JP5659479B2 - Cell counting method and cell counting apparatus in culture vessel - Google Patents

Description

The present invention relates to a counting method for counting objects in a container, and more particularly to a method for counting cells in a culture container and a cell counting device for counting the number of cells in a culture solution in a sealed culture container .

In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, and the like, it has been required to efficiently culture a large amount of cells, tissues, microorganisms, and the like in an artificial environment.
In such cell culture, it is necessary to maintain the density of the cells in the culture solution within an appropriate range as the cells grow.

That is, if the cell density in the culture solution is too high, sufficient oxygen and nutrients cannot be supplied to each cell, and the cell growth efficiency decreases. Moreover, sufficient proliferation efficiency cannot be obtained even if the cell density in the culture solution is too small.
Therefore, in cell culture, in order to grasp the cell density during culture, it was necessary to appropriately measure the number of cells in the culture solution in the culture vessel.

For example, Patent Literature 1 discloses a cell culture device that can appropriately maintain the cell density in a culture solution in conjunction with cell proliferation.
In the case of measuring the number of cells using such a cell culture device, conventionally, a culture solution containing cultured cells is collected from a tube connected to the culture vessel, and a predetermined buffer is added to measure the cells in the culture solution. After adjusting the density to be suitable for the cell, it was injected into a counting board, and the number of cells was read by a person or machine to calculate the cell density.

  Further, Patent Literature 2 discloses a culture apparatus provided with photographing means. According to this culture apparatus, cell images can be periodically acquired and stored.

International Publication No. 2008/136371 Pamphlet International Publication No. 2007/052718 Pamphlet

  However, in the method of counting cells in the culture solution using the cell culture device described in Patent Document 1, it is necessary to collect the culture solution from the inside of the culture vessel, and the culture system is opened, so that various germs and the like are mixed. There was a risk of contamination.

Moreover, in the culture apparatus described in Patent Document 2, a cell image can be acquired, but it has been difficult to accurately measure the number of cells based on the image and obtain a cell density.
That is, when photographing the cells in the culture vessel by the photographing means described in Patent Document 2, the number of cells in the cell image is measured and divided by the volume of the culture solution in the field of view of the photographing means, Cell density can be calculated.
However, when the cells in the cell container are directly observed in this way, as shown in FIG. 17, when the cell density is large and the cells overlap, the number of cells cannot be measured accurately. Further, when the cell density is too small, it is difficult to predict the whole density, and there is a problem that the accuracy of the calculated cell density is lowered.

In this way, when directly measuring the cells in the culture vessel using the imaging means, the thickness of the counter is 0.1 mm as compared with the case of measuring using the conventional counter. The thickness is about 1 to 2 cm, and the thickness is 100 to 200 times.
For this reason, the number of cells that can be seen from the imaging means is 100 to 200 times that of actual measurement using a counter even at the same volume density. Therefore, the cells in the culture container often overlap each other, and it is difficult to directly measure the number of cells by directly observing the cells in the culture container.

Therefore, as a result of diligent research, the present inventors have adjusted the thickness of the culture vessel so that the number of cells visible from the imaging means can be measured, and then directly measuring the number of cells in the culture vessel. The present invention was completed by successfully obtaining a cell density close to the actually measured value.
That is, the present invention is a method for counting cells in a culture vessel capable of measuring the number of cells in any density range in a culture environment without opening the culture system and irrespective of the density of the proliferated cells, And it aims at providing the apparatus for cell counting.

The method for counting cells in a culture vessel according to the present invention is a method for measuring the number of cells in a liquid in a sealed culture vessel , wherein the thickness of at least a part of the culture vessel is adjusted and adjusted. This is a method of measuring the number of cells in the measurement target range with at least a part as the measurement target range.
In addition, the cell counting method in the culture container of the present invention is a method of adjusting the thickness of at least a part of the culture container after stirring the liquid in the culture container and equalizing the cells in the liquid.

Further, cell counting device of the present invention is a cell for counting apparatus for counting the number of cells in a liquid of a sealed culture vessel, and stacking plate for stacking the culture vessel, measured in the culture vessel An adjustment member that adjusts at least a part including the target range to a predetermined thickness is provided.
In addition, the cell counting apparatus of the present invention may further include a stirring member that stirs the liquid in the culture container before the thickness of the culture container is adjusted by the adjustment member.

