JP7709015B2 - Device for determining detachment of cultured cells - Google Patents

Description

The present invention relates to a device for determining whether cultured cells have been detached, and more specifically, to a device for determining whether cultured cells have been detached from the bottom surface of a culture vessel.

Conventionally, a cultured cell detachment determination device is known that includes a support member that supports a culture vessel, an imaging means that images the cultured cells in the culture vessel, and a control means that inputs a signal from the imaging means and determines the adhesion state of the cultured cells adhered to the bottom surface of the culture vessel (Patent Document 1).
The photographing means is incorporated in a phase-contrast microscope, and the control means is adapted to judge whether or not the cultured cells are attached to the bottom surface by phase-contrast observation utilizing the diffraction and interference of light.
Furthermore, when determining whether or not the cultured cells are attached to the bottom surface of the culture vessel, the support member supporting the culture vessel is vibrated by the vibration means, and the determination is made after the vibration.

Patent No. 5446082

The determination device of Patent Document 1 mentioned above can determine whether or not cultured cells are adhered to the bottom surface of the culture vessel by vibrating the culture vessel. Therefore, when a release agent is injected into the culture vessel and the vessel is vibrated for a predetermined period of time to release the cultured cells adhered to the bottom surface from the bottom surface, it becomes possible to determine whether or not the cells have been detached after the predetermined period of vibration.
However, the above-mentioned assessment device requires a phase-contrast microscope, which makes it expensive, and since there is a risk that the release agent and vibrations may damage the cells, it is desirable to minimize the effects of these.
In view of the above circumstances, the present invention provides an apparatus for determining detachment of cultured cells which is inexpensive and has a low risk of damaging the cells.

The present invention provides an apparatus for determining whether cultured cells have been detached, the apparatus comprising: a support member for supporting a culture vessel; an imaging means for imaging cultured cells in the culture vessel; and a control means for inputting a signal from the imaging means and determining an adhesion state of the cultured cells adhered to the bottom surface of the culture vessel,
The photographing means is provided integrally with the support member, and a shaking means is provided for shaking the support member to shake the culture vessel supported by it, the shaking means shakes the support member to shake and tilt the culture vessel and the photographing means together, and the control means photographs the cultured cells in the culture vessel that is shaken and tilted by the photographing means, and determines that the cultured cells have detached from the bottom surface from the difference in the image while the culture vessel is being shaken.

According to the invention of claim 1, when the cultured cells are attached to the bottom surface of the culture vessel, the cultured cells do not move significantly within the culture vessel even when the culture vessel is agitated by a shaking means. However, when the cultured cells detach from the bottom surface of the culture vessel, the cultured cells begin to move significantly within the culture vessel. Therefore, it becomes possible to easily determine whether or not the cultured cells have detached from the bottom surface from the difference in images taken by the photographing means before and after the movement.
In other words, it is not necessary to use an expensive phase contrast microscope as a photographing means, and it is possible to easily determine the presence or absence of detachment by an inexpensive photographing means such as a CCD. In addition, since the shaking means rocks the culture vessel instead of vibrating it, the risk of damaging the cultured cells can be reduced compared to when vibration is applied.
Furthermore, according to the present invention, the shaking means rocks the support member to rock the culture vessel and the imaging means together, so that imaging can be performed at any timing without being concerned about the rocking position of the culture vessel, thereby making it possible to immediately detect that the cultured cells have been detached.
In other words, if the imaging means is fixed and only the culture vessel is rocked, differences will occur in the captured images depending on whether the orientation of the culture vessel is horizontal or tilted, but if the culture vessel and the imaging means are rocked together, the orientation of the captured culture vessel is always constant, so that when comparing images, it is not necessary to align the orientation of the culture vessel before taking the image, making it easier to set the timing of the image capture. Moreover, since it is possible to determine whether or not the cultured cells have detached even while the rocking means is rocking, it is possible to quickly detect that the cultured cells have detached, and as a result, the detachment operation can be stopped immediately, making it possible to reduce the adverse effects of the detachment agent on the cultured cells.

FIG. 1 is a perspective view showing an embodiment of the present invention;

The present invention will now be described with reference to the illustrated embodiment. In FIG. 1, a cultured cell detachment determination device 1 has a support member 3 for supporting a culture vessel 2, and the culture vessel 2 is placed on a mounting portion 3A of the support member 3 and is detachably held on the mounting portion 3A by holding means 4, such as engaging claws, provided at multiple points around the culture vessel 2.
The culture vessel 2 contains adhesive cultured cells that adhere to the bottom surface of the culture vessel 2 and grow therein, and the entire detachment determination device 1 is contained in an incubator (not shown) so that the cells in the culture vessel 2 are cultured.
The culture vessel 2 is, for example, a transparent vessel made of glass or resin, and may be a dish-type vessel called a petri dish or a petri dish, or may be a horizontal flask-type vessel.

