JP2019000001A - Cell culture device - Google Patents

Abstract

To provide methods for controlling the diameter of a cell mass in view of the actual condition that a diameter control method of a cell cluster suitable for the suspension culture aiming at mass production is not present now, and to provide cell culture devices using the controlling method.SOLUTION: The cell culture device according to the present invention controls the diameter of a cell mass by imparting an ultrasonic wave to a comparatively large cell mass floating in a culture solution.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell culture apparatus for culturing cells such as artificial pluripotent cells.

  Animal cells are mainly used in the medical and pharmaceutical industries. Stem cells (such as induced pluripotent cells), which are a kind of animal cells, can differentiate into various cells. In recent years, stem cells have begun to be used as cellular medicines for regenerative medicine that treat damaged organs and the like. Cellular pharmaceutical production can be obtained by culturing and proliferating stem cells.

  Conventionally, adhesion culture has been used as a method for culturing animal cells used in regenerative medicine. Adhesion culture is a method in which cells are adhered to a culture vessel and cultured. Until now, regenerative medicine has been carried out in small-scale production using adhesion culture such as cell sheets. Artificial pluripotent cells, which are a type of stem cell, form a cell mass and proliferate. The diameter of the cell mass affects the properties (undifferentiated state, survival rate) of the induced pluripotent cells. Therefore, in some cells such as artificial pluripotent cells, it is necessary to control the diameter of the cell mass. This diameter control is performed by applying physical force to the cell mass. The physical force referred to here is a shear stress applied to cells by pipetting operation. In adhesion culture for the purpose of small-scale production, manual pipetting has been used to apply force to the cell mass of artificial pluripotent cells.

In general, when treating heart disease or the like by regenerative medicine, 10 ⁹ cells differentiated from stem cells into cardiomyocytes are required. In the case of adhesion culture, which is currently used mainly for culturing stem cells, the limit is that about 10 ⁷ stem cells can be cultured in one commonly used culture dish having a diameter of 10 cm. If 10 ⁹ cells are produced by adhesion culture, 100 culture dishes are required. Adhesive culture is not suitable for mass production because it takes a lot of labor to culture and maintain 100 culture dishes.

  As one of the mass culture techniques, application of a suspension culture technique is being studied. Even in suspension culture, in the case of induced pluripotent cells, the diameter of the cell mass is involved in maintaining the characteristics. Therefore, in artificial pluripotent cells, it is necessary to control the diameter of the cell mass even in suspension culture. In suspension culture of induced pluripotent cells, it is necessary to efficiently control the diameter of a large amount of cell mass.

  In the prior art, mesh is applied as a method for controlling the diameter of a cell mass in suspension culture. This control method can be applied to many cell clusters at once, but mesh clogging becomes a problem. When put into practical use, there is a high possibility that the cost and work efficiency will deteriorate due to the replacement work of the mesh.

  As a means for applying a physical force to the cell mass, a method of irradiating cells with ultrasonic waves can be considered. The example of the process of the ultrasonic wave with respect to the conventional cell is as follows. Patent Document 1 describes a cell culture device having a culture tank, a stirring blade, and an ultrasonic irradiation means, and discloses a method of detaching cells by irradiating the cells attached to the wall of the culture tank with ultrasonic waves. . However, since this method applies ultrasonic waves to the entire cells in the culture tank, some cells are subjected to extra physical stress.

  As an example of irradiating cells with ultrasonic waves, Patent Document 2 describes a method in which ultrasonic waves are locally applied to an adhesion culture attached to a container and desired cells are detached from the culture container. This ultrasonic irradiation method can apply a local ultrasonic wave and does not apply extra stress to the cells. However, as in Patent Document 1, it is an effective technique for the purpose of peeling attached cells, and it is not possible to irradiate only specific cells floating in the culture solution with ultrasonic waves.

JP-A-6-141850 JP 2014-18185 A

  Currently, there is no method for controlling the diameter of cell mass suitable for suspension culture for mass production. Then, in view of the above situation, an object of the present invention is to provide a method for controlling the diameter of a cell mass and a cell culture device to which the control method is applied.

  The culture apparatus according to the present invention controls the diameter of a cell mass by applying ultrasonic waves to a relatively large cell mass while floating in a culture solution.

  A cell culture device capable of easily controlling the diameter of a cell mass during culture can be provided.

It is an example of the cell culture apparatus which concerns on this embodiment. 2 is a cell culture device according to Example 2. 3 is a cell culture device according to Example 3. 4 is a cell culture device according to Example 4; 10 is a cell culture device according to Example 5.