Further, the cell count device of the present invention, further counting means for counting the number of cells in the captured image, the result of measurement by the counting means, when number of cells is not within the predetermined range, the image of A driving device that drives the adjusting member to adjust at least a part of the culture vessel to a predetermined thickness so that the number of cells is within a predetermined range may be provided.

  According to the present invention, the number of cells can be measured without opening the culture system and irrespective of the density of the proliferated cells.

It is a figure which shows the principle of the counting method of the counting target object in the container of this invention. It is a figure which shows the structure of the apparatus for counting of 1st embodiment of this invention. It is a figure which shows the thickness adjustment method (when reducing thickness) of the container by the counting apparatus of 1st embodiment of this invention. It is a figure which shows the thickness adjustment method (when increasing thickness) of the container by the apparatus for counting of 1st embodiment of this invention. It is a figure which shows the structure of the apparatus for counting of 2nd embodiment of this invention. It is a figure which shows the structure of the drive device (for stirring members) in the apparatus for counting of 2nd embodiment of this invention. It is a figure which shows the basic position of the apparatus for counting of 3rd embodiment of this invention. It is a figure which shows the stirring state of the apparatus for counting of 3rd embodiment of this invention. It is a figure which shows the thickness adjustment of the counting apparatus of 3rd embodiment of this invention, and the waiting state for sedimentation (when reducing thickness). It is a figure which shows the microscope observation state of the apparatus for counting of 3rd embodiment of this invention. It is a figure which shows the thickness adjustment of the apparatus for counting of 3rd embodiment of this invention, and the waiting state for precipitation (when increasing thickness). It is a figure which shows the culture container used by the Example and comparative example of this invention. It is a figure which shows the image of the cell in Example 1 and Comparative Example 1 of this invention. It is a figure which shows the result of Example 1 and Comparative Example 1 of this invention. It is a figure which shows the image of the cell in Examples 2-5 of this invention. It is a figure which shows the result of Examples 2-5 of this invention. It is a figure which shows the counting method of the counting target object in the conventional container.

Hereinafter, embodiments of the present invention will be specifically described.
First, the principle of the counting method for counting objects in the container of the present invention will be described with reference to FIG.
FIG. 1 shows a state in which cells in a culture container are directly observed using a microscope and the number thereof is measured. FIG. 1 (A) adjusts the thickness of the container as in the prior art. It shows the case of observing without. In addition, by using a culture medium whose specific gravity is smaller than the specific gravity of the cells, the cells can be submerged in the bottom of the container and can be made suitable for microscopic observation. On the contrary, a culture solution having a large specific gravity may be used to collect cells at the top of the container for observation.

  In the case of FIG. 1 (A), as the number of cells in the container increases, overlapping gradually occurs when suspended and allowed to stand. Therefore, this method cannot accurately measure the number of cells when culturing a large amount of cells.

Therefore, in the present invention, when the number of cells is too large to be measured accurately, as shown in FIG. 1 (B), by reducing the thickness of the container, in the observation range (measurement target range). Reduce the number of cells and adjust to the number of cells suitable for measurement.
Thereby, even when culturing a large amount of cells in a culture container, the number of cells can be measured without opening the culture system by directly observing the cells in the culture container.
In addition, even when the number of cells in the culture vessel is small and it is difficult to predict the density of the entire culture vessel, increasing the vessel thickness increases the number of cells in the observation range and adjusts the number of cells suitable for measurement. can do.

[First embodiment]
Next, a counting method and counting device for counting objects in a container according to the first embodiment of the present invention will be described with reference to FIG. This figure is a diagram showing the configuration of the counting apparatus of the present embodiment.
As shown in the figure, the counting device 1 of the present embodiment includes a thickness adjusting member 2, a container loading table 3, a photographing means 4, a driving device (for adjusting member) 5, a driving device (for photographing means) 6, In addition, the culture vessel 8 is arranged on the vessel loading table 3 and the number of cultured cells in the culture vessel 8 is measured.

  The thickness adjusting member 2 adjusts the culture vessel 8 on the vessel loading table 3. In the example of the figure, the thickness adjusting member 2 is composed of a pressing plate having a flat portion for pressing the culture vessel 8, and the thickness of the thickness adjusting member 2 is determined by pressing a part of the culture vessel 8 made of soft packaging material from above. It is possible to reduce this.

