JP2021048778A - Cell recovery method and cell culture apparatus - Google Patents

Abstract

To provide a cell recovery method capable of recovering a cultured cell without damaging the cell in the case of performing a process of separating a cell culture part and a covering part.SOLUTION: The cell recovery method comprises a step of realizing a state that cells 101 and culture solution are present in wells 2, the wells 2 are covered by a seal member 4 (covering part), and the cell culture part 1 is housed in a container 6, and saline 102 (aqueous liquid) is stored in the container 6 to a position higher than a well-forming face 1a (surface) of the cell culture part 1 being housed in the container 6. Then, the cell recovery method comprises a step of separating the seal member 4 from the well-forming face 1a of the cell culture part 1 in the state where saline 102 is stored in the container 6 to the position higher than the well-forming face 1a of the cell culture part 1.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cell recovery method and a cell culture device, and more particularly to a cell recovery method and a cell culture device in which a cell culture section including a well-forming surface on which concave wells are formed is used.

Conventionally, a cell recovery method and a cell culture apparatus using a cell culture unit including a well-forming surface on which a concave well is formed are known (see, for example, Non-Patent Document 1).

The cell culture apparatus of Non-Patent Document 1 includes a plate-shaped cell culture plate (cell culture section) in which a plurality of concave wells are formed on the surface. Further, on the surface of the cell culture plate, a plurality of wells are connected to each other, and a flow path through which the culture solution flows is formed. In the cell culture device, cells are seeded in each of the plurality of wells, and the cells are cultured in the culture medium. Here, when the culture solution is introduced into each well, in order to prevent the culture solution from overflowing from the wells (channels), the plate-shaped covering portion is arranged on the surface on which the wells are formed. The culture is introduced into the channels and wells.

Further, although not specified in Non-Patent Document 1, in a conventional cell culture apparatus as described in Non-Patent Document 1, after cell culture is completed, the covering portion and the cell culture plate are used. Is separated and agglomerates of cells are collected from the wells. The step of separating the cell culture plate and the covering portion is performed in air.

K.Hirano et al. “Closed-channel culture system for efficient and reproducible differentiation of human pluripotent stem cells into islet cells” Biochemical and Biophysical Research Communications, 2017, vol.487, p.344-350

However, in the conventional cell culture apparatus as described in Non-Patent Document 1, the coating portion and the cell culture plate (cell culture portion) are separated in the air, so that the coating portion is separated from the surface of the cell culture plate. A layer of air may enter between the surface of the cell culture plate and the coating when the cells are separated. In addition, the covering portion is generally hydrophilic. Therefore, when the coating portion is separated from the surface of the cell culture plate, the culture solution and cells existing between the surface of the cell culture plate and the coating portion (that is, the well and the culture solution flow path) adhere to the surface of the coating portion. May be done. Further, even when only the culture solution in the well adheres to the coating portion, air (air bubbles) is mixed in the well due to the removal of the culture solution from the well, and the cells in the well come into contact with the air. It can be damaged by interfacial tension. Therefore, the conventional cell culture apparatus as described in Non-Patent Document 1 has a problem that it is difficult to recover the cultured cells without damaging them.

The present invention has been made to solve the above-mentioned problems, and one object of the present invention is to use cultured cells when a step of separating a cell culture part and a coating part is performed. It is an object of the present invention to provide a cell recovery method and a cell culture apparatus capable of recovering without damaging them.

In order to achieve the above object, the cell recovery method in the first aspect of the present invention comprises a cell culture section having a surface provided with concave wells for culturing cells and a coating covering the surface of the cell culture section. The step of preparing the part and the container for accommodating the cell culture part, the cells and the culture solution are present in the well, the well is covered with the coating part, and the cell culture part is contained in the container, and the container is filled with the cell culture part. A step to realize a state in which the aqueous liquid is stored in the container to a position higher than the surface of the cell culture part in the contained state, and a state in which the aqueous liquid is stored in the container to a position higher than the surface of the cell culture part. The step of separating the coating portion from the surface of the cell culture portion is provided.

The cell culture apparatus according to the second aspect of the present invention includes a cell culture part including a well-forming surface on which cells are cultured and a concave well is provided, a covering part provided so as to cover the wells on the well-forming surface, and the like. It comprises a container for accommodating the cell culture part, and at least one of the cell culture part and the coating part has flexibility.

According to the cell recovery method according to the first aspect of the present invention, as described above, the coating portion is formed from the surface of the cell culture portion in a state where the aqueous liquid is stored in the container to a position higher than the surface of the cell culture portion. Be separated. Here, in the conventional cell recovery method, when the coating portion is separated from the surface of the cell culture portion in the air, an air layer enters between the surface of the cell culture portion and the coating portion. In addition, the covering portion is generally hydrophilic. Therefore, when the coating portion is separated from the surface of the cell culture portion, the culture solution and cells existing between the surface of the cell culture portion and the coating portion (that is, in the well) may adhere to the surface of the coating portion. .. On the other hand, in the cell recovery method according to the first aspect, since the air layer does not enter between the surface of the cell culture portion and the coating portion, the cells in the well and the culture solution adhere to the coating portion. Can be suppressed. Further, since it is possible to suppress the culture solution in the well from adhering to the coating portion, it is possible to prevent the cells from being damaged by contact with air in the well. As a result, when the step of separating the cell culture part and the coating part is performed, the cultured cells can be recovered without being damaged. The aqueous liquid means a liquid (aqueous solution) in which a certain substance is dissolved in water.

Further, according to the cell culture apparatus according to the second aspect, at least one of the cell culture part and the coating part has flexibility as described above. Thereby, the coating parts can be gradually peeled off from the surface of the cell culture part to separate them from each other. As a result, it is possible to separate the cells from each other while gradually increasing the area of the non-contact portion of the interface between the cell culture portion and the coating portion. As a result, the liquid flow when the water-based liquid stored in the container flows into the non-contact portion can be made relatively gentle as compared with the case where the area is rapidly increased. As a result, when the coating portion is separated from the surface of the cell culture portion, it is possible to prevent the cells in the well from being washed away by the above-mentioned liquid flow and being discharged from the well.

It is sectional drawing which showed the structure of the cell culture apparatus by 1st Embodiment. It is a top view of the cell culture part by 1st Embodiment. It is sectional drawing of the state in the process of peeling off the seal member of the cell culture apparatus by 1st Embodiment. It is sectional drawing which shows the state at the time of cell culture of the cell culture apparatus by 1st Embodiment. It is a flow chart which showed the cell recovery method by 1st Embodiment. It is sectional drawing which shows the state which housed the cell culture part by 1st Embodiment in a container. It is sectional drawing which shows the state which stored the physiological saline in the container by 1st Embodiment. It is sectional drawing in the state which removed the lid member by 1st Embodiment. It is a figure which showed the state which collected the cell by 1st Embodiment. It is a perspective view which showed the structure of the cell culture apparatus by 2nd Embodiment. FIG. 5 is an enlarged cross-sectional view showing a state in which a plurality of layers of tape according to the second embodiment are wound around a first core and then unwound. FIG. 5 is an enlarged cross-sectional view showing a state in which a single-layer tape according to the second embodiment is wound from a state in which the first core is wound. It is a flow chart which showed the cell recovery method by 2nd Embodiment. It is the schematic which shows the state which the tape by 2nd Embodiment is housed in the housing part. It is a schematic diagram which shows the cell seeding method by 2nd Embodiment. It is a schematic diagram which shows the cell culture method by 2nd Embodiment. It is the schematic which shows the state which stored the physiological saline solution by 2nd Embodiment in a housing part. It is the schematic which shows the state which collects the cell by 2nd Embodiment. FIG. 18 is a partially enlarged cross-sectional view of FIG. It is sectional drawing which shows the structure of the cell culture apparatus by the modification of 1st Embodiment.

Hereinafter, embodiments embodying the present invention will be described with reference to the drawings.

[First Embodiment]
(Structure of cell culture device)
The configuration of the cell culture apparatus 100 according to the first embodiment will be described with reference to FIGS. 1 to 4. In the first embodiment, the Z direction means the vertical direction, and the Z1 direction and the Z2 direction mean the upper side and the lower side of the vertical direction, respectively.