  Hereinafter, the cell culture device according to the present embodiment will be described in detail with reference to the drawings. The cell culture device according to the present embodiment can be applied, for example, to culture of cells in which the cells themselves are pharmaceuticals. Examples of cells to be cultured include animal cells, plant cells, insect cells, bacteria, yeasts, fungi, and algae. In particular, cells in which cell clusters such as iPS cells are aggregated and cultured are preferred.

  FIG. 1 schematically shows an example of a cell culture device according to the present embodiment. The cell culture device 20 includes a container body 1. The container body 1 includes a culture solution 3 including a cell mass that is an aggregate of a plurality of cells having different sizes, an agitation unit 2 that agitates the culture solution 3, and an ultrasonic transmission terminal 4 that is a terminal that transmits ultrasonic waves. And have. The ultrasonic transmission terminal 4 is in contact with the liquid surface of the culture solution 3 and is disposed on the lid body 8.

  Outside the container main body 1, an ultrasonic irradiation unit 5 that irradiates ultrasonic waves, a measuring device 6 that measures the size of ultrasonic waves, and a cell observation unit 7 that observes cells are provided.

  A cell mass is photographed by the cell observation means 7 to obtain a photographed image. The measurement device 6 analyzes the captured image, and the ultrasonic irradiation means 5 performs measurement when the cell mass captured in the captured image is in a position to receive the ultrasonic wave from the ultrasonic transmission terminal 4 based on the analysis result. A signal is sent from the device 6, and an ultrasonic wave is transmitted from the ultrasonic transmission terminal 4.

  The cell observation means 7 is constituted by a camera using a CCD element disposed outside the container body 1. A cell or a cell mass existing in a predetermined region at the tip of the ultrasonic wave transmitting terminal 4 is photographed to acquire image data.

  The cell observation means 7 may be fixed so as to photograph a predetermined area. Since the culture solution 3 is stirred and circulated by the stirring means 2, even if the cell observation means 7 is fixed, each cell mass contained in the culture solution can be regularly observed.

  The cell observation means 7 may be movable without being fixed. In that case, the cell observation means 7 is arranged at a position where the entire cell mass can be observed so that it can be found as soon as a large cell mass is generated in the culture solution 3.

  The measuring device 6 is a computer equipped with image analysis software that has a function of recognizing a cell mass and measuring its diameter. The cell observation means 7 and the ultrasonic irradiation means 5 are connected, and the image data taken by the cell observation means 7 is received, the diameter of the cell mass in the image data is measured, and exceeds a predetermined size. When a cell mass is found, a signal is sent to the ultrasonic irradiation means 5 to activate the ultrasonic irradiation means 5.

  The ultrasonic irradiation means 5 is connected to the measuring device 6 and the ultrasonic transmission terminal 4. The ultrasonic irradiation means 5 is a method using piezoelectric ceramics. Moreover, the output of an ultrasonic wave is used so that the ultrasonic wave reaches only a part of the culture tank. Alternatively, a reflector may be attached to the ultrasonic transmission terminal 4 so that ultrasonic waves are transmitted locally.

  Thus, the cell mass exceeding the threshold in the whole culture solution is included in the ultrasonic irradiation range by the ultrasonic irradiation means 5, and the cell mass not exceeding the threshold is in the ultrasonic irradiation range by the ultrasonic irradiation means 5. The configuration is not included. Since the cell mass irradiated with the ultrasonic wave is subjected to stress, the cell mass whose size does not exceed the threshold value is not irradiated with the ultrasonic wave so as to avoid applying an extra physical stress.

  The frequency of the ultrasonic wave emitted by the ultrasonic wave irradiation means 5 is desirably 20 Hz to 10 GHz. This frequency is appropriate for the desired cell mass to be subdivided. It is further desirable to use a range in which ultrasonic waves are transmitted only to a part of the culture tank. Since the time required for subdividing the cell mass is mainly related to the frequency of the ultrasonic wave, it can be determined according to the size of the desired cell mass.

  A part or all of the container body 1 is a transparent container, and uses glass or plastic. Further, in order to minimize the influence of ultrasonic waves, the flow path 15 may be attached as shown in FIG. 4, or the plastic support 17 and the plastic wall 18 may be attached as shown in FIG. good. The channel 16 is made of a transparent material such as glass or plastic.