In addition, at least the upper part of the measurement target range of the culture vessel 8 in the thickness adjusting member 2 is made of a transparent material, the part is illuminated from above by the illumination 7, and observed from below by the photographing means 4 comprising a microscope and a CCD camera. It is like that.
Note that the thickness adjusting member 2 is not limited to a pressing plate that presses the culture vessel 8 with a flat portion as shown in FIG. 2, and can also be configured using a roller or a tension member.

  For example, the thickness of the culture vessel 8 can be increased by reducing the horizontal area of the culture vessel 8 by moving the culture vessel 8 from both the upper and lower sides or from one side with a roller. Moreover, the thickness of the culture container 8 can also be reduced by extending the culture container 8 in the horizontal direction using a tension member.

The container loading table 3 is a flat table on which the culture vessel 8 is placed, and constitutes a loading device for counting together with the thickness adjusting member 2.
The lower part of the measurement target range in the container loading table 3 is made of a transparent member such as a glass plate, and the culture vessel 8 can be observed from below by the photographing means 4.

As shown in FIG. 2, the drive device (for adjusting member) 5 moves the thickness adjusting member 2 up and down by a ball screw screw. As a result, the thickness adjusting member 2 is pressed against the culture vessel 8 on the vessel loading table 3 so that the thickness of the culture vessel 8 can be adjusted.
For example, a rod-type electric cylinder (vertical operation actuator) can be used for the drive device (for adjusting member) 5, and thereby the thickness of the culture vessel 8 can be finely adjusted by 0.1 mm. Is possible.

Further, as shown in FIG. 2, the driving device (for photographing means) 6 moves the photographing means 4 in the horizontal direction with respect to the container loading table 3 by a ball screw screw. By this driving device (for photographing means) 6, the photographing means 4 is arranged outside the container loading table 3 except when photographing the cells in the culture vessel 8, and the photographing means 4 is measured in the measurement target range of the culture container 8 at the time of photographing. Can be moved down.
The driving device (for adjusting member) 5 and the driving device (for photographing means) 6 are configured using an actuator using air pressure, hydraulic pressure, and electromagnetic force in addition to an electric actuator, or using a motor or a cam. It can also be configured.

The illumination 7 illuminates the measurement target range in the culture vessel 8 through the thickness adjusting member 2 and provides the brightness necessary for photographing the cells by the photographing means 4.
The culture container 8 is a container formed in a bag shape (back shape) using a soft packaging material as a material. Thus, by using a soft wrapping material as the material of the culture vessel 8, flexibility and softness can be imparted to the culture vessel 8. Examples of the soft packaging material include, for example, Japanese Patent Application Laid-Open No. 2002-255277 (a food packaging body using a soft packaging material film sheet and a method for taking out food), and Japanese Patent Application Laid-Open No. 2004-323077 (a pressure extraction type bag shape). Those described in (Containers) can be used.

  The culture vessel 8 has gas permeability necessary for cell culture, and part or all of the culture vessel 8 is transparent so that the contents can be confirmed. Examples of the culture vessel material that satisfies such conditions include polyolefins, ethylene-vinyl acetate copolymers, styrene elastomers, polyester thermoplastic elastomers, silicone thermoplastic elastomers, and silicone rubber.

  Next, an example of adjusting the thickness of the culture vessel 8 by the counting device of the present embodiment will be described with reference to FIGS. FIG. 3 shows a case where the thickness of the container is reduced, and FIG. 4 shows a case where the thickness of the container is increased.

  When the thickness of the culture vessel 8 is reduced, the thickness adjustment of the vessel is performed by pressing the thickness adjusting member 2 from below the culture vessel 8 as shown in FIG. Can be performed. Although not shown, it is preferable to fix the position of the upper surface of the culture vessel 8 with a glass plate or the like at this time.

  When the culture vessel 8 is made of a material that can be extended horizontally, the culture vessel 8 is pressed using the thickness adjusting member 2 that can cover the entire surface of the culture vessel 8 as shown in FIG. The thickness of the culture vessel 8 can also be reduced. In addition, as shown in FIG. 3C, the thickness of the culture vessel 8 can be extended by the thickness adjusting member 2 to reduce the thickness thereof. In this case, one end of the edge of the culture vessel 8 is fixed, and the edge on the opposite side in the horizontal direction is pulled by the thickness adjusting member 2 made of a tensile member, or two thickness adjusting members 2 are used. The culture vessel 8 can be stretched by pulling both ends in the horizontal direction of the culture vessel 8. As a material for such a flexible culture vessel 8, for example, silicone rubber or the like can be used.