As shown in FIG. 1, the cell culture device 100 includes a cell culture unit 1 for culturing cells 101. The cell culture unit 1 has a plate shape (see FIG. 2). The cell culture unit 1 is made of resin (for example, made of silicon rubber). The cell culture unit 1 has flexibility.

The cell culture unit 1 includes a well forming surface 1a provided with a concave well 2 in which cells 101 are cultured. The well-forming surface 1a is a surface on the upper side (Z1 direction side) of the cell culture unit 1.

A plurality of wells 2 are provided on the well forming surface 1a. For example, as shown in FIG. 2, the plurality of wells 2 are arranged in a matrix. In FIG. 2, for the sake of simplification, the lid member 5 described later is not shown.

Further, the inner wall 2a of the well 2 is coated with a non-adhesive polymer to the cells 101. Further, the bottom surface 2b of the inner wall 2a of the well 2 has a spherical shape. As a result, the cells 101 in the well 2 gather together to form one agglomerate.

Further, on the well forming surface 1a, a culture solution flow path 3 connected to the well 2 and through which the culture solution flows is formed. The culture solution flow path 3 is provided so as to connect the wells 2. Specifically, the culture solution flow path 3 is provided so as to connect the wells 2 arranged in a predetermined direction (X direction in FIG. 2) among the plurality of wells 2 arranged in a matrix. Teru. The arrangement of the plurality of wells 2 and the culture solution flow path 3 is not limited to this.

Further, the cell culture device 100 includes a seal member 4 provided so as to cover the well 2 of the well forming surface 1a. The sealing member 4 has flexibility (flexibility) and seals the well-forming surface 1a of the cell culture unit 1. The sealing member 4 is made of plastic (for example, polyethylene terephthalate or polyethylene) or rubber (for example, silicon rubber). Further, the seal member 4 is an example of a "covered portion" within the scope of claims.

Here, in the first embodiment, the seal member 4 is provided so as to cover all of the plurality of wells 2. Specifically, the seal member 4 has an area substantially equal to the well-forming surface 1a of the cell culture unit 1 in a plan view (viewed from the Z1 direction side), and covers the entire area of the well-forming surface 1a. It is provided as follows. Although the seal member 4 (see the broken line) is shown slightly smaller than the well forming surface 1a in FIG. 2 for easy understanding, it is actually the same size.

Further, the cells 101 are collected through the opening of the well 2 in a state where the seal member 4 is peeled off from the cell culture unit 1. As a result, the cells 101 can be individually collected while maintaining the state in which the cells 101 are aligned according to the arrangement of the wells 2. Further, unlike the case where the cells 101 are collected via the culture solution flow path 3, it is not necessary to design the culture solution flow path 3 according to the size of the agglomerates of the cells 101, and the culture solution flow path 3 can be designed. It will be easier.

Further, the cell culture device 100 includes a lid member 5 arranged on the surface 3a of the seal member 4 on the side opposite to the cell culture unit 1 (Z1 direction side). The lid member 5 is not flexible and is configured to be openable and closable independently of the seal member 4. That is, the seal member 4 is sandwiched between the lid member 5 and the cell culture unit 1. Further, the seal member 4 and the lid member 5 are in contact with each other without being adhered to each other. The lid member 5 is made of, for example, polycarbonate, acrylic, glass, or the like. Further, each of the cell culture unit 1, the seal member 4, and the lid member 5 is transparent. The lid member 5 is an example of the "first lid member" in the claims.

Further, the cell culture device 100 includes a container 6 for accommodating the cell culture unit 1 in a state where the well 2 is covered with the seal member 4. Specifically, the container 6 is configured to accommodate both the cell culture unit 1 and the seal member 4.

Specifically, the container 6 has a concave shape in which the side opposite to the bottom surface portion 6a (Z2 direction side) on which the cell culture portion 1 is arranged is open. The surface 4a on the Z1 direction side of the seal member 4 is on the bottom surface 6a side (Z2 direction side) of the contact portion 6c formed by the tip end portion 6b on the side opposite to the bottom surface portion 6a (Z2 direction side) of the container 6. Is located in. The contact portion 6c is provided in a circumferential shape (see FIG. 2).

Here, in the first embodiment, as shown in FIG. 1, the container 6 is in a state where the interface between the cell culture unit 1 and the sealing member 4 is immersed in the physiological saline 102 stored in the container 6. The cells 101 are configured to be recovered. Specifically, the entire interface between the cell culture unit 1 and the sealing member 4 is immersed in the physiological saline 102 stored in the container 6 at the time of collecting the cells 101. The physiological saline 102 is an example of the "aqueous liquid" in the claims.

Further, the container 6 is configured such that the cells 101 are collected in a state where the entire cell culture unit 1 and the sealing member 4 are immersed in the physiological saline 102 stored in the container 6. Specifically, at the time of collecting the cells 101, the entire cell culture unit 1 and the seal member 4 and a part of the lid member 5 on the seal member 4 side (Z2 direction side) are made of physiological saline. Immersed in 102.

That is, the depth d1 (in the Z direction) of the container 6 is larger than the sum of the thickness t1 (for example, 2 mm) of the cell culture unit 1 and the thickness t2 (for example, 100 μm) of the seal member 4 (d1> t1 + t2). Further, the depth d2 of the physiological saline 102 stored in the container 6 at the time of collecting the cells 101 is larger than t1 + t2 and smaller than d1 (t1 + t2 <d2 <d1). The depth d2 of the physiological saline 102 may be t1 + t2 or less as long as it is larger than t1.

Further, in the first embodiment, as shown in FIG. 3, the seal member 4 has flexibility so that it can be gradually peeled off from the cell culture unit 1. Specifically, the seal member 4 is configured to be able to be gradually peeled off (turned over) from the well-forming surface 1a of the cell culture unit 1 while being deformed (bent). Note that FIG. 3 shows how the seal member 4 is gradually peeled off from the end on one side (right side in FIG. 3) in the X direction. In this case, of the plurality of wells 2 on the well forming surface 1a, the wells 2 on one side in the X direction are opened in order. The method of peeling the seal member 4 (peeling direction) is not limited to this, and may be from any direction. Further, the well forming surface 1a is an example of the "surface" in the claims.

Further, as shown in FIG. 1, the cell culture apparatus 100 includes a screw member 7 that penetrates the lid member 5 and is fastened to the container 6. As a result, the screw member 7 presses the lid member 5 against the seal member 4 to fix the seal member 4. Specifically, the screw member 7 penetrates the lid member 5 and is fastened to the bottom surface portion 6a of the container 6. Further, a plurality of screw members 7 are provided, for example, they are provided near both ends of the lid member 5 in the X direction. The screw member 7 is an example of a "fixing member" within the scope of the claims.

Further, in the screw member 7, the lid member 5 is relatively firmly fastened to the container 6 so that a pressing force that slightly deforms the seal member 4 and the cell culture unit 1 is generated. As a result, the well-forming surface 1a of the cell culture unit 1 is reliably (stable) sealed by the sealing member 4.

Further, in the first embodiment, as shown in FIG. 4, the cell culture apparatus 100 includes a lid member 8 that seals the inside of the container 6 by abutting on the contact portion 6c of the container 6. Specifically, the lid member 8 has a concave shape in which the cell culture unit 1 side (Z2 direction side) is open. The lid member 8 is composed of a tip portion 8a on the cell culture portion 1 side and includes a contact portion 8b provided in a circumferential shape. The container 6 is sealed by the contact portion 6c of the container 6 and the contact portion 8b of the lid member 8 contacting each other in a circumferential shape. As will be described later, the lid member 8 is used only when the cells 101 are cultured. Further, the lid member 8 is an example of the "second lid member" in the claims.

