Device for determining detachment of cultured cells

Abstract

To provide a removal determination device of a cultured cell, capable of peeling a cultured cell by oscillating a culture vessel 2, thereby suppressing damage of the cell, the device being inexpensive.SOLUTION: A removal determination device 1 of a cultured cell comprises: a support member 3 for supporting a culture vessel 2; imaging means 5 for imaging a cultured cell in the culture vessel 2; and control means 7 for inputting a signal from the imaging means 5 for determining that, the cultured cell adhered to a bottom face of the culture vessel 2 is detached from the same face. Shaking means 11 oscillates the support member 3 for integrally oscillating the culture vessel 2 and the imaging means 5 supported thereto, the control means 7 determines that, the cultured cell is detached from the bottom face of the culture vessel, on the basis of a difference of images during oscillation of the culture vessel 2.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a device for determining detachment of cultured cells, and more particularly to a device for determining the adhesion state of cultured cells adhered to the bottom surface of a culture vessel.

Background Art

[0002] Conventionally, there is known a device for determining detachment of cultured cells, which includes a support member for supporting a culture vessel, imaging means for imaging cultured cells in the culture vessel, and control means for inputting a signal from the imaging means and determining the adhesion state of the cultured cells adhered to the bottom surface of the culture vessel (Patent Document 1). The imaging means is incorporated in a phase-contrast microscope, and the control means determines whether or not the cultured cells are adhered to the bottom surface by phase-contrast observation using diffraction and interference of light. In addition, when determining whether or not the cultured cells are adhered to the bottom surface of the culture vessel, the support member for supporting the culture vessel is vibrated by vibration means, and the determination is made after the vibration.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the determination device of Patent Document 1, since the culture vessel can be vibrated to determine whether or not the cultured cells are adhered to the bottom surface of the culture vessel, when a detachment agent is injected into the culture vessel and vibrated for a predetermined time to detach the cultured cells adhered to the bottom surface from the bottom surface, it is possible to determine the presence or absence of detachment after the vibration for the predetermined time. However, since the above-described determination device requires a phase contrast microscope, it is expensive. In addition, since a release agent or vibration may damage cells, it is desirable that these effects be minimized. In view of the above circumstances, the present invention provides a culture cell detachment determination device that is inexpensive and has a low risk of damaging cells.

Means for Solving the Problems

[0005] The invention according to claim 1 is a culture cell detachment determination device comprising a support member for supporting a culture vessel, imaging means for imaging the culture cells in the culture vessel, and control means for inputting a signal from the imaging means and determining the adhesion state of the culture cells adhered to the bottom surface of the culture vessel. The imaging means is provided integrally with the support member, and an oscillating means is provided for oscillating the support member to thereby oscillate the culture vessel supported thereby. The oscillating means oscillates the support member to integrally oscillate the culture vessel and the imaging means. tilt The control means... is tilted ...images the culture cells in the culture vessel by the imaging means, and determines that the culture cells have detached from the bottom surface based on the difference in the images during the oscillation of the culture vessel.

Effects of the Invention

[0006] According to the invention of claim 1, when the culture cells are adhered to the bottom surface of the culture vessel, the culture cells do not move significantly in the culture vessel even when the culture vessel is oscillated by the oscillating means. However, when the culture cells detach from the bottom surface of the culture vessel, the culture cells move significantly in the culture vessel. Therefore, it is possible to easily determine whether the culture cells have detached from the bottom surface based on the difference in the images before and after the movement by the imaging means. That is, it is not necessary to use an expensive phase contrast microscope as the imaging means, and it is possible to easily determine the presence or absence of detachment by using an inexpensive imaging means such as a CCD. In addition, since the oscillating means oscillates the culture vessel instead of vibrating it, the risk of damaging the culture cells can be reduced as compared with the case of applying vibration. Furthermore, according to the present invention, since the oscillation means swings the support member to integrally swing the culture vessel and the imaging means, it is possible to take an image at an arbitrary timing without worrying about the swinging position of the culture vessel, and thereby it is possible to immediately detect that the cultured cells have detached. That is, when the imaging means is fixed and only the culture vessel is swung, there will be a difference in the captured image depending only on whether the posture of the culture vessel is in the horizontal position or the inclined position. However, if the culture vessel and the imaging means are integrally swung, the posture of the captured culture vessel will always be constant. Therefore, it is not necessary to align the posture of the culture vessel before taking an image when comparing images, and the timing setting of imaging becomes easy. Moreover, since it is possible to determine the presence or absence of detachment even during the swinging of the oscillation means, it is possible to quickly detect that the cultured cells have detached, and thereby it is possible to immediately stop the detachment operation, so that it is possible to reduce the adverse effect on the cultured cells by the detachment agent.