On the other hand, when increasing the thickness of the culture vessel 8, for example, as shown in FIG. 4D, a part of the upper surface of the culture vessel 8 is pressed by the thickness adjusting member 2 and pressed in the culture vessel 8. The thickness other than the portion can be increased.
Further, as shown in FIG. 4 (e), a roller or the like is used as the thickness adjusting member 2, and this is moved to press the culture vessel 8, thereby reducing the horizontal area of the culture vessel 8 and culturing. The thickness of the container 8 can also be increased.

Next, the counting method of the counting object in the container of this embodiment will be described in more detail with reference to FIG.
First, prior to the step of measuring the number of cells in the culture vessel 8, it is preferable to stir the culture solution in the culture vessel 8. In addition, about the stirring means of a culture solution, it demonstrates in detail in 2nd embodiment.

Next, using the driving device (for imaging means) 6, the imaging means 4 is moved below the measurement target range of the culture vessel 8.
Next, the thickness adjusting member 2 is lowered using the driving device (for adjusting member) 5 to adjust the thickness of the culture vessel 8 to a predetermined thickness.
At this time, the predetermined thickness can take various values depending on the cell type, the size of the culture vessel, the area of the measurement target range, the culture period, and the like.

  Next, the measurement object range is illuminated by the illumination 7 and the photographing unit 4 captures an image. Then, the cells on the photographed image are counted. At this time, the photographed image can be transmitted from the photographing means 4 to the counting means, and the number of cells can be automatically measured by the counting means. As such a counting means, a known cell counting analyzer, cell number measuring device, or the like can be used.

Here, in the present embodiment, by using a culture solution having a specific gravity smaller than that of the cultured cells, the cultured cells are precipitated on the bottom of the culture vessel 8, and the photographing means is focused on the precipitated cells. Can be taken. However, if the cell density is large and the cells in the measurement target range overlap, the number of cells cannot be counted correctly.
Therefore, in the counting method of the counting object in the container according to the present embodiment, when cells in the measurement target range overlap in this way, the thickness adjustment member 2 is moved to reduce the thickness of the culture container 8, and the measurement is performed. By reducing the number of target cells, the cells can be measured.

  Note that the maximum number of cells that can be observed without overlapping in the measurement target range can be considered to be less than the number obtained by dividing the area of the measurement target range by the average horizontal area of the cultured cells. Therefore, when the number of cells is calculated to be greater than or equal to the maximum number of cells as a result of counting the cells, it is preferable to further reduce the thickness of the culture vessel 8 and perform counting again. Whether or not the thickness of the culture vessel 8 is adjusted with other values, such as more accurately estimating the number when the cells are densely arranged in the measurement target range and using the estimated value as the maximum number of cells. It can also be used for judgment.

  In addition, when the number of cells is less than a certain value as a result of counting the cells, the accuracy of the obtained cell density is lowered. Therefore, it is preferable to count again after increasing the thickness of the culture vessel 8. The measuring device for increasing the thickness of the culture vessel 8 will be described in detail in the third embodiment.

When the number of cells in the measurement target range is measured in this manner, the cell density can be calculated by dividing the number of cells by the volume of the measurement target range. Furthermore, the number of cells in the entire culture vessel 8 can be calculated by multiplying the obtained cell density by the volume of the culture vessel 8.
Such cell density and the number of cells in the entire culture vessel 8 can also be automatically calculated by the counting means.

  Thus, according to this embodiment, when the number of cells in the culture vessel 8 is large and the culture vessel 8 is directly observed, the thickness of the culture vessel is reduced even when the number of cells cannot be measured accurately. Thus, the number of cells can be measured, and the cell density in the culture vessel 8 can be calculated.

