Cell culture apparatus

The present invention relates to a cell culture device, and more particularly to a cell culture device for separating and proliferating a target cell from a cell-containing solution containing a plurality of different types of cells.

When proliferating cells by cell culture, it may be necessary to culture in a state containing multiple different types of cells, then separate these different types of cells and perform culture to proliferate only the target cells. is there.

For example, when mesenchymal stem cells (hereinafter abbreviated as MSC), which are adherent cells, are separated from the bone marrow fluid of animals such as mammals and obtained as a cell line, the extracted bone marrow cells are diluted with a cell culture medium. After culturing, the MSCs, which are the cells of interest, are separated and further cultured. Therefore, it is necessary to separate the MSC, which is the target cell, from other cells. In culturing cells such as embryonic stem cells (hereinafter abbreviated as ES cells), after co-culturing with feeder cells, the separated ES cells can be transplanted into fertilized or unfertilized eggs, or differentiation and growth factors. In some cases, the cells are differentiated by adding the above and then transplanted to another cell, or the ES cells are grown alone by adding a growth factor. Therefore, when performing such a culture, it is necessary to separate the ES cells and the feeder cells because the ES cells are cultured alone after the co-culture with the feeder cells.

In this way, when it is necessary to separate different types of cells and perform culture to proliferate only the target cells, for example, in MSC, the adherence is utilized to culture cells other than adherent cells. Alternatively, after performing a separation operation of rinsing with a washing solution or the like to remove the cells, an operation of planting the cells in a medium in order to proliferate the cells in the culture solution is performed. Then, the operation of separating these cells and the operation of planting the cells in the medium are manually performed by the operator using a pipette or the like. As another method, it is also possible to carry out culturing in which the target cells are separated and proliferated by a device such as a cell sorter based on the cell size, surface antigen, or the like. Even when the separation is performed using the cell sorter in this way, the separation operation can be performed by the cell sorter, but the operation of planting the separated cells in the medium is manually performed by the operator using a pipette or the like.

In addition, in ES cells that need to be co-cultured with feeder cells, there is a difference in adhesion between ES cells and feeder cells, and the adhesion of feeder cells is stronger. For this reason, when the ES cells and the feeder cells are separated and the feeder cells are removed, the ES cells are co-cultured with the feeder cells, and then only the ES cells that are not attached to the culture bed are taken out and cultured. , Such cell separation and culturing operations are also manually performed by the operator using a pipette or the like.

In this way, in the conventional cell culture for separating and proliferating a target cell from a cell-containing medium containing a plurality of different types of cells, an operation for separating the target cell from a plurality of different types of cells and the separated purpose The operator manually uses a pipette or the like to plant the cells in a medium in order to proliferate the cells. For this reason, the occurrence of contamination has become a problem. In addition, even if the target cells can be separated by a device such as a cell sorter, the operator manually performs the operation of planting the separated target cells in a medium in order to proliferate them by using a pipette or the like. In addition, when the device processes a large number of samples, cross-contamination may occur between the samples, so that the occurrence of contamination is also a problem.

On the other hand, in order to prevent contamination from the outside in the operation of subculture, the medium and cultured cells are supplied, collected, discharged, etc. without the need for the operator to manually operate with a pipette or the like. As a cell culture device capable of performing subculture, a device for performing two culture beds in which subculture is integrated has been proposed (see, for example, Patent Document 1).<patcit num="1"><text>Japanese Patent Application Laid-Open No. 2001-275659 (pages 4-9, Fig. 1)</text></patcit>

<p> However, the cell culture apparatus as in Patent Document 1 is for the purpose of preventing external contamination in the operation of subculture, and when it is necessary to separate the target cells from a plurality of different types of cells. , It is necessary for the operator to do it manually using a pipette or the like. Further, in a cell culture apparatus as in Patent Document 1, it is difficult to perform an operation for separating target cells from a plurality of different types of cells in the apparatus. Therefore, in a cell culture apparatus as in Patent Document 1, it is difficult to suppress the occurrence of contamination when the target cells are separated and proliferated from the cell-containing solution containing a plurality of types of cells. Therefore, there is a need for a cell culture device capable of suppressing the occurrence of contamination when separating and proliferating target cells from a cell-containing solution containing a plurality of different types of cells.</p><p> An object of the present invention is to suppress the occurrence of contamination in a cell culture for separating and proliferating a target cell from a cell-containing solution containing a plurality of different types of cells.</p>