Further, as shown in FIG. 1, the cell culture unit 1 and the container 6 are provided with an introduction side flow path 9 which is connected to the culture solution flow path 3 and introduces the culture solution into the culture solution flow path 3. .. Further, the cell culture unit 1 and the container 6 are provided with a discharge side flow path 10 which is connected to the culture solution flow path 3 and discharges the culture solution from the culture solution flow path 3. Each of the introduction side flow path 9 and the discharge side flow path 10 is provided on the side opposite to the seal member 4 (Z2 direction side) with respect to the culture solution flow path 3. The introduction-side flow path 9 and the discharge-side flow path 10 are examples of the "first introduction-side flow path" and the "first discharge-side flow path" in the claims, respectively.

Specifically, the introduction side flow path 9 includes a flow path 9a formed in the cell culture unit 1 and a flow path 9b formed in the container 6. The flow path 9a of the cell culture unit 1 is connected to the culture solution flow path 3. The flow path 9b of the container 6 communicates with the flow path 9a of the cell culture unit 1. Further, the culture solution is introduced into the flow path 9b of the container 6 from the introduction side tube 103. The opening 9c of the flow path 9b of the container 6 connected to the introduction side tube 103 is provided on the side surface 6d on one side (left side in FIG. 1) of the container 6 in the X direction.

Further, the discharge side flow path 10 includes a flow path 10a formed in the cell culture unit 1 and a flow path 10b formed in the container 6. The flow path 10a of the cell culture unit 1 is connected to the culture solution flow path 3. The flow path 10b of the container 6 communicates with the flow path 10a of the cell culture unit 1. Further, the culture solution flowing through the flow path 10b of the container 6 is discharged to the outside of the container 6 by the discharge side tube 104. The opening 10c of the flow path 10b of the container 6 connected to the discharge side tube 104 is provided on the side surface 6e on one side (right side in FIG. 1) of the container 6 in the X direction.

(Cell recovery method)
Next, the cell recovery method (and the cell culture method) of the cell culture apparatus 100 will be described with reference to FIGS. 3 to 9.

As shown in FIG. 5, the cell recovery method of the cell culture apparatus 100 includes a step (step 801) of preparing the cell culture unit 1, the seal member 4, and the container 6.

Next, in the cell recovery method of the cell culture apparatus 100, the cells 101 and the culture solution are present in the well 2, the well 2 is covered with the seal member 4, and the cell culture unit 1 is housed in the container 6. A step (steps 802 to 804) for realizing a state in which the physiological saline 102 is stored in the container 6 up to a position higher than the well forming surface 1a of the cell culture unit 1 in the state of being housed in the container 6 is provided. Details will be described in the description of steps 802 to 804 below.

Specifically, in this step 802, the well-forming surface 1a of the cell culture unit 1 is sealed by the flexible sealing member 4. Then, the cells 101 are housed in the well 2, and the cell culture unit 1 in a state where the well 2 is covered (sealed) by the sealing member 4 is housed in the container 6. That is, both the cell culture unit 1 and the seal member 4 are housed in the container 6. In this step 802, the seal member 4 may be arranged on the well-forming surface 1a after the cell culture unit 1 is housed in the container 6, or the cell culture unit 1 in which the seal member 4 is arranged on the well-forming surface 1a in advance. May be contained in the container 6. The seeding of the cells 101 in the well 2 may be performed before or after the cell culture unit 1 is housed in the container 6.

Next, in the first embodiment, in step 803, the cells 101 and the culture solution are housed in the well 2 and the cells 101 are cultured. Here, the step of culturing the cells 101 (step 803) is a step of increasing the sealing property of the sealing member 4 with respect to the well forming surface 1a of the cell culture unit 1 and a step of increasing the sealing property of the sealing member 4 in a state where the sealing property of the sealing member 4 is increased. Including the step of culturing.

Specifically, the step of increasing the sealing property of the sealing member 4 with respect to the well forming surface 1a of the cell culture unit 1 is the surface of the sealing member 4 with the cell culture unit 1 and the sealing member 4 housed in the container 6. A step of arranging the lid member 5 (see FIG. 6) on the 4a and a step of pressing the lid member 5 against the seal member 4 by the screw member 7 (see FIG. 6) to fix the seal member 4 to the container 6 are included. The lid member 5 (see FIG. 6) may be arranged on the surface 4a of the seal member 4 before the cell culture unit 1 and the seal member 4 are housed in the container 6.

Then, in the step of culturing the cells 101, after the lid member 5 and the screw member 7 are attached to the cell culture unit 1, the introduction side tube 103 and the discharge side tube 104 are connected to the container 6 (see FIG. 4). Then, the culture solution is introduced into the culture solution flow path 3 by the introduction side flow path 9, and the culture solution is discharged from the discharge side flow path 10. Specifically, the culture solution is flowed in the order of the introduction side tube 103, the introduction side flow path 9, the culture solution flow path 3, the discharge side flow path 10, and the discharge side tube 104, and the culture solution is always fresh in the well 2. Is supplied. Then, the cells 101 in the well 2 are cultured by performing the flow of the culture solution for a certain period of time.

Further, in the first embodiment, in step 803 (see FIG. 4), the inside of the container 6 is sealed by bringing the lid member 8 into contact with the contact portion 6c of the container 6. That is, in step 803, the culture solution flows in a state in which the lid member 8 is arranged so as to cover the container 6 (a state in which the contact portion 6c of the container 6 and the contact portion 8b of the lid member 8 are in contact with each other). Will be done.

Next, in step 804, the physiological saline 102 is stored in the container 6 up to a position higher than the well forming surface 1a of the cell culture unit 1 in the state of being housed in the container 6. In other words, the physiological saline 102 is stored in the container 6 so that the interface between the cell culture unit 1 and the sealing member 4 is immersed in the physiological saline 102. Specifically, the physiological saline 102 is stored in the container 6 so that the entire cell culture unit 1 and the sealing member 4 are immersed in the physiological saline 102. For example, the saline 102 is injected into the container 6 by a pipette 105 (see FIG. 7).

Here, in the first embodiment, in this step 804, the container 6 is opened (the sealed state is released) by removing the lid member 8 used in the step 803 from the container 6. That is, the physiological saline 102 is injected into the container 6 with the lid member 8 removed from the container 6.

Next, in the cell recovery method, the seal member 4 is pressed from the well-forming surface 1a of the cell culture unit 1 in a state where the physiological saline 102 is stored in the container 6 to a position higher than the well-forming surface 1a of the cell culture unit 1. It comprises a step of separation (step 805).

Here, in the first embodiment, the cell recovery method includes a step of releasing the state in which the sealing property of the sealing member 4 is increased before the step of separating the sealing member 4 from the well forming surface 1a of the cell culture unit 1. Be prepared. Specifically, in step 805, as shown in FIG. 8, the screw member 7 (see FIG. 7) is unfastened and the lid is released before the seal member 4 is separated from the well-forming surface 1a of the cell culture unit 1. The member 5 is removed from the seal member 4.

Next, as shown in FIG. 3, in a state where the interface between the cell culture unit 1 and the seal member 4 is immersed in the physiological saline 102, the seal member 4 having flexibility (flexibility) is the cell culture unit 1. Is gradually peeled off from the well-forming surface 1a. For example, the seal member 4 is slowly peeled off from the well forming surface 1a at a speed of 1 cm / s. The diameter of the well 2 is, for example, about 400 μm.

Then, as shown in FIG. 5, in the cell recovery method, after the step of separating the cell culture unit 1 and the seal member 4 (step 805), the cell 101 is recovered (acquired) from the well 2 (step 806). To be equipped. For example, as shown in FIG. 9, in step 806, a step of collecting each cell 101 of the plurality of wells 2 is performed by the pipette 106. The recovery step of the cells 101 is performed in a state where the cell culture unit 1 is immersed in the physiological saline 102 in the container 6. This completes the recovery of cells 101.

(Effect of the first embodiment)
In the first embodiment, the following effects can be obtained.