[Second Embodiment]
Next, a counting method and counting device for counting objects in a container according to a second embodiment of the present invention will be described with reference to FIGS. FIG. 5 is a diagram illustrating the configuration of the counting device according to the present embodiment, and FIG. 6 is a diagram illustrating the configuration of the driving device (for the stirring member) in the counting device according to the present embodiment.
The counting device of the present embodiment further includes a stirring member in addition to the configuration of the counting device of the first embodiment. In the present embodiment, the agitation member is used to agitate the culture solution in the culture vessel 8 so that the cells can be dispersed so as to be easily observed. Other points can be the same as those in the first embodiment.

The stirring member 9 presses and moves the culture vessel 8, stirs the culture solution in the culture vessel 8, and disperses the cultured cells in the culture solution. As the stirring member 9, for example, a roller can be used as shown in FIG.
In the example of the figure, while the culture vessel 8 is pressed by the stirring member 9 with a predetermined pressing amount, the stirring member 9 is repeatedly moved at a predetermined speed and a predetermined cycle in parallel with the container loading table 3, thereby culturing. The liquid is agitated.

As shown in FIG. 5, the support base 10 is connected to a bearing portion that is erected upward on one side on both sides of the container stacking base 3 and rotatably supports both ends of the stirring member 9. Formed by a connecting portion.
Further, as shown in FIG. 6, the support base 10 can be moved up and down by a rod-type electric cylinder 12 (vertical direction actuator) attached below the connecting portion, and a stirring member attached to the support base 10. It is possible to finely adjust the pressing amount against the culture vessel 8 by 9 in units of 0.1 mm.

The rod-type electric cylinder 12 is attached to the moving table 11 on the slider-type electric cylinder 16 (horizontal operation actuator), and moves in the horizontal direction with respect to the container stacking table 3. Further, the moving speed of the stirring member 9 attached to the support table 10 is adjusted by controlling the moving speed of the moving table 11 in the horizontal direction.
For the operation control of the stirring member, an actuator using air pressure, hydraulic pressure, electromagnetic force, or a motor or cam is used instead of the electric actuator such as the rod type electric cylinder 12 or the slider type electric cylinder 16. It is also possible to make a configuration that was suitable.

According to the counting method for counting objects in the container and the counting device of the present embodiment, the stirring member 9 is pressed against the culture container 8 with a predetermined pressing amount before the number of cells in the culture container 8 is measured. In this state, the stirring member 9 can be reciprocated in the horizontal direction for a certain time.
As a result, the culture solution in the culture vessel 8 can be stirred, and the cultured cells in the culture vessel 8 can be dispersed so as to be easily counted.

[Third embodiment]
Next, a counting method and counting device for counting objects in a container according to a third embodiment of the present invention will be described with reference to FIGS. These figures respectively show the basic position of the counting device (FIG. 7), the stirring state (FIG. 8), the thickness adjustment and the waiting state for precipitation (the cells to be counted are counted in the counting object counting method in the container of this embodiment. FIG. 9 is a schematic diagram showing a case where it is desired to reduce FIG. 9), a microscope observation state (FIG. 10), a thickness adjustment and a waiting state for precipitation (when it is desired to increase the number of cells to be counted).

In addition to the configuration of the counting device of the second embodiment, the counting device of the present embodiment further includes a thickness adjusting member for increasing the thickness of the culture vessel 8. Other points can be the same as in the second embodiment.
Hereinafter, the operation procedure of the counting apparatus in the counting method of the counting object in the container according to the present embodiment will be described including the operation for increasing the thickness of the culture container 8 and the operation for decreasing the thickness.

(1) Basic position First, with reference to FIG. 7, the basic position of the component of the counting apparatus of this embodiment is demonstrated.
In the figure, an observation hole for microscopic observation is provided in the container loading table 3, and a glass plate constituting a part of the upper surface of the container loading table 3 is fitted above the observation hole.

The culture vessel 8 is arranged on the vessel loading table 3, and a thickness adjusting member 2-1 (pressing plate) is arranged above the observation hole of the vessel loading table 3. The thickness adjusting member 2-1 is moved downward to press the culture vessel 8 to reduce its thickness.
In addition, a thickness adjusting member 2-2 (roller) is arranged at one end of the edge of the culture vessel 8, and by moving this, the horizontal area of the culture vessel 8 can be reduced and the thickness can be increased. It has become.