Brief Description of the Drawings

[0007]

Figure 1

Modes for Carrying Out the Invention

[0008] Hereinafter, the present invention will be described with reference to the illustrated embodiments. In FIG. 1, a detachment determination device 1 for cultured cells includes a support member 3 that supports a culture vessel 2. The culture vessel 2 is placed on a placement portion 3A of the support member 3 and is detachably held on the placement portion 3A by holding means 4 such as engagement claws provided at a plurality of locations around the culture vessel 2. Adhesive cultured cells that adhere and grow on the bottom surface of the culture vessel 2 are accommodated in the culture vessel 2. The entire detachment determination device 1 is accommodated in an incubator (not shown), and the cells in the culture vessel 2 are cultured. The culture vessel 2 is, for example, a transparent container made of glass or resin, and may be a dish-shaped container called a petri dish or a Petri dish, or may be a horizontally placed flask-shaped container.

[0009] In the illustrated embodiment, the support member 3 is integrally formed in a U-shaped cross section as a whole from a horizontally plate-shaped placement portion 3A on which the culture vessel 2 is placed, a side wall portion 3B extending upward from one side of the placement portion 3A, and a horizontally plate-shaped top surface portion 3C facing the upper surface of the placement portion 3A from the upper end portion of the side wall portion 3B. At the center of the portion of the placement portion 3A where the culture vessel 2 is placed, imaging means 5 for imaging the bottom of the culture vessel 2 placed on the placement portion 3A, more specifically, adherent culture cells that adhere to and grow on the bottom of the culture vessel 2, is provided. As the imaging means 5, an imaging element, for example, a CCD camera can be used. On the other hand, on the top surface portion 3C, illumination means 6 for illuminating the culture vessel 2 from the top surface is provided at a position directly above and facing the imaging means 5 provided on the placement portion 3A. As the illumination means 6, a light emitting element, for example, an LED can be used. The signal from the imaging means 5 is transmitted to control means 7 provided outside the incubator by wire or wirelessly, and image analysis input from the imaging means 5 is performed by the control means 7 so that it is possible to determine the adhesion state of whether the culture cells adhered to the bottom surface of the culture vessel 2 have peeled off from the bottom surface.

[0010] The peeling determination device 1 includes shaking means 11 for shaking the support member 3 to shake the culture vessel 2 supported thereby. In the shaking means 11 of the illustrated embodiment, the support member 3 and the culture vessel 2 can be shaken in a seesaw shape. The shaking means 11 includes a box-shaped base 12 disposed in the incubator, and two support columns 13 erected on both sides of the upper surface of the base 12. Rotating shafts 14 horizontally protruding and fixed on both sides of the placement portion 3A of the support member 3 are rotatably supported by the respective support columns 13. A driven pulley 15 is fixed to the tip of one of the rotating shafts 14, and an endless drive belt 16 is looped around the driven pulley 15. Inside the box-shaped base 12, a drive motor 17 is provided, and the drive belt 16 is looped around a drive pulley 18 provided at the tip of the drive shaft 17A of the drive motor 17.