  The cell culture apparatus 20 shown in FIG. 1 has a configuration suitable for batch culture (a method in which a new medium is prepared every time, a strain is planted there, and no medium is added until harvest). However, the present invention is not limited to this configuration. In the case of adapting to fed-batch culture (a method in which the medium itself or a specific component in the medium is added during cultivation and the product is not extracted until the end of the culture), an addition medium apparatus can be added. When applying continuous culture (a method of supplying a culture medium to a culture system at a constant speed and simultaneously extracting the same amount of culture solution), a line for extracting the culture solution may be provided, or an airlift type culture device may be provided. You may provide an ultrasonic irradiation means, an ultrasonic transmission terminal, and a culture solution observation means.

<Example 1>
Example 1 is an example in which the culture apparatus of FIG. 1 is used on a commercially available temperature control heater and this embodiment is adapted to batch culture. The container body 1 is composed of a small-scale spinner flask or a glass container.

  Using a flat blade type blade as the stirring means 2, the culture solution was stirred at a rotation speed of 30 rpm. Commercially available induced pluripotent cells were used as the cells. In addition, a medium mainly composed of a commercially available DMEM / F12 medium was used as a cell culture solution.

  A digital microscope with an enlarged photographing function was used as the culture medium observation means 7 outside. It is installed outside the container body 1 and photographs the cells in the culture medium 3. Based on the enlarged image of the cell mass recorded by the culture medium observation means 7, the size of the cell mass was measured by the measurement means 6. A computer and commercially available image analysis software were used as the measuring device 6. The acquired image was binarized with commercially available image analysis software, and the cell mass was recognized. The portion where the cells were dense was defined as a cell mass, and the portion where only a single cell was detected was not regarded as a cell mass. The diameter of the part recognized as a cell mass was measured, and it was judged whether it was larger than the specification. When the cell mass was excessively large, the ultrasonic irradiation means 5 was operated to subdivide the cell mass.

  The cell culture device 20 applies ultrasonic waves to the cell mass by the ultrasonic wave irradiation means 5 and the ultrasonic wave transmission terminal 4 and subdivides them. The ultrasonic transmission terminal 4 is installed so that the tip is in contact with the culture solution 3. In addition, when the measurement device 6 processes the enlarged image data of the animal cell obtained by the culture medium observation means 7 and determines whether or not to apply ultrasonic waves, the tip of the tip is the cell on the processed image data. Located in a location where ultrasonic waves can reach. The cells in the culture medium being stirred are moving at about 0.3 m / s, and ultrasonic waves are irradiated from the culture medium observation means 7 in the direction of rotation of the stirring blade 3 seconds later. Install one. The frequency used by the ultrasonic irradiation means 5 is 20 Hz to 10 GHz.

50 mL of a commercially available medium was put into this apparatus, and artificial pluripotent cells were cultured for 7 days. In 7 days, the cells grew from 3 × 10 ⁵ cells to 7 × 10 ⁷ cells.

  Before the start of culturing, the diameter of the cell mass is 50-100 μm. When culturing is started, the cell mass becomes large, but if it becomes too large, cell characteristics (undifferentiated state, survival rate) are adversely affected. Irradiate sound waves. This threshold value is, for example, 300 μm. The cell mass irradiated with ultrasonic waves was subdivided and became 200 μm to 300 μm at the end of the culture.

<Example 2>
FIG. 2 shows a cell culture apparatus according to the second embodiment. In Example 2, the cell culture apparatus is adapted for fed-batch culture. The lid 8 is provided with a 5 ng / mL fibroblast growth factor additive conduit 9 and a glass bottle for aseptically storing the additive connected to the additive conduit 9. 5 ng / mL fibroblast growth factor was added every day. 50 mL of a commercially available medium was put into this apparatus, and artificial pluripotent cells were cultured for 7 days. Cells grew from 3 × 10 ⁵ cells to 7.4 × 10 ⁷ cells.

  When the size of the cell mass was controlled by the ultrasonic irradiation means 5 in the same manner as in Example 1, the diameter of the cell mass cell mass was changed from 50 to 100 μm at the start of the culture to 200 μm to 300 μm at the end of the culture. Furthermore, by adapting the apparatus to fed-batch culture, the work of adding fibroblast growth factor, which was performed every day for cell maintenance, was eliminated, and work efficiency was improved.