Further, a stirring member 9 (roller) is disposed above the culture vessel 8, and the stirring member 9 is moved downward and moved in the horizontal direction while pressing the culture vessel 8. It is possible to agitate the culture solution.
At the basic position, the microscope lens 4-1, the CCD camera 4-2, and the illumination 7 that constitute the imaging device 4 for imaging cells are arranged outside the container stack 3.

(2) Stirring state Next, with reference to FIG. 8, a procedure for stirring the culture solution in the culture vessel 8 before counting the number of objects to be counted in the vessel will be described.
First, the stirring member 9 is moved downward from the basic position in FIG. 7 to press the culture vessel 8 with a predetermined pressing amount. Next, the stirring member 9 is repeatedly moved at a predetermined speed and a predetermined cycle in parallel with the container loading table 3.
Thereby, it is possible to agitate the culture solution in the culture vessel 8 and equalize the cells in the culture solution.

(3) Thickness adjustment and waiting for settling (when reducing thickness)
Next, a procedure for reducing the thickness of the culture vessel 8 and reducing the number of cells to be counted will be described with reference to FIG.
First, the stirring member 9 is moved upward to return to the basic position in FIG. 7, and the thickness adjusting member 2-1 is moved downward to reduce the thickness of the culture vessel. At this time, a part of the culture vessel 8 including the region on the observation hole of the vessel loading table 3 is pressed by the thickness adjusting member 2-1, and the thickness of the measurement target range of the culture vessel 8 is adjusted to a predetermined size. To do. At this time, the number of cells to be counted can be reduced as the thickness of the culture vessel 8 is smaller in the culture environment.
After reducing the thickness of the culture vessel 8, the culture vessel 8 is allowed to stand until the cells in the culture vessel 8 are precipitated.

(4) Microscope Observation State Next, a procedure for performing cell counting will be described with reference to FIG.
After the cells in the culture vessel 8 are precipitated in a state where the thickness of the culture vessel 8 is reduced, as shown in FIG. 10, the photographing means 4 including the microscope 4-1 and the CCD camera 4-2, and illumination 7 is moved in the horizontal direction from the basic position in FIG. 7, and the photographing means 4 is arranged directly below the observation hole and the illumination 7 is arranged above the observation hole. Then, the cells precipitated in the culture vessel 8 are photographed by the CCD camera 4-2.

The image thus obtained is input to a counting device (not shown), the number of cells in the image is measured by this counting device, and the obtained number of cells is measured by a measurement target range (observed by the CCD camera 4-2). The cell density in the culture vessel 8 can be calculated by dividing by the volume of the region in the culture vessel 8).
As a result, if cells in the culture vessel 8 grow and settle with the same thickness, even if they are overlapped and cannot be counted accurately, the thickness can be adjusted by adjusting the thickness of the culture vessel 8. It is possible to calculate the cell density in the culture vessel 8 without opening the culture system.

(5) Thickness adjustment and waiting for precipitation (when increasing thickness)
On the other hand, in the initial stage of culture and the like, since the number of cells is small, counting can be performed, but the accuracy of the obtained cell density may be lowered.
For this reason, when the number of observed cells is small, as shown in FIG. 11, the thickness of the culture vessel 8 is increased to increase the number of cells in the measurement target range, and then the microscopic observation described in (4) is performed. Do.

  That is, after stirring in (2), the stirring member 9 is moved upward to return to the basic position in FIG. 7, and the thickness adjusting member 2-2 made of a roller is moved in the direction of the measurement target range to press the culture vessel 8. Then, the thickness of the culture vessel 8 is increased. At this time, in order to make the thickness of the measurement target range in the culture vessel 8 constant, the thickness adjusting member 2-1 is moved to a position where the upper surface of the culture vessel 8 is brought into contact with the measurement target range. And it is left still until the cell in the culture container 8 precipitates.

Thereby, the number of cells in the measurement target range can be increased, and the cell density can be calculated more accurately when the number of cells in the culture vessel 8 is small.
The specific number of cells when the number of cells in the culture vessel 8 is small is appropriately determined based on the type of cells, the area of the measurement target range, etc., but can be a number such as 0 or less than 10, for example.

  In addition, after adjusting the thickness of the culture vessel 8 according to the procedure of (1) to (5) described above, the cells in the measurement target range are overlapped, or the cells are not seen or almost seen. If not, the thickness of the culture vessel 8 is adjusted again according to the procedures (1) to (5), and the number of cells is counted.