[0011] Therefore, by rotating the drive motor 17 forward and backward, the support member 3 can be rocked in a seesaw shape via the drive shaft 17A of the drive motor 17, the drive pulley 18, the drive belt 16, the driven pulley 15, and the rotating shaft 14. The drive motor 17 is controlled in operation by control means 7. The control means 7 can rock the support member 3 by rotating the drive motor 17 forward and backward, thereby rocking the culture vessel 2 placed on the support member 3. The culture vessel 2 can be rocked integrally with photographing means 5 and lighting means 6 provided integrally with the support member 3. At that time, the control means 7 can rock the culture vessel 2 by means of a "conditioning operation" so that the culture solution in the culture vessel 2 is evenly distributed to all cells to promote good cell culture, or so that a dissociating agent such as trypsin is evenly distributed to all cells to promote good dissociation. Also, the control means 7 can rock the culture vessel 2 to promote the dissociation when dissociating the cultured cells adhered to the bottom surface of the culture vessel 2 by means of a "dissociation operation". The culture vessel 2 is rocked relatively slowly in the "conditioning operation" and rocked with a larger movement width and more abruptly in the "dissociation operation". The "conditioning operation" can also be used during PBS washing.

[0012] In the above configuration, as described above, the detachment determination device 1 is arranged in the incubator, and the placement portion 3A of the support member 3 is maintained in a horizontal state. In this state, the culture vessel 2 is placed on the placement portion 3A, and the culturing of the cells in the cell vessel 2 is started. The inside of the incubator is maintained at, for example, 37°C and a humidity of 95% RH, and the "acclimation operation" is executed by the control means 7 at a predetermined timing, so that the culture solution in the culture vessel 2 is evenly distributed to all the cells.

[0013] When removing the cultured cells from the culture vessel 2 after the cell culture is completed, a detachment agent such as trypsin is injected into the culture vessel 2. This injection may be performed manually or automatically. When the detachment agent is injected into the culture vessel 2, the control means 7 first activates the drive motor 17 of the shaking means 11 based on the "acclimation operation" to rock the culture vessel 2 and evenly distribute the detachment agent in the culture vessel 2 to all the cells. Thereafter, the control means 7 controls the drive motor 17 at a predetermined timing based on the "detachment operation" to rock the culture vessel 2 under conditions suitable for detaching the cultured cells adhered to the bottom surface of the culture vessel 2 from the bottom surface.

[0014] At the same time, the control means 7 illuminates the cultured cells adhered to the bottom surface of the culture vessel 2 from the top surface by the illumination means 6 and photographs them from the bottom surface by the photographing means 5, inputs the signal from the photographing means 5, and compares the images before and after the posture change of the culture vessel 2 by the shaking means 11. For example, the pre-image taken with the culture vessel 2 in a horizontal state and the post-image taken with the culture vessel 2 in an inclined state are compared. At this time, since the photographing means 5 is swung integrally with the culture vessel 2, the posture of the culture vessel 2 in the pre-image taken with the culture vessel 2 in a horizontal state is the same as the posture of the culture vessel 2 in the post-image taken with the culture vessel 2 in an inclined state. That is, regardless of the posture of the culture vessel 2 by the shaking means 11, even if the culture vessel 2 is photographed at an arbitrary timing, the posture of the culture vessel 2 in the pre-image and the post-image is the same. Therefore, it is possible to detect the displacement of only the cultured cells from the pre-image and the post-image. And for example, by binarizing the image to grasp the contour of the cells and recognizing the change in the contour position, it is possible to determine the detachment. Of course, as the post-image, an image when the culture vessel 2 is tilted from the horizontal state and then returns to the horizontal state, or an image when it is tilted in the reverse direction may be adopted. Also, since the illumination means 6 is also swung integrally with the imaging means 5 and the culture vessel 2, the direction of illumination does not change with the swinging, and the difference in the images can be accurately grasped.

