unit 59 may include an LED light source 61, a collimating lens 63, and a diffuser plate 65 arranged in an array as described above, but the function of switching the emission position of the illumination light is unnecessary, and the opening 69a A light source having an arbitrary light source may be adopted as long as it is a light source capable of emitting illumination light from a wider range.

13 (a) to 13 (c) are examples having circular openings 69a, and show examples in which the radial direction and the number of openings 69a are different. FIG. 14A shows a case where the opening 69a has a fan shape, and FIG. 14B shows a case where the opening 69a has an annular shape. Any size, position and shape of the opening 69a can be adopted.

Further, in the present embodiment, the cells A are housed in the culture vessel C having the top plate C2 such as a cell culture flask, and the illumination light is reflected on the inner surface of the top plate C2 of the culture vessel C. It is not limited to this. For example, when the cell A is housed in a container 71 having no top plate such as a petri dish (without a lid) as the culture container C, as shown in FIG. 15, a mirror is placed at a position to close the upper opening of the petri dish. The reflection member 73 as described above may be arranged, and the reflection member 73 may reflect the illumination light transmitted from the bottom to the top of the bottom surface 71b of the container 71. The reflective member 73 may be provided so as to be removable at an upper position of the cell A by linear motion or rocking.

Further, in the present embodiment, as shown in FIG. 16, a light-shielding member 75 made of a material that shields light may be provided above the top plate C2 of the culture container C.
By doing so, since the external light from the outside is shielded by the light-shielding member 75, it is possible to suppress the external light from entering the culture vessel C from the top plate C2 and to perform the observation efficiently. ..

Further, in the present embodiment, as the light source unit 59, the LED light source 61, the collimating lens 63, and the diffuser plate 65 are arranged substantially horizontally along the glass plate 53a, but instead of this, FIG. 17 As shown in the above, the LED light source 61, the collimating lens 63, and the diffuser plate 65 may be arranged so as to be tilted toward the optical axis S.
By doing so, it is possible to suppress the loss of the illumination light emitted from the LED light source 61 and efficiently irradiate the cell A with the illumination light.

Further, in the present embodiment, the light source unit 59 includes a diffuser plate 65, but the diffuser plate 65 may not be provided.
Further, in the present embodiment, the light source unit 59 including the collimating lens 63 is illustrated, but the collimating lens 63 may not be provided.
Further, in the present embodiment, the light source unit 59 including the collimating lens 63 and the diffuser plate 65 is illustrated, but the arrangement of the collimating lens 63 and the diffuser plate 65 may be reversed. The collimating lens 63 and the diffuser 65 may not be provided.

Further, in the present embodiment, the configuration in which the light source unit 59 is arranged below the culture container C has been described as an example. However, for example, the light source unit 59 is arranged above the culture container C with respect to the cell A. The illumination light may be irradiated from above.
Further, in the present embodiment, the photographing optical system 57 may be moved in the X and Y directions by the drive mechanism. In this case, the light source unit 59 may be moved together with the photographing optical system 57 by the drive mechanism.

Further, in the present embodiment, a configuration in which a plurality of LED light sources (light sources) 61 are arranged around the objective lens 55 at intervals in the circumferential direction and the radial direction as the light source unit 59 has been described as an example. , A plurality of LED light sources (light sources) may be arranged at intervals only in the circumferential direction. For example, four LED light sources (light sources) may be arranged at intervals of 90 degrees in the circumferential direction. One LED light source (light source) may be arranged.

Although the embodiments of the present invention have been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and design changes and the like within a range not deviating from the gist of the present invention are also included. For example, the present invention is not limited to the one applied to each of the above embodiments and modifications, and may be applied to an embodiment in which these embodiments and modifications are appropriately combined, and the present invention is not particularly limited. .. Further, transmission / reception between the transmission unit 17 and the reception unit 21 may be wired or wireless.

1,31,41,51 Cell observation system 3 Culture observation device (image acquisition unit)
7 Monitor (display)
23 Cell analysis department (image analysis department, statistical analysis department, comparison department, quality evaluation department)
A cell C culture vessel

Claims (6)

An image acquisition unit that acquires images in the culture vessel in which cells are cultured over time,
Based on each of the images acquired by the image acquisition unit, an image analysis unit that quantitatively analyzes the culture state of the cells cultured in the culture vessel, and an image analysis unit.
A statistical analysis unit that statistically analyzes the data analyzed by the image analysis unit,
It is provided with a display unit for displaying the statistical analysis results in the culture vessel in a plurality of passage cycles obtained by the statistical analysis unit in a contrastable manner .
A cell observation system in which the display unit displays the growth curve of the cells within each passage cycle as the result of the statistical analysis. The cell observation system according to claim 1 , wherein the display unit superimposes and displays the growth curves of the plurality of passage cycles. The cell observation system according to claim 1 or 2 , further comprising a comparison unit that compares the time change of the number of the cells in each passage cycle and outputs the change in the proliferation rate of the cells. The cell observation system according to any one of claims 1 to 3 , further comprising a quality evaluation unit for evaluating the quality of the cells based on the proliferation rate of the cells in the passage cycle. The cell observation system according to claim 1 , wherein the display unit displays the growth curves of the plurality of passage cycles in series along a time axis. Images of the inside of the culture vessel in which the cells are cultured are acquired over time.
Based on each of the acquired images, the culture status of the cells cultured in the culture vessel was quantitatively analyzed.
Statistical analysis of the analyzed data
A cell observation method for displaying the growth curve of the cells in each of the passage cycles as a result of statistical analysis in the culture vessel in the plurality of passage cycles obtained by the statistical analysis.

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