<Example 3>
FIG. 3 schematically shows Example 3 in which this embodiment is applied to continuous culture. A fresh medium conduit 11 on the lid 8, a fresh medium storage container 14 that can store the fresh medium connected to the fresh medium conduit 11 in an aseptic state, a waste liquid conduit 12, and a waste liquid glass container 13. installed. A sterilization filter 10 was installed in the fresh medium pipe 11 and the waste liquid pipe 12. The culture apparatus was agitated for 20 minutes every 24 hours, and the cell mass was submerged in the bottom of the culture solution. Further, the supernatant was aspirated through the waste liquid line 12, and a new medium was added through the fresh medium line 11. 50 mL of a commercially available medium was put into this apparatus, and artificial pluripotent cells were cultured for 7 days. Cells grew from 3 × 10 ⁵ cells to 6.5 × 10 ⁷ cells.

  When the size of the cell mass was controlled by the ultrasonic irradiation means 5 as in Example 1, the diameter of the cell mass was changed from 50 to 100 μm at the start of the culture to 200 μm to 300 μm at the end of the culture. Furthermore, adapting the device to continuous culture eliminates the need for medium change necessary for cell maintenance, and improves labor efficiency.

<Example 4>
FIG. 4 schematically shows Example 4 in the present embodiment. A flow path 15 is attached to the container body 1, the culture solution observation means 7 is attached to the outside of the flow path 15, and the ultrasonic transmission terminal 4 is attached to the flow path 15. 50 mL of a commercially available medium was put into this apparatus, and artificial pluripotent cells were cultured for 7 days. Cells grew from 3 × 10 ⁵ cells to 7.5 × 10 ⁷ cells.

  When the size of the cell mass was controlled by the ultrasonic irradiation means 5 as in Example 1, the diameter of the cell mass was changed from 50 to 100 μm at the start of the culture to 200 μm to 300 μm at the end of the culture. A part of the cell mass contained in the culture fluid flows through the flow path 15.

  By attaching the flow path 15, it becomes easier to irradiate only a part of the cells with ultrasonic waves than when it is not attached, and the influence on the cells can be minimized.

<Example 5>
FIG. 5 schematically shows Example 5 in the present embodiment. The container body 1 and a 10 cm ² square plate 17 were attached to the inside of the container body 1 by using a support column 16 having a length of 5 cm. The ultrasonic transmission terminal 4 was installed on the wall surface of the plate 17 and the container body 1. 50 mL of a commercially available medium was put into this apparatus, and artificial pluripotent cells were cultured for 7 days. Cells grew from 3 × 10 ⁵ cells to 8.0 × 10 ⁷ cells.

  When the size of the cell mass was controlled by the ultrasonic irradiation means 5 as in Example 1, the diameter of the cell mass was changed from 50 to 100 μm at the start of the culture to 200 μm to 300 μm at the end of the culture.

DESCRIPTION OF SYMBOLS 1 ... Container body, 2 ... Stirring means, 3 ... Culture solution, 4 ... Ultrasonic transmission terminal,
5 ... Ultrasonic irradiation means, 6 ... Measuring device, 7 ... Culture medium observation means,
8 ... Lid, 9 ... Pipe for additive, 10 ... Disinfection filter,
11 ... Pipe for fresh medium, 12 ... Pipe for waste liquid, 13 ... Glass container for waste liquid,
14 ... Fresh medium storage container, 15 ... Channel, 16 ... Post, 17 ... Plate

Claims (7)

A cell culture device for culturing cells,
A container having a culture solution;
Cell observation means for acquiring an image of the culture solution containing a plurality of cell masses;
Based on the image acquired by the cell observation means, measurement means for measuring the size of the cell mass,
An ultrasonic irradiation means for irradiating an ultrasonic wave to the cell mass exceeding the threshold when the size of the cell mass measured by the measurement means exceeds the threshold. The cell culture device according to claim 1,
A stirring means for stirring the culture solution;
The cell culturing device, wherein the cell observation means acquires an image of the culture solution stirred by the stirring means. The cell culture device according to claim 2,
The cell culturing apparatus, wherein the cell observation means is fixed so as to photograph a predetermined region. In the cell culture device according to any one of claims 1 to 3,
The cell culture apparatus characterized in that the size of the cell mass measured by the measuring means is the diameter of the cell mass. In the cell culture device according to any one of claims 1 to 4,
The cell mass that exceeds the threshold value in the whole culture solution is included in the ultrasonic irradiation range by the ultrasonic irradiation means,
A cell mass that does not exceed the threshold in the whole culture solution is not included in the ultrasonic irradiation range by the ultrasonic irradiation means. In the cell culture device according to any one of claims 1 to 5,
Having a flow path for circulating a part of the culture solution;
A cell culture device characterized by acquiring an image of a culture solution flowing through the flow path. In the cell culture device according to any one of claims 1 to 6,
A cell culture apparatus, wherein a part or the whole of the container is transparent.

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