[0015] In a state where the cultured cells remain adhered to the bottom surface of the culture vessel 2 and have not peeled off, the cultured cells adhered to the bottom surface of the culture vessel 2 do not move before and after the swinging, so there is substantially no change between the pre-image and the post-image during the swinging. Therefore, from this, the control means 7 can determine that the cultured cells have not peeled off from the bottom surface of the culture vessel 2. When it is determined that the cultured cells have not peeled off from the bottom surface of the culture vessel 2, the control means 7 continues the "peeling operation" at a predetermined timing. On the other hand, when the cultured cells peel off from the bottom surface of the culture vessel 2, the cultured cells will move greatly due to the tilting of the culture vessel 2, so a large change will occur between the pre-image and the post-image during the swinging. From this, the control means 7 can easily confirm that the cultured cells have peeled off from the bottom surface of the culture vessel 2. When the control means 7 confirms that the cultured cells have peeled off from the bottom surface of the culture vessel 2, it displays and warns to that effect, returns the culture vessel 2 to the horizontal state to end the peeling operation, and then, quickly injects a trypsin inactivator into the culture vessel 2 manually or automatically.

[0016] In the above-described embodiment, the photographing means 5 is provided on the placement portion 3A of the support member 3 so as to photograph the cultured cells from the bottom surface of the culture vessel 2. However, the present invention is not limited to this. The photographing means 5 may be provided on the top surface portion 3C of the support member 3. In this case, the photographing means 5 is arranged at the position of the illumination means 6 on the top surface portion 3C in FIG. 1, and the illumination means 6 is a ring illumination surrounding the periphery of the photographing means 5. That is, when photographing the cultured cells adhered to the bottom surface of the culture vessel 2 from the top surface of the culture vessel 2, adjustment of the focal length is required. In order to omit this, illumination is performed from the top surface in the same manner as photographing to obtain a reflected image of the cultured cells. Even if the image is slightly out of focus, the change in the reflected luminance is captured to recognize the difference between the pre-image and the post-image during swinging, and the adhesion state of the cultured cells is determined. Also, in the above-described embodiment, the shaking means 11 inclines and swings the support member 3 in a seesaw shape. However, the present invention is not necessarily limited to this. As the shaking means 11, various configurations have already been proposed in the past. As presented there, the culture vessel 2 may be rotated or reciprocated on a plane, or may perform these combined operations.

Explanation of Reference Numerals

[0017] 1 Cell detachment determination device for cultured cells 2 Culture vessel 3 Support member 3A Placement portion 3B Top surface portion 5 Photographing means 6 Illumination means 7 Control means 11 Shaking means

Claims

1. A detachment determination device for cultured cells, comprising: a support member for supporting a culture vessel; imaging means for imaging the cultured cells in the culture vessel; and control means for inputting a signal from the imaging means and determining the adhesion state of the cultured cells adhered to the bottom surface of the culture vessel. In this device, the imaging means is provided integrally with the support member, and there is provided a shaking means for shaking the support member to shake the culture vessel supported thereby. The shaking means shakes the support member to integrally shake and incline the culture vessel and the imaging means. The control means images the cultured cells in the culture vessel that is shaken and inclined by the imaging means, and determines that the cultured cells have detached from the bottom surface based on the difference in the images during the shaking of the culture vessel. This is the detachment determination device for cultured cells, characterized by the above.

2. Illumination means for illuminating the culture vessel supported by the support means is provided integrally with the support member. The shaking means shakes the support member to integrally shake the culture vessel and the illumination means. The detachment determination device for cultured cells according to Claim 1, characterized by the above.

3. The support member includes a placement portion on which the culture vessel is placed, and a top surface portion facing the placement portion from above the placement portion. The imaging means is provided on the placement portion to image the cultured cells from the bottom surface of the culture vessel placed on the placement portion, and the illumination means is provided on the top surface portion to illuminate the culture vessel placed on the placement portion. The detachment determination device for cultured cells according to Claim 2, characterized by the above.

4. The support member includes a placement portion on which the culture vessel is placed, and a top surface portion facing the placement portion from above the placement portion. The imaging means is provided on the top surface portion to image the cultured cells from the top surface of the culture vessel placed on the placement portion, and the illumination means is provided on the top surface portion to illuminate the culture vessel placed on the placement portion. The detachment determination device for cultured cells according to Claim 2, characterized by the above.

Images