In the illustrated embodiment, the support member 3 is integrally formed with the horizontal plate-shaped mounting portion 3A on which the culture vessel 2 is placed, a side wall portion 3B extending upward from one side of the mounting portion 3A, and a horizontal plate-shaped top surface portion 3C facing the upper surface of the mounting portion 3A from the upper end portion of the side wall portion 3B, so as to have an overall U-shaped cross section.
An imaging means 5 is provided in the center of the area where the culture vessel 2 is placed on the mounting portion 3A, for photographing the bottom of the culture vessel 2 placed on the mounting portion 3A, more specifically, the adhesive cultured cells adhering to and growing on the bottom of the culture vessel 2. The imaging means 5 can be an image sensor, for example a CCD camera.
On the other hand, the top surface portion 3C is provided with an illumination means 6 for illuminating the culture vessel 2 from the top surface, directly above and opposite the photographing means 5 provided on the mounting portion 3A, and the illumination means 6 can be a light-emitting element, for example an LED.
The signal from the imaging means 5 is transmitted by wire or wirelessly to a control means 7 provided outside the incubator, and the control means 7 performs image analysis of the image input from the imaging means 5, thereby making it possible to determine the adhesion state of the cultured cells adhered to the bottom surface of the culture vessel 2, i.e., whether or not they have detached from the bottom surface.

The detachment determination device 1 is equipped with a shaking means 11 that rocks the support member 3 to rock the culture vessel 2 supported by it, and in the shaking means 11 of the illustrated embodiment, the support member 3 and the culture vessel 2 can be rocked in a seesaw-like manner.
The shaking means 11 comprises a box-shaped base 12 to be placed inside the incubator, and two pillars 13 erected on both sides of the upper surface of the base 12, and a rotating shaft 14 which protrudes horizontally and is fixed to both sides of the mounting portion 3A of the support member 3 is supported by each of the pillars 13 so as to be freely rotatable.
A driven pulley 15 is fixed to the tip of one of the rotating shafts 14, and an endless drive belt 16 is stretched around this driven pulley 15.
A drive motor 17 is provided inside the box-shaped base 12, and the drive belt 16 is stretched around a drive pulley 18 provided at the tip of a drive shaft 17A of the drive motor 17.

Therefore, by rotating the drive motor 17 forward and backward, the support member 3 can be swung in a seesaw manner 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 operation of the drive motor 17 is controlled by the control means 7, and the control means 7 can rotate the drive motor 17 forward and backward to oscillate the support member 3 and thereby oscillate the culture vessel 2 placed on the support member 3, and can oscillate the culture vessel 2 together with the imaging means 5 and lighting means 6 which are provided integrally with the support member 3.
At this time, the control means 7 is capable of rocking the culture vessel 2 through a "familiarization operation" to ensure that the culture fluid in the culture vessel 2 is distributed evenly to all the cells, thereby promoting good cell cultivation, or that a detachment agent such as trypsin is distributed evenly to all the cells, thereby promoting good detachment.
In addition, the control means 7 is capable of performing a "detachment operation" to rock the culture vessel 2 to promote detachment of cultured cells adhered to the bottom surface of the culture vessel 2 from the bottom surface.
The culture vessel 2 is swung relatively slowly in the "acclimatization operation", and is swung a little more rapidly with a larger movement range in the "peeling operation". The "acclimatization operation" can also be used during PBS washing.

In the above configuration, the detachment determination device 1 is placed in an incubator as described above, and the mounting portion 3A of the support member 3 is maintained in a horizontal state. In this state, the culture vessel 2 is placed on the mounting portion 3A, and the culture of cells in the cell vessel 2 is started.
The inside of the incubator is maintained at, for example, 37° C. and humidity of 95% RH, and the control means 7 executes an “acclimatization operation” at a predetermined timing, so that the culture fluid in the culture container 2 is distributed uniformly over all the cells.

When the cell culture is completed and the cultured cells are to be removed from the culture vessel 2, a detachment agent such as trypsin is injected into the culture vessel 2. This injection may be performed manually or automatically.
When the release agent is injected into the culture vessel 2, the control means 7 first starts the drive motor 17 of the shaking means 11 based on the ``acclimation operation'' to rock the culture vessel 2 and distribute the release agent evenly to all of the cells in the culture vessel 2.
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.