In the first embodiment, as described above, the cell culture unit 1 is in a state where the physiological saline 102 (aqueous liquid) is stored in the container 6 to a position higher than the well forming surface 1a (surface) of the cell culture unit 1. The cell recovery method is configured to include a step of separating the seal member 4 (covered portion) from the well-forming surface 1a of the cell. Here, in the conventional cell recovery method, when the coating portion is separated from the surface of the cell culture portion in the air, an air layer enters between the surface of the cell culture portion and the coating portion. In addition, the covering portion is generally hydrophilic. Therefore, when the coating portion is separated from the surface of the cell culture portion, the culture solution and cells existing between the surface of the cell culture portion and the coating portion (that is, the well and the culture solution flow path) adhere to the surface of the coating portion. May be done. On the other hand, in the cell recovery method of the first embodiment, since the air layer does not enter between the well forming surface 1a of the cell culture unit 1 and the seal member 4, the cells 101 in the well 2 and the culture are cultured. It is possible to prevent the liquid from adhering to the sealing member 4. Further, since it is possible to prevent the culture solution in the well 2 from adhering to the seal member 4, it is possible to prevent the cells 101 from being damaged by contact with air in the well 2. As a result, when the step of separating the cell culture unit 1 and the seal member 4 is performed, the cultured cells 101 can be recovered without being damaged.

Further, since the well 2 is covered (sealed) by the seal member 4 (covered portion), it is possible to suppress the formation of a gap between the seal member 4 and the well forming surface 1a (surface) as much as possible. Can be done. As a result, the well 2 (well forming surface 1a) is covered with the seal member 4, so that the cells 101 can be suppressed from being discharged from the gap between the well 2 and the seal member 4.

Further, here, the physiological saline solution is a liquid that does not adversely affect the cells as much as possible. Therefore, the cells 101 are recovered by recovering the cells 101 in a state where the interface between the cell culture section 1 and the sealing member 4 (coating section) is immersed in the physiological saline 102 (aqueous liquid) stored in the container 6. Occasionally, it is possible to prevent the cells 101 from being adversely affected by the liquid (saline 102) in the container 6.

Further, in the first embodiment, as described above, after separating the seal member 4 (coating portion) from the well forming surface 1a (surface) of the cell culture unit 1, the step of acquiring the cells 101 from the well 2 is provided. In addition, a cell recovery method is constructed. Thereby, the cells 101 can be easily obtained from the well 2.

Further, in the first embodiment, as described above, the step of separating the seal member 4 (coating portion) from the well forming surface 1a (surface) of the cell culture unit 1 is a step of separating the sealing member 4 (covering portion) from the well forming surface 1a (surface) of the cell culture unit 1, but at least one of the sealing members 4 has flexibility. The cell recovery method is configured to include a step of separating the cell culture unit 1 from the cell culture unit 1. As a result, the seal members 4 can be separated from each other by gradually peeling the seal members 4 from the well-forming surface 1a of the cell culture unit 1. As a result, the interface between the cell culture unit 1 and the seal member 4 can be separated from each other while gradually increasing the area of the non-contact portion. As a result, the liquid flow when the physiological saline 102 stored in the container 6 flows into the non-contact portion can be made relatively gentle as compared with the case where the area is rapidly increased. .. As a result, when the seal member 4 is separated from the well-forming surface 1a of the cell culture unit 1, it is possible to prevent the cells 101 in the well 2 from being washed away by the above-mentioned liquid flow and discharged from the well 2. ..

Further, in the first embodiment, as described above, the step of realizing the state in which the physiological saline 102 (aqueous liquid) is stored in the container 6 accommodates the cells 101 and the culture solution in the well 2 and the cells. The cell recovery method is configured to include the step of culturing 101. As a result, the cells 101 can be cultured in the cell culture unit 1 before the cells 101 are collected (before the seal member 4 (coating portion) is separated from the well forming surface 1a (surface)).

Further, in the first embodiment, as described above, the step of culturing the cell 101 is a step of increasing the sealing property of the sealing member 4 (covering portion) with respect to the well forming surface 1a (surface) of the cell culturing portion 1. The step includes culturing the cells 101 in a state where the sealing property of the sealing member 4 is increased. Further, the cell recovery method is configured so as to include a step of releasing the state in which the sealing property of the sealing member 4 is increased before the step of separating the sealing member 4 from the well forming surface 1a of the cell culture unit 1. Thereby, by culturing the cells 101 in a state where the sealing property of the sealing member 4 is increased, it is possible to suppress the discharge of the cells 101 from the well 2 during the culturing of the cells 101. Further, before the step of separating the seal member 4 from the well forming surface 1a, the sealed member 4 can be easily separated from the well forming surface 1a by releasing the state in which the sealing property of the sealing member 4 is increased. it can.

Further, in the first embodiment, as described above, the step of realizing the state in which the physiological saline 102 (aqueous liquid) is stored in the container 6 is a cell culture by the flexible sealing member 4 (coating portion). A step of sealing the well-forming surface 1a (surface) of the part 1 is included. Then, the step of increasing the sealing property of the sealing member 4 is formed on the surface 4a of the sealing member 4 opposite to the cell culture unit 1 so that the lid member 5 (which can be opened and closed independently of the sealing member 4). The cell recovery method is configured to include the step of arranging the first lid member). As a result, the seal member 4 is fixed by the lid member 5, so that the seal member 4 can be stably fixed to the cell culture unit 1. Further, since the lid member 5 can be opened and closed independently of the seal member 4, the lid member 5 can be removed while the seal member 4 is arranged in the cell culture unit 1. As a result, when the lid member 5 is removed, it is possible to prevent the physiological saline 102 from flowing into the interface between the seal member 4 and the cell culture unit 1.

Further, in the first embodiment, as described above, the step of increasing the sealing property of the sealing member 4 (covered portion) penetrates the lid member 5 (first lid member) and is fastened to the container 6. The cell collection method is configured so as to include a step of pressing the lid member 5 against the seal member 4 to fix the seal member 4 by the 7 (fixing member). As a result, the seal member 4 can be stably fixed by the cell culture unit 1 by the lid member 5 and the screw member 7.

Further, in the first embodiment, as described above, the step of culturing the cells 101 is provided in the container 6 having a concave shape whose side opposite to the bottom surface portion 6a where the cell culture unit 1 is arranged is open. A step of sealing the inside of the container 6 by bringing the lid member 8 (second lid member) into contact with the contact portion 6c which is composed of the tip portion 6b on the opposite side of the bottom surface portion 6a and is provided in a circumferential shape. The cell recovery method is configured to include. Further, before the step of separating the seal member 4 (coating portion) from the well forming surface 1a (surface) of the cell culture unit 1, a step of opening the container 6 by removing the lid member 8 from the container 6 is performed. Configure the cell recovery method to be prepared. As a result, the cells 101 can be cultured in a state where the inside of the container 6 is sealed by the lid member 8, so that it is possible to prevent dust and the like from being mixed into the container 6 when the cells 101 are cultured. As a result, it is possible to suppress the contamination of the physiological saline 102 (water-based liquid) in the container 6, so that it is possible to prevent the cells 101 from being adversely affected by the contamination of the physiological saline 102 when the cells 101 are collected. Can be done. Further, since the lid member 8 is removed from the container 6 before the step of separating the seal member 4 (covering portion) from the well forming surface 1a (surface), the sealing member 4 (covering portion) is removed from the well forming surface 1a (surface). Can be easily separated.

Further, in the first embodiment, as described above, the step of culturing the cells 101 is provided in the cell culture unit 1 and the container 6 on the side opposite to the seal member 4 with respect to the culture solution flow path 3, and the culture solution is provided. The step of introducing the culture solution into the culture solution flow path 3 by the introduction side flow path 9 (first introduction side flow path) connected to the flow path 3 and the opposite of the sealing member 4 to the culture solution flow path 3 A step of discharging the culture solution from the culture solution flow path 3 by the discharge side flow path 10 (first discharge side flow path) provided in the cell culture unit 1 and the container 6 on the side and connected to the culture solution flow path 3. The cell recovery method is configured to include. As a result, the well 2 is formed by the culture solution flowing through the introduction side flow path 9, the culture solution flow path 3, and the discharge side flow path 10 in a state where the well forming surface 1a of the cell culture unit 1 is covered with the seal member 4. The cells 101 in the cell 101 can be easily cultured. Further, since each of the introduction side flow path 9 and the discharge side flow path 10 is provided in the cell culture unit 1 and the container 6, the cell culture unit 1 (container 6) is provided with respect to the seal member 4 and the lid member 5. Even if there are obstacles on the opposite side, the tubes (103, 104) are introduced while suppressing the interference between the tubes (103, 104) through which the culture solution is circulated and the obstacles, etc. It can be connected to each of the side flow path 9 and the discharge side flow path 10.