  As described above, according to the present embodiment, when the number of cells in the culture vessel 8 is small and there is a possibility that an accurate cell density may not be obtained when the culture vessel 8 is directly observed, the thickness of the culture vessel is reduced. By increasing the cell density, the cell density in the culture vessel 8 can be calculated more accurately.

  Hereinafter, the embodiment of measuring the number of cells by adjusting the thickness of the culture vessel 8 and the thickness of the culture vessel 8 by the counting method and the counting device of the counting object in the vessel of the present invention are adjusted. A comparative example in which the number of cells was measured will be described.

Example 1
As the culture container 8, a bag made of LDPE (linear low density polyethylene) and having a film thickness of 0.15 mm was used. As shown in FIG. 12, the culture vessel 8 is partitioned by a partition member (rubber roller) so that the long side of the culture part, which is the culture area, is 250 mm, the short side is 210 mm, and 640 ml of the culture solution is placed in the culture part. I put it in and used it. In addition, the container thickness when the culture container 8 was laid flat at this time was approximately 16 mm, although the upper surface of the container was not completely horizontal. The size of the horizontal plane of the observation range (measurement target range) is a square with a side of 0.5 mm.

As a culture solution, AlyS505N-0 medium of Cell Science Laboratory was used, and as a cultured cell, a human leukemia T lymphoid Jurkat E6.1 strain grown in a required amount in a cell culture dish was used.
A roller having a diameter of 12 mm was used as the agitating member 9 and agitation was performed under the agitation conditions of a pressing amount of 13 mm, a speed of 50 mm / s, and a reciprocation frequency of 10 times.

Immediately after stirring, the thickness adjusting member made of an acrylic plate having a width of 50 mm, a thickness of 3 mm, and a length larger than the long side of the culture vessel 8 was lowered to adjust the thickness of the bag to 3.1 mm. And after leaving still for 12 minutes, the observation range was image | photographed and the number of cells was counted. The cell density was calculated based on the number of cells and the volume of the observation range. The photograph taken is shown in FIG. 13, and the number of cells in the observation range, cell density, measured density, and measured value ratio are shown in FIG.
The actual density was obtained by measuring the number of cells in the culture medium collected from the bag using a counting board by a conventional method and dividing this by the volume of the observation range.

(Comparative Example 1)
Stirring was performed under the same stirring conditions using the same culture vessel 8 and culture solution as in Example 1.
Next, after allowing to stand for 60 minutes without adjusting the thickness of the bag, the observation range was photographed. Since the upper surface of the bag was not pressed, it was wavy and the thickness of the bag was approximately 16 mm.
In this case, the cells were overlapped, the number of cells could not be counted, and the density could not be calculated. The results are shown in FIGS.

(Example 2)
Similar to Example 1, except that a culture solution having a cell density different from that of Example 1 was used, and that the number of cells was measured, the bag thickness was 11.0 mm, and the resting time was 45 minutes thereafter. The experiment was conducted. The types of the culture solution and the cultured cells are the same as those in Example 1. However, in this example, those having fewer cultured cells than Example 1 were used. The results are shown in FIGS.

Example 3
In measuring the number of cells, the experiment was conducted in the same manner as in Example 2 except that the thickness of the bag was 7.0 mm and the resting time was 30 minutes. The results are shown in FIGS.

Example 4
In measuring the number of cells, the experiment was performed in the same manner as in Example 2 except that the thickness of the bag was 4.0 mm and the subsequent standing time was 12 minutes. The results are shown in FIGS.

(Example 5)
In measuring the number of cells, the experiment was performed in the same manner as in Example 2 except that the thickness of the bag was 3.1 mm and the resting time was 12 minutes. The results are shown in FIGS.

As shown in FIGS. 13 and 14, in Comparative Example 1 in which the thickness of the bag was not adjusted, cells overlapped and could not be counted accurately. Note that “uncountable” refers to a case where the count result is equal to or greater than a predetermined value (the maximum number of cells visible within the observation range), in addition to the case where the calculation is not actually possible.
On the other hand, in Example 1, the number of cells could be measured by reducing the thickness of the bag, and the cell density in the culture vessel 8 could be calculated.