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 with the lighting means 6 and photographs them from the bottom surface with the imaging means 5, inputs a signal from the imaging means 5, and compares the images before and after the change in the attitude of the culture vessel 2 caused by the shaking means 11. For example, a pre-image taken when the culture vessel 2 was horizontal and a post-image taken when the culture vessel 2 was tilted 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 front image photographed when the culture vessel 2 is horizontal is the same as the posture of the culture vessel 2 in the rear image photographed when the culture vessel 2 is tilted. In other words, even if the photographing of the culture vessel 2 is performed at any timing without taking into account the posture of the culture vessel 2 by the shaking means 11, the posture of the culture vessel 2 in the front image and the rear image will be the same, so it becomes possible to detect the displacement of only the cultured cells from the front image and the rear image.
For example, the image can be binarized to grasp the outline of the cell, and detachment can be determined by recognizing the change in the position of the outline.
Of course, the subsequent image may be an image taken when the culture vessel 2 is tilted from a horizontal state and then returned to a horizontal state, or an image taken when the culture vessel 2 is tilted in the opposite direction.
Furthermore, since the illumination means 6 is also swung integrally with the imaging means 5 and the incubation container 2, the direction of illumination does not change with the swinging, and the difference between the images can be accurately grasped.

When the cultured cells remain adhered to the bottom surface of the culture vessel 2 and have not been detached, the cultured cells adhered to the bottom surface of the culture vessel 2 do not move before and after the rocking motion, so there is essentially no change between the before and after images. Therefore, from this, the control means 7 can determine that the cultured cells have not been detached from the bottom surface of the culture vessel 2.
When it is determined that the cultured cells have not been detached from the bottom surface of the culture vessel 2, the control means 7 continues the "detachment operation" at a predetermined timing.
On the other hand, when the cultured cells are detached from the bottom surface of the culture vessel 2, the cultured cells move significantly due to the tilt of the culture vessel 2, resulting in a significant change between the before and after images during the above-mentioned rocking. This enables the control means 7 to easily confirm that the cultured cells have detached from the bottom surface of the culture vessel 2.
When the control means 7 confirms that the cultured cells have been detached from the bottom surface of the culture vessel 2, it displays and warns the user of this fact, returns the culture vessel 2 to a horizontal position, and ends the detachment operation. After that, a trypsin inactivator is promptly injected into the culture vessel 2 manually or automatically.

In the above embodiment, the photographing means 5 is provided on the mounting portion 3A of the support member 3 to photograph the cultured cells from the bottom surface of the culture vessel 2, but the present invention is not limited to this. The photographing means 5 may be provided on the top surface 3C of the support member 3. In this case, the photographing means 5 is placed at the position of the illumination means 6 on the top surface 3C in FIG. 1, and the illumination means 6 is a ring illumination surrounding 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, it is necessary to adjust the focal length. In order to omit this, illumination is performed from the top surface as in the photographing, and a reflected image of the cultured cells is obtained. Even if the image is slightly out of focus, the change in reflected brightness is captured, and the difference between the front image and the back image during the rocking is recognized, and the adhesion state of the cultured cells is judged.
In the above embodiment, the shaking means 11 tilts the support member 3 like a seesaw and shakes it, but the present invention is not limited to this. Various configurations of the shaking means 11 have been proposed so far, and the shaking means 11 may be one that rotates or reciprocates the culture vessel 2 on a plane as proposed therein, or one that performs a combination of these operations.

REFERENCE SIGNS LIST 1 Cultured cell detachment determination device 2 Culture vessel 3 Support member 3A Mounting section 3B Top surface section 5 Photography means 6 Illumination means 7 Control means 11 Shaking means

Claims (4)

An apparatus for determining detachment of cultured cells, comprising: a support member for supporting a culture vessel; an imaging means for imaging cultured cells in the culture vessel; and a control means for inputting a signal from the imaging means and determining an adhesion state of the cultured cells adhered to a bottom surface of the culture vessel,
The imaging means is provided integrally with the support member, and a shaking means is provided for shaking the support member to shake the culture vessel supported thereby, the shaking means shakes the support member to shake and tilt the culture vessel and the imaging means together, and the control means photographs the cultured cells in the shaken and tilted culture vessel with the imaging means, and determines whether the cultured cells have detached from the bottom surface based on the difference in the images during the shaking of the culture vessel. The device for determining detachment of cultured cells according to claim 1, characterized in that an illumination means for illuminating the culture vessel supported by the support means is provided integrally with the support member, and the shaking means rocks the support member to rock the culture vessel and the illumination means integrally. The support member includes a mounting section on which the culture vessel is placed and a top surface section facing the mounting section from above, the imaging means is provided on the mounting section to image the cultured cells from the bottom surface of the culture vessel placed on the mounting section, and the illumination means is provided on the top surface section to illuminate the culture vessel placed on the mounting section. The device for determining detachment of cultured cells according to claim 2. The support member includes a mounting section on which the culture vessel is placed and a top surface section facing the mounting section from above, the imaging means is provided on the top surface section to image the cultured cells from the top surface of the culture vessel placed on the mounting section, and the illumination means is provided on the top surface section to illuminate the culture vessel placed on the mounting section. The device for determining detachment of cultured cells according to claim 2.