Further, in the first embodiment, as described above, the cell culture device 100 is configured so that at least one of the cell culture unit 1 and the seal member 4 (coating unit) has flexibility. As a result, the seal members 4 can be separated from each other by gradually peeling the seal members 4 from the well-forming surface 1a (surface) of the cell culture unit 1. As a result, the interface between the cell culture unit 1 and the seal member 4 can be separated from each other while gradually increasing the area of the non-contact portion. As a result, the liquid flow when the physiological saline 102 (aqueous liquid) stored in the container 6 flows into the non-contact state is relatively gentle as compared with the case where the area is rapidly increased. can do. As a result, when the seal member 4 is separated from the well-forming surface 1a of the cell culture unit 1, it is possible to prevent the cells 101 in the well 2 from being washed away by the above-mentioned liquid flow and discharged from the well 2. Cell culture apparatus 100 can be provided.

[Second Embodiment]
Next, the cell culture apparatus 200 according to the second embodiment will be described with reference to FIGS. 10 to 19. The cell culture device 200 of the second embodiment includes a strip-shaped tape 11 unlike the above-mentioned first embodiment including the cell culture unit 1 having a plate shape. The same configuration as that of the first embodiment is illustrated with the same reference numerals as those of the first embodiment, and the description thereof will be omitted.

(Structure of cell culture device)
The configuration of the cell culture apparatus 200 according to the second embodiment will be described with reference to FIGS. 10 to 12. In the second embodiment, the Z direction means the vertical direction, and the Z1 direction and the Z2 direction mean the upper side and the lower side of the vertical direction, respectively.

As shown in FIG. 10, the cell culture apparatus 200 includes a microarray tape (hereinafter, simply referred to as tape 11) that is flexible and can be wound around the circumference. The tape 11 is formed in a band shape. The tape 11 is an example of a "cell culture unit" within the scope of the claims.

The tape 11 includes a well forming surface 11a provided with a plurality of wells 2. Specifically, as shown in FIG. 10, on the well forming surface 11a, a well arrangement region 11b (shaded portion in FIG. 10) in which a plurality of wells 2 are densely arranged is provided along the direction in which the tape 11 extends. It is provided at predetermined distances. The well-forming surface 11a is an example of the "surface" in the claims.

The cell culture device 200 includes a housing portion 20 that houses the tape 11. The housing portion 20 has a substantially rectangular parallelepiped shape. The housing portion 20 is made of, for example, resin. The housing portion 20 is an example of a “container” within the scope of claims.

Further, the cell culture apparatus 200 includes a first core 30 extending along the central axis α1 of the winding of the tape 11. Further, the cell culture device 200 is provided separately from the first core 30, and includes a second core 40 extending along the central axis α2 of the winding of the tape 11. The second core 40 is configured to wind up the tape 11 unwound from the first core 30 (in the cell recovery step). The central axis α1 of the first core 30 and the central axis α2 of the second core 40 extend substantially in parallel. The tape 11 wound around the first core 30 is wound with the well forming surface 11a facing the first core 30 side. Further, the tape 11 wound around the second core 40 is wound with the well forming surface 11a facing the opposite side to the second core 40.

Further, the housing portion 20 is provided so as to accommodate the first core 30 and the second core 40. Further, the housing portion 20 is configured to be capable of storing the physiological saline solution 209, which will be described later.

Further, the first core 30 includes a protruding end 30a projecting from the side surface 21 of the housing portion 20 in the direction in which the central axis line α1 (α2) extends to the outside of the housing portion 20. Further, the second core 40 includes a protruding end 40a projecting from the side surface 21 of the housing portion 20 to the outside of the housing portion 20.

Further, the housing portion 20 includes a window portion 22a provided on a side surface 22 (side surface on the Z1 direction side) extending along the direction in which the central axis line α1 (α2) extends. The window portion 22a is configured to be openable and closable. When the window portion 22a is closed, the inside of the housing portion 20 is sealed (confidentiality is maintained). The window portion 22a has a substantially rectangular shape. The window portion 22a may have a shape other than the rectangular shape (for example, a circular shape).

Further, in the housing portion 20, two guide portions 23 provided between the first core 30 and the second core 40 are provided. By rotating the first core 30 and the second core 40 while the tape 11 is guided by the two guide portions 23, the tape 11 wound around the first core 30 is wound around the second core 40 ( Alternatively, the tape 11 wound around the first core 30 is wound around the second core 40). The protruding end 30a of the first core 30 is provided with a groove portion (not shown) to which a jig (driver or the like) (not shown) for rotating the first core 30 is attached. Further, the protruding end 40a of the second core 40 is provided with a groove portion (not shown) to which a jig (driver or the like) (not shown) for rotating the second core 40 is attached. By rotating the first core 30 (second core 40) by rotating the jig with a motor or the like, the tape 11 can be wound around the first core 30 (second core 40). There is. The motors that drive each of the first core 30 and the second core 40 are configured to be driven at the same time.

Further, when the tape 11 is wound by the first core 30 or the second core 40 while being guided by the guide portion 23, the well forming surface 11a of the tape 11 faces the window portion 22a along the window portion 22a. It is configured to be moved (transported). That is, in this case, the window portion 22a and the well forming surface 11a of the tape 11 are configured to overlap when viewed from the direction perpendicular to the side surface 22.

Further, the tape 11 unwound from the first core 30 is used for introducing the cell suspension 101a (for seeding the cells 101) at a position where the entire region of the well arrangement region 11b is exposed from the window portion 22a. It will be temporarily stopped. The flat tape 11 between the two guide portions 23 is not immersed in the culture solution in the housing portion 20, but is held in the air inside the housing portion 20.

Here, in the second embodiment, as shown in FIGS. 11 and 12, the cell culture apparatus 200 is provided by the tape 11 wound around the inner peripheral side of the tape 11 on the outer peripheral side or the first core 30. The covering portion 14 is provided. Specifically, as shown in FIG. 11, when the plurality of layers of tape 11 are wound around the first core 30, the outermost tape 11 is on the inner peripheral side of the outermost tape 11 at that time. The tape 11 wound in contact with the tape 11 serves as a covering portion 14 provided so as to cover the well 2 provided on the well forming surface 11a of the outermost tape 11. Further, as shown in FIG. 12, when only one layer of tape 11 is wound around the first core 30, the first core 30 covers the well 2 provided on the well forming surface 11a of the tape 11. It becomes the covering portion 14 provided.

(Cell recovery method)
Next, the cell recovery method (further, the cell seeding method and the cell culture method) of the cell culture apparatus 200 will be described with reference to FIGS. 13 to 19.

As shown in FIG. 13, the cell recovery method of the cell culture apparatus 200 includes a step (step 901) of preparing the tape 11, the first core 30, the second core 40, and the housing portion 20. ..

Next, in the cell recovery method of the cell culture apparatus 200, the cells 101 and the culture solution are present in the well 2, the well 2 is covered with the covering portion 14, and the tape 11 is housed in the housing portion 20. A step (steps 902 to 905) for realizing a state in which the physiological saline 209 is stored in the housing portion 20 up to a position higher than the well forming surface 11a of the tape 11 housed in the housing portion 20 is provided. .. Details will be described in the description of steps 902 to 905 below.

Specifically, in step 902, the tape 11 is housed in the housing portion 20. At this point, the tape 11 is wound around the second core 40. Further, in step 902, the physiological saline 203 (see FIG. 14) is stored in the housing portion 20. The physiological saline 203 is introduced into the housing 20 from the distribution unit 24 (see FIG. 14) provided in the housing 20. The distribution unit 24 is provided below the second core 40 (on the Z2 direction side). At this time, the first core 30 and the second core 40 are immersed in the physiological saline 203, and the two guide portions 23 are not immersed in the physiological saline 203.