Moreover, as shown in FIG.15 and FIG.16, in Examples 2-5, it turns out that the number of measured cells decreases as thickness of a bag is reduced. In addition, the calculated cell density was not significantly different from the actually measured density.
Therefore, it was found that when the number of cells is large and the count is not possible, the cells can be measured by decreasing the bag thickness more greatly as the cell density increases.
From these experimental results, according to the counting method of the counting object in the container using the counting device of the present invention, the number of cells in the culture container was determined without opening the culture system and irrespective of the cell density. It was revealed that the cell density could be measured and the cell density could be calculated.

The present invention is not limited to the above-described embodiments and examples, and it goes without saying that various modifications can be made within the scope of the present invention.
For example, in the above-described embodiment and examples, the cultured cell is a measurement target. However, the measurement target is not limited to this, and other organic substances such as plankton or inorganic substances can be the measurement target. The “liquid” in the culture vessel includes a liquid such as a culture solution and a semi-fluid. In addition, by using a culture solution in the culture vessel 8 that is larger than the specific gravity of the cultured cells, the cultured cells can be positioned in the upper portion of the culture vessel 8, and this may be counted. . In addition to counting the cells in the above embodiment and examples, it is also possible to observe the growth state of the cells. Furthermore, the agitating member 9 can be appropriately changed, for example, by forming the cross-section in various shapes such as a star shape instead of a columnar shape so that various shaking effects can be obtained.

  The present invention can be suitably used for culturing cells in large quantities using a cell culture vessel.

1 Counting device 2 Thickness adjustment member 2-1 Thickness adjustment member (for thickness reduction)
2-2 Thickness adjusting member (for increasing thickness)
3 Container loading table 4 Imaging means 4-1 Microscope 4-2 CCD camera 5 Driving device (for adjusting member)
6 Drive unit (for photographing means)
7 Illumination 8 Culture vessel 9 Stirring member 10 Support base 11 Moving base 12 Rod-type electric cylinder (actuator for vertical operation)
13 Ball screw 14 Guide rod 15 Base 16 Slide type electric cylinder (actuator for horizontal operation)
17 Glass plate

Claims (11)

A method for measuring the number of cells in a liquid in a sealed culture container , wherein the thickness of at least a part of the culture container is adjusted, and at least a part of the adjusted range is set as a measurement target range. A method for counting cells in a culture vessel , comprising measuring the number of cells in a range. Stirring the liquid in the culture vessel, after equalizing the cells in the liquid, the cells of claim 1 culture vessel, wherein adjusting at least a portion of the thickness of said culture vessel Counting method. By using the number of measured said cells, to calculate the density of cells in the liquid, and / or the culture container according to claim 1 or 2, wherein calculating the number of cells in the entire liquid Cell counting method. Measurement of the number of the cells, the cells in the culture vessel according to claim 1, characterized in that it is carried out by counting the number of said cells in an image obtained by photographing the measuring object range Counting method. The number of the cells is measured after reducing the thickness of at least a part of the culture container when the number of the cells in the measurement target range is equal to or greater than a predetermined value. The method for counting cells in the culture vessel according to any one of the above. Using the thickness adjusting member, presses the culture vessel, or the stretching of the culture vessel, the cell of claim 5 culture vessel, wherein the reducing at least a portion of the thickness of said culture vessel Counting method. The number of the cells is measured after increasing the thickness of at least a part of the culture container when the number of cells in the measurement target range is less than a predetermined value. The method for counting cells in the culture vessel according to any one of the above. The method of counting cells in a culture container according to claim 7, wherein the thickness of the culture container is increased by pressing the culture container using a thickness adjusting member. A cell counting device for measuring the number of cells in a liquid in a sealed culture vessel ,
An apparatus for counting cells , comprising: a loading table on which the culture container is loaded; and an adjustment member that adjusts at least a part of the culture container including a measurement target range to a predetermined thickness. The cell counting apparatus according to claim 9 , further comprising a stirring member that stirs the liquid in the culture container before the thickness of the culture container is adjusted by the adjustment member. A photographing means for photographing the cells in said culture vessel, and counting means for counting the number of the cells in the photographed image, the result of measurement by the counting means, the number of the cells is not within a predetermined range A drive device that drives the adjustment member to adjust at least a part of the culture vessel to a predetermined thickness so that the number of cells in the image falls within a predetermined range. The device for counting cells according to claim 9 or 10 , wherein the device is used for counting cells .