Next, in step 903, a step of seeding the cells 101 into the well 2 is performed. Specifically, as shown in FIG. 15, on a flat tape 11 (between two guide portions 23) while being unwound from the second core 40 and wound up by the first core 30. The cell suspension 204a is discharged from the dispenser 204 into the well 2 provided. The cell suspension 204a discharged from the dispenser 204 is discharged to the well 2 through the window portion 22a of the housing portion 20. As a result, the cells 101 are seeded in the well 2.

Next, as shown in FIG. 13, in step 904, a step of culturing the cells 101 is performed. Specifically, as shown in FIG. 16, the culture solution stored in the liquid storage unit 205 is introduced by the pump 206 into each of the plurality of wells 2 of the tape 11 wound around the first core 30. To. Then, the culture solution introduced into each of the plurality of wells 2 is stored in the liquid storage unit 205 again. The culture solution circulates in the liquid storage unit 205, the pump 206, and the tape 11 for a certain period of time, so that the cells 101 seeded in the well 2 of the tape 11 are cultured.

Specifically, the culture solution stored in the liquid storage unit 205 is introduced into the first core 30 by the pump 206 through the introduction side tube 207 connected to the first core 30. The culture broth introduced into the first core 30 is introduced into each of the plurality of wells 2 of the tape 11 through a flow path (not shown) provided in the first core 30. Further, the culture solution introduced into each of the plurality of wells 2 is discharged to the outside of the first core 30 through a flow path (not shown) provided in the first core 30. The culture solution discharged to the outside of the first core 30 is introduced into the liquid storage unit 205 through the discharge side tube 208 connected to the first core 30. The step of culturing the cells 101 is performed in a state in which the physiological saline 203 (see FIG. 15) in the housing portion 20 is discharged (a state in which the housing portion 20 is filled with air).

Next, as shown in FIG. 13, in step 905, a step of storing the physiological saline 209 (see FIG. 17) in the housing portion 20 is performed. Specifically, the physiological saline 209 is stored in the housing 20 up to a position higher than the well forming surface 11a of the tape 11 housed in the housing 20. Specifically, physiological saline is applied to the housing portion 20 so that the interface between the tape 11 and the covering portion 14 (tape 11 on the inner peripheral side or the first core 30) is immersed in physiological saline 209 (see FIG. 17). Water 209 is stored. The physiological saline 209 is an example of the "aqueous liquid" in the claims.

Further, as shown in FIG. 17, the housing is provided so that the entire 1st core 30, the tape 11 wound around the 1st core 30, and the 2nd core 40 are immersed in the physiological saline 209. Saline 209 is stored in part 20. The physiological saline 209 is introduced into the housing 20 from the distribution unit 24 of the housing 20.

Next, as shown in FIG. 13, in the cell recovery method, physiological saline 209 (see FIG. 17) is placed in the housing portion up to a position higher than the well forming surface 11a of the tape 11 housed in the housing portion 20. In the state of being stored in the tape 20 (the interface between the tape 11 and the covering portion 14 (the tape 11 on the inner peripheral side or the first core 30) is immersed in the physiological saline 209), the tape 11 and the covering portion 14 (Step 906) is provided.

Specifically, as shown in FIG. 18, in step 906, the tape 11 wound around the first core 30 is unwound (the tape 11 is wound by the second core 40), so that the covering portion is covered. 14 (tape 11 on the inner peripheral side or the first core 30) is separated from the well forming surface 11a of the tape 11. At this time, the tape 11 is slowly unwound at a speed of, for example, 1 cm / s.

Then, as shown in FIG. 13, in the cell recovery method, after the step (step 906) of separating the tape 11 and the covering portion 14 (tape 11 on the inner peripheral side or the first core 30), the cells 101 from the well 2 (Step 907) is provided. Specifically, as shown in FIG. 18, the tape 11 unwound from the first core 30 has a second well-forming surface 11a facing the opposite side of the second core 40 (see FIG. 19). It is wound up on the core 40. As a result, when the well-forming surface 11a of the tape 11 wound around the second core 40 faces downward (Z2 direction side) (see FIG. 19), the cells 101 in the well 2 settle downward due to their own weight. .. Then, the cells 101 that have settled downward are discharged to the outside of the housing portion 20 through the distribution portion 24. As a result, the cells 101 are collected.

Other configurations of the second embodiment are the same as those of the first embodiment.

(Effect of the second embodiment)
In the second embodiment, the following effects can be obtained.

Further, in the second embodiment, as described above, the step of realizing the state in which the physiological saline 209 (aqueous liquid) is stored in the housing portion 20 (container) is flexible and wound in a circumferential shape. A step of accommodating the tape 11 (cell culture unit), the first core 30, and the second core 40, which are configured to be rotatable, in the housing portion 20 is included. Then, the step of separating the covering portion 14 from the well forming surface 11a (surface) of the tape 11 is to unwind the tape 11 from the first core 30 so that the tape 11 is wound on the outer peripheral side in the first core 30. The cell recovery method is configured to include a step of separating the tape 11 wound on the inner peripheral side or the covering portion 14 composed of the first core 30 from the well forming surface 11a of the tape 11. As a result, the tape 11 wound around the first core 30 is unwound while being immersed in the physiological saline 209 in the housing portion 20, so that the cells 101 cultured in the well 2 are contained therein. It is possible to prevent the tape 11 or the first core 30 on the peripheral side from adhering to the tape 11. Further, since the tape 11 has flexibility, when it is unwound from the first core 30, it is gradually peeled off from the tape 11 on the inner peripheral side or the first core 30. As a result, when the cells 101 in the well 2 are unwound from the first core 30, they are prevented from being washed away by the flow of the physiological saline 209 in the housing 20 and discharged from the well 2. be able to.

Of the other effects of the second embodiment, the same effects as those obtained in the first embodiment will be omitted.

(Modification example)
It should be noted that the embodiments disclosed this time are exemplary in all respects and are not considered to be restrictive. The scope of the present invention is shown by the scope of claims, not the description of the above-described embodiment, and further includes all modifications (modifications) within the meaning and scope equivalent to the scope of claims.

For example, in the first embodiment, the introduction side flow path 9 and the discharge side flow path 10 are provided in the container 6 and the cell culture unit 1, but the present invention is not limited to this. The introduction side flow path and the discharge side flow path may be provided in different members.

For example, as shown in FIG. 20, in the step of culturing the cells 101 of the cell recovery method in the cell culture apparatus 300, the culture solution is cultivated in the culture solution flow path 3 by the introduction side flow path 49 (second introduction side flow path) described later. Including a step of introducing the culture solution into the culture solution and a step of discharging the culture solution from the culture solution flow path 3 by the discharge side flow path 50 described later.

Specifically, the cell culture apparatus 300 includes a cell culture unit 31 including a well-forming surface 31a and a container 16 for accommodating the cell culture unit 31. A culture solution flow path 3 connected to the well 2 and through which the culture solution flows is formed on the well forming surface 31a. The cell culture device 300 includes a seal member 34 and a lid member 35 arranged on the surface 34a of the seal member 34 opposite to the cell culture unit 31. The seal member 34 and the lid member 35 are provided with an introduction side flow path 49 which is connected to the culture solution flow path 3 and introduces the culture solution into the culture solution flow path 3. Further, the seal member 34 and the lid member 35 are provided with a discharge side flow path 50 which is connected to the culture solution flow path 3 and discharges the culture solution from the culture solution flow path 3. The container 16 is not provided with a flow path through which the culture solution flows. Further, the seal member 34 and the lid member 35 are examples of the "covered portion" and the "first lid member" in the claims, respectively. Further, the introduction side flow path 49 and the discharge side flow path 50 are examples of the “second introduction side flow path” and the “second discharge side flow path” in the claims, respectively. Further, the well forming surface 31a is an example of the "surface" in the claims.

Specifically, the introduction side flow path 49 (second introduction side flow path) is formed in the flow path 49a formed in the seal member 34 (covered portion) and the lid member 35 (first lid member). Includes flow path 49b. The flow path 49a of the seal member 34 is connected to the culture solution flow path 3. The flow path 49b of the lid member 35 communicates with the flow path 49a of the seal member 34. Further, the culture solution is introduced into the flow path 49b of the lid member 35 from the introduction side tube 103.

Further, the discharge side flow path 50 (second discharge side flow path) is a flow path 50a formed in the seal member 34 (covered portion) and a flow path formed in the lid member 35 (first lid member). Includes 50b and. The flow path 50a of the seal member 34 is connected to the culture solution flow path 3. The flow path 50b of the lid member 35 communicates with the flow path 50a of the seal member 34. Further, the culture solution flowing through the flow path 50b of the lid member 35 is discharged to the outside of the lid member 35 by the discharge side tube 104.

As a result, the well 2 of the cell culture section 31 is covered with the seal member 34 (coating section), and the introduction side flow path 49 (second introduction side flow path), the culture solution flow path 3, and the discharge side flow. The cells 101 in the well 2 can be easily cultured by the culture solution flowing through the passage 50 (second discharge side flow path). Further, each of the introduction side flow path 49 (second introduction side flow path) and the discharge side flow path 50 (second discharge side flow path) is provided on the seal member 34 and the lid member 35 (first lid member). As a result, when there is an obstacle or the like on the side opposite to the seal member 34 (lid member 35) with respect to the cell culture unit 31 and the container 16, the tube (103, 104) through which the culture solution is circulated is provided. The tubes (103, 104) can be connected to each of the introduction side flow path 49 and the discharge side flow path 50 while suppressing interference with the obstacles and the like.

Further, in the first embodiment, the seal member 4 is immersed in the physiological saline 102 (aqueous liquid) stored in the container 6 as a whole of the cell culture unit 1 and the seal member 4 (coating unit). Is peeled off from the cell culture unit 1, but the present invention is not limited to this. If the interface between the cell culture unit 1 and the seal member 4 is immersed in the physiological saline 102, only a part of the seal member 4 may be immersed in the physiological saline 102. In this case, only the cell culture unit 1 of the cell culture unit 1 and the seal member 4 is housed in the container 6.

Further, in the first embodiment, an example in which the seal member 4 (coating portion) is peeled off from the cell culture portion 1 is shown, but the present invention is not limited to this. The cell culture unit 1 may be peeled off from the seal member 4.

Further, in the first and second embodiments, the sealing member 4 (coating portion) and the cell culture portion are stored in the container 6 (housing portion 20) with the physiological saline 102 (209) (aqueous liquid). Although an example in which the step of separating from 1 is performed is shown, the present invention is not limited to this. The above step may be performed in a state where the culture solution is stored in the container 6 (housing portion 20).

Further, in the first embodiment, an example is shown in which the lid member 5 (first lid member) is pressed against the seal member 4 (covered portion) by the screw member 7 (fixing member) to fix the seal member 4. The present invention is not limited to this. For example, by placing a weight on the lid member 5, the lid member 5 may be pressed against the seal member 4 to fix the seal member 4.

Further, in the first embodiment, the cell 101 is cultured in a state where the container 6 is sealed by the lid member 8 (second lid member), but the present invention is not limited to this. The cell 101 may be cultured without using the lid member 8.

Further, in the first embodiment, an example is shown in which the seal member 4 (coating portion) is gradually peeled off from the cell culture portion 1 while being deformed (bent), but the present invention is not limited to this. Neither the cell culture unit 1 nor the seal member 4 has flexibility (flexibility), and one of the cell culture unit 1 and the seal member 4 is not deformed (without bending), and the cell culture is performed. It may be gradually peeled off from the other of the part 1 and the seal member 4. In this case, one of the cell culture unit 1 and the seal member 4 may be gradually moved by a deceleration mechanism or the like.

[Aspect]
It will be understood by those skilled in the art that the above exemplary embodiments are specific examples of the following embodiments.

(Item 1)
A cell culture section (1, 11, 31) having a surface (1a, 11a, 31a) provided with a concave well 2 for culturing cells 101, and the cell culture section (1, 11, 31). A step of preparing a covering portion (4, 14, 34) covering the surface (1a, 11a, 31a) and a container (6, 16, 20) containing the cell culture portion (1, 11, 31).
The cells 101 and the culture solution are present in the well 2, the well 2 is covered with the covering portion (4, 14, 34), and the cell culture portion (1, 11, 31) is the container (6). , 16, 20) and higher than the surface (1a, 11a, 31a) of the cell culture unit (1, 11, 31) in the state of being housed in the container (6, 16, 20). The step of realizing the state in which the aqueous liquid (102, 209) is stored in the container (6, 16, 20), and
In a state where the aqueous liquid (102, 209) is stored in the container (6, 16, 20) up to a position higher than the surface (1a, 11a, 31a) of the cell culture unit (1, 11, 31). , A cell recovery method comprising a step of separating the coating portion (4, 14, 34) from the surface (1a, 11a, 31a) of the cell culture portion (1, 11, 31).

(Item 2)
After separating the coating part (4, 14, 34) from the surface (1a, 11a, 31a) of the cell culture part (1, 11, 31), a step of acquiring the cell 101 from the well 2 is further added. Item 1 is a cell recovery method.

(Item 3)
The step of separating the coating portion (4, 14, 34) from the surface (1a, 11a, 31a) of the cell culture portion (1, 11, 31) is a step of separating the coating portion (4, 14, 34) having at least one of the flexible coating portions (1, 11, 31). Item 1 or 2, wherein the cell recovery method comprises a step of separating 4, 14, 34) and the cell culture section (1, 11, 31).

(Item 4)
In the step of realizing the state in which the aqueous liquid (102, 209) is stored in the container (6, 16, 20), the cell 101 and the culture solution are housed in the well 2 to store the cell 101. The cell recovery method according to any one of items 1 to 3, which comprises a step of culturing.

(Item 5)
The step of culturing the cell 101 is
A step of increasing the sealing property of the covering portion (4, 34) with respect to the surface (1a, 31a) of the cell culture portion (1, 31), and
Including the step of culturing the cells 101 in a state where the sealing property of the covering portion (4, 34) is increased.
A state in which the sealing property of the coating portion (4, 34) is increased before the step of separating the coating portion (4, 34) from the surface (1a, 31a) of the cell culture portion (1, 31). The cell recovery method according to any one of items 1 to 4, further comprising a step of releasing the cell.

(Item 6)
The step of realizing the state in which the aqueous liquid 102 is stored in the container (6, 16) is the cell culture section by the sealing member (4, 34) as the covering portion (4, 34) having flexibility. Including the step of sealing the surface (1a, 31a) of (1, 31).
The step of increasing the sealing property of the covering portion (4, 34) is carried out on the surface (4a, 34a) of the sealing member (4, 34) opposite to the cell culture portion (1, 31). The cell recovery method according to item 5, which comprises a step of arranging the first lid member (5, 35) which is configured to be openable and closable independently of the seal member (4, 34).

(Item 7)
The step of increasing the sealing property of the covering portion (4, 34) is the step of penetrating the first lid member (5, 35) and fastening the fixing member 7 to the container (6, 16). 1. The cell recovery method according to item 6, further comprising a step of pressing the lid member (5, 35) against the seal member (4, 34) to fix the seal member (4, 34).

(Item 8)
The step of culturing the cell 101 is
The cell culture unit is connected to the well 2 on the surface 1a of the cell culture unit 1 and is opposite to the seal member (4, 34) with respect to the culture solution flow path 3 through which the culture solution flows. 1 and the step of introducing the culture solution into the culture solution flow path 3 by the first introduction side flow path 9 provided in the container (6, 16) and connected to the culture solution flow path 3.
A second that is provided in the cell culture unit 1 and the container (6, 16) on the side opposite to the seal member (4, 34) with respect to the culture solution flow path 3 and is connected to the culture solution flow path 3. 1 The cell recovery method according to item 6 or 7, comprising a step of discharging the culture solution from the culture solution flow path 3 by the discharge side flow path 10.

(Item 9)
The step of culturing the cell 101 is
A second that is provided on the seal member 34 and the first lid member 35, is connected to the well 2 on the surface 31a of the cell culture unit 31, and is connected to the culture solution flow path 3 through which the culture solution flows. 2 The step of introducing the culture solution into the culture solution flow path 3 by the introduction side flow path 49, and
A step of discharging the culture solution from the culture solution flow path 3 by a second discharge side flow path 50 provided on the seal member 34 and the first lid member 35 and connected to the culture solution flow path 3. 6. The cell recovery method according to item 6 or 7.

(Item 10)
The step of culturing the cells 101 is provided in the container (6, 16) having a concave shape whose side opposite to the bottom surface portion 6a on which the cell culture unit (1, 31) is arranged is open, and the bottom surface thereof. A step of sealing the inside of the container (6, 16) by bringing the second lid member 8 into contact with the contact portion 6c, which is composed of the tip portion 6b on the opposite side of the portion 6a and is provided in a circumferential shape. Including
The second lid member 8 is removed from the container (6, 16) prior to the step of separating the coating (4, 34) from the surface (1a, 31a) of the cell culture unit (1, 31). The cell recovery method according to any one of items 5 to 9, further comprising a step of opening the container (6, 16).

(Item 11)
The step of realizing the state in which the aqueous liquid 209 is stored in the container 20 is the winding of the cell culture unit 11 which is flexible and is configured to be rotatable in a circumferential shape, and the cell culture unit 11. The first core 30 extending along the central axis α1 of the times and the first core 30 are provided separately, and extend along the central axis α2 of the winding of the cell culture unit 11 and from the first core 30. A step of accommodating the second core 40 for winding the unwound cell culture unit 11 in the container 20 is included.
The step of separating the covering portion 14 from the surface 11a of the cell culture portion 11 is wound around the outer periphery in the first core 30 by unwinding the cell culture portion 11 from the first core 30. The cell culture unit 11 wound around the inner circumference side of the cell culture unit 11 or the coating unit 14 composed of the first core 30 is separated from the surface 11a of the cell culture unit 11. The cell recovery method according to any one of items 1 to 4, which comprises a step.

(Item 12)
A cell culture section (1, 11, 31) having a surface (1a, 11a, 31a) provided with a concave well 2 for culturing the cell 101, and a cell culture section (1, 11, 31).
A covering portion (4, 14, 34) covering the surface (1a, 11a, 31a) of the cell culture portion (1, 11, 31) and a coating portion (4, 14, 34).
A container (6, 16, 20) for accommodating the cell culture unit (1, 11, 31) is provided.
A cell culture apparatus (100, 200, 300) in which at least one of the cell culture section (1, 11, 31) and the coating section (4, 14, 34) has flexibility.

1,31 Cell culture part 1a, 11a, 31a Well forming surface (surface)
2 wells 3 culture solution flow path 4, 34 Sealing member (covering part)
4a, 34a surfaces (seal member surfaces)
5, 35 Lid member (1st lid member)
6, 16 Container 6a Bottom part 6b Tip part 6c Abutment part 7 Screw member (fixing member)
8 Lid member (second lid member)
9 Introduction side flow path (1st introduction side flow path)
10 Discharge side flow path (first discharge side flow path)
11 Tape (cell culture section)
14 Cover 20 Housing (container)
30 1st core 40 2nd core 49 Introduction side flow path (2nd introduction side flow path)
50 Discharge side flow path (second discharge side flow path)
100, 200, 300 cell culture device 101 cells 102, 209 Saline (aqueous liquid)
α1 central axis (central axis of the first core)
α2 center axis (center axis of the second core)

Claims (12)

A step of preparing a cell culture part having a surface provided with a concave well for culturing cells, a coating part covering the surface of the cell culture part, and a container for accommodating the cell culture part.
The cells and the culture solution are present in the well, the well is covered with the coating portion, and the cell culture portion is housed in the container, and the cell culture part is housed in the container. A step to realize a state in which the aqueous liquid is stored in the container up to a position higher than the surface, and
A cell recovery method comprising a step of separating the coating portion from the surface of the cell culture portion in a state where the aqueous liquid is stored in the container to a position higher than the surface of the cell culture portion. The cell recovery method according to claim 1, further comprising a step of acquiring the cells from the well after separating the covering portion from the surface of the cell culture portion. The cell according to claim 1 or 2, wherein the step of separating the coating portion from the surface of the cell culture portion includes a step of separating the coating portion having at least one of them flexible and the cell culture portion. Collection method. Any one of claims 1 to 3, wherein the step of realizing the state in which the aqueous liquid is stored in the container includes a step of accommodating the cell and the culture solution in the well and culturing the cell. The cell recovery method according to the section. The step of culturing the cells is
A step of increasing the sealing property of the covering portion with respect to the surface of the cell culture portion, and
Including the step of culturing the cells in a state where the sealing property of the covering portion is increased.
The invention according to any one of claims 1 to 4, further comprising a step of releasing the state in which the sealing property of the coating portion is increased before the step of separating the coating portion from the surface of the cell culture portion. Cell recovery method. The step of realizing the state in which the aqueous liquid is stored in the container includes a step of sealing the surface of the cell culture part with a sealing member as the covering part having flexibility.
In the step of increasing the sealing property of the covering portion, a first lid member configured to be openable and closable independently of the sealing member is arranged on the surface of the sealing member opposite to the cell culture portion. The cell recovery method according to claim 5, which comprises the step of performing. The step of increasing the sealing property of the covering portion is to press the first lid member against the sealing member by a fixing member that penetrates the first lid member and is fastened to the container to fix the sealing member. The cell recovery method according to claim 6, which comprises a step. The step of culturing the cells is
The cell culture section and the container are provided on the surface of the cell culture section on the opposite side of the seal member with respect to the culture solution flow path in which the culture solution flows while being connected to the well, and the culture is provided. A step of introducing the culture solution into the culture solution flow path by the first introduction side flow path connected to the liquid flow path, and a step of introducing the culture solution into the culture solution flow path.
From the culture solution flow path, a first discharge side flow path provided in the cell culture section and the container on the side opposite to the seal member with respect to the culture solution flow path and connected to the culture solution flow path. The cell recovery method according to claim 6 or 7, wherein the step of draining the culture solution is included. The step of culturing the cells is
By a second introduction side flow path provided on the seal member and the first lid member, connected to the well on the surface of the cell culture unit and connected to the culture solution flow path through which the culture solution flows. , The step of introducing the culture solution into the culture solution flow path,
The claim includes a step of discharging the culture solution from the culture solution flow path by a second discharge side flow path provided on the seal member and the first lid member and connected to the culture solution flow path. The cell recovery method according to 6 or 7. The step of culturing the cells is provided in the container having a concave shape whose side opposite to the bottom surface portion on which the cell culture portion is arranged is open, and is composed of a tip portion on the side opposite to the bottom surface portion. Including a step of sealing the inside of the container by bringing the second lid member into contact with the contact portion provided in a circumferential shape.
Claims 5 to 9, further comprising a step of opening the container by removing the second lid member from the container before the step of separating the coating from the surface of the cell culture unit. The cell recovery method according to any one item. The steps to realize the state in which the aqueous liquid is stored in the container are the cell culture section, which is flexible and is configured to be rotatable in a circumferential shape, and the central axis of winding of the cell culture section. A first core extending along the first core and the first core are provided separately, and the cell culture part extending along the central axis of the winding of the cell culture part and winding the cell culture part unwound from the first core is wound. Including a step of accommodating the second core to be taken in the container.
The step of separating the coating portion from the surface of the cell culture portion is within the cell culture portion wound around the outer periphery in the first core by unwinding the cell culture portion from the first core. Any of claims 1 to 4, which comprises a step of separating the cell culture part wound around the peripheral side or the coating part composed of the first core from the surface of the cell culture part. The cell recovery method according to item 1. A cell culture section having a surface provided with concave wells for culturing cells,
A covering portion covering the surface of the cell culture portion and
A container for accommodating the cell culture unit is provided.
A cell culture apparatus in which at least one of the cell culture part and the coating part has flexibility.

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