<p> The cell culture apparatus of the present invention contains a cell-containing solution containing a plurality of different types of cells, and separates a target cell from the cells contained in the cell-containing solution into a cell separation chamber and a purpose of separation in the cell separation chamber. A cell proliferation chamber for culturing cells to proliferate the cells, a movement channel for moving the target cells separated in the cell separation chamber to the cell proliferation chamber, and a liquid connected to the cell separation chamber to flow liquid into the cell separation chamber. The first injection channel for injection, the first out-flow channel for deriving the liquid in the cell separation chamber, the second injection channel connected to the cell growth chamber and injecting the liquid into the cell growth chamber, and the cell growth chamber. The cell separation chamber is equipped with a second derivation channel for deriving the liquid of the cell, and among the cells contained in the contained cell-containing liquid, the cells having or having stronger adhesion than other cells are selected. The above-mentioned problems are solved by having a structure in which a moving flow path has a bonding portion to be attached and the moving flow path has a moving flow path opening / closing means for opening / closing the moving flow path.</p><p> With such a configuration, cells contained in the cell-containing solution that have adhesiveness or are stronger than other cells are attached to the attachment part in the cell separation chamber. It is possible to separate the cells attached to the attached portion from the cells not attached to the attached portion. Furthermore, the first injection flow path and the first lead-out flow path connected to the cell separation chamber, the movement flow path connecting the cell separation chamber and the cell growth chamber, the second injection flow path and the second flow passage connected to the cell growth chamber. Through the derivation flow path, injection of cell-containing fluid or buffer solution containing multiple different types of cells into the cell separation chamber, derivation of unnecessary cells or fluid from the cell separation chamber, cells of cells separated in the cell separation chamber It is possible to move to the growth chamber, inject a culture solution or buffer into the cell growth chamber, collect cells from the cell growth chamber, and the like. For this reason, a consistent space isolated from the outside without the need for the operator to manually perform operations such as separation of the target cells and implantation of the separated target cells in the medium using a pipette or the like. Can be done in. Therefore, it is possible to suppress the occurrence of contamination in a cell culture for separating and proliferating a target cell from a cell-containing solution containing a plurality of different types of cells.</p><p> Further, the first injection flow path has a first injection flow path opening / closing means for opening / closing the first injection flow path, and the first lead-out flow path has a first lead-out for opening / closing the first lead-out flow path. Each of the flow path opening / closing means is provided, the second injection flow path is provided with a second injection flow path opening / closing means for opening / closing the second injection flow path, and the second lead-out flow path is provided with the second lead-out flow path. The configuration is such that each of the second lead-out flow path opening / closing means for opening / closing the road is provided.</p><p> With such a configuration, all the flow paths for flowing in and out of the cell separation chamber and the cell proliferation chamber can be blocked by each flow path opening / closing means, and only the necessary flow paths can be opened to allow the liquid to flow in and out. It becomes easier to maintain a sterilized state in a space such as a cell separation chamber or a cell proliferation chamber, and the occurrence of contamination can be suppressed more reliably.</p><p> By the way, when adherent cells are cultured, a proteolytic enzyme such as trypsin is added in order to detach the cells from the inner wall surface of the culture vessel after the culture. However, animal cell-derived proteolytic enzymes such as trypsin may reduce the cell's ability to proliferate and differentiate. Therefore, when the cells attached to the attachment part of the cell separation chamber are the target cells, it is necessary to separate the target cells from the attachment part. For this separation of the target cells, a protein derived from an animal cell such as trypsin When degrading enzymes are used, the ability of the cells to be reduced may make it impossible to obtain cells of the quality required for subsequent culture in the cell proliferation chamber.</p><p> On the other hand, the adhesion part in the cell separation chamber is formed of a cell adhesion material whose adhesion to cells changes according to temperature changes and cell adhesion and detachment can be controlled by changes in the ambient temperature of the cells. The configuration is as follows. Further, the attachment portion in the cell separation chamber is a component peculiar to plant cells and is formed of a cell adhesion material containing an enzymatically decomposable component. With these configurations, it is not necessary to use animal cell-derived proteolytic enzymes such as trypsin for cell detachment from the attachment part in the cell separation chamber, and the purpose is to use a cell-containing solution containing multiple different types of cells. In cell culture for separating and proliferating cells, it is possible to suppress the occurrence of contamination while suppressing the decrease in the ability of the cells.</p><p> Further, when the cells not attached to the attachment part of the cell separation chamber are the target cells, the attachment part in the cell separation chamber promotes the attachment of the cells to at least a part of the inner wall surface of the cell separation chamber. If the configuration is formed by subjecting the surface treatment of the above cells, the adhesion of cells other than the target cells to the attachment portion is promoted, and the separation ability of the target cells in the cell separation chamber can be improved.</p>

<p> According to the present invention, it is possible to suppress the occurrence of contamination in a cell culture for separating and proliferating a target cell from a cell-containing solution containing a plurality of different types of cells.</p>

Hereinafter, an embodiment of a cell culture apparatus to which the present invention is applied will be described with reference to FIGS. 1 to 10. In the following description, the expression ES cell includes not only embryonic stem cells but also cells established from cloned embryos and fertilized egg clones.

FIG. 1 is a diagram schematically showing a schematic configuration of a cell culture apparatus to which the present invention is applied, in which (a) is the overall configuration, and (b) is a cell separation chamber, a moving tube, and a cell proliferation chamber. It is a perspective view which shows the state which took out and disassembled the bag and tube which make up. FIG. 2 is a diagram schematically explaining the principle of cell separation in the cell separation chamber of the cell culture device to which the present invention is applied. FIG. 3 is a diagram schematically showing an operation when a target cell is an adherent cell in a cell culture apparatus to which the present invention is applied. FIG. 4 is a flow chart showing an operation when the target cell is an adherent cell in the cell culture apparatus to which the present invention is applied. FIG. 5 is a diagram schematically showing an operation when the target cell is a cell that is not attached to the attachment portion in the cell culture apparatus to which the present invention is applied. FIG. 6 is a flow chart showing the operation of cells in which the target cells are not attached to the attachment portion in the cell culture apparatus to which the present invention is applied. 7 to 9 are diagrams schematically showing another schematic configuration of a cell culture apparatus to which the present invention is applied. FIG. 10 is a perspective view schematically showing a schematic configuration when a culture operation in a cell culture apparatus to which the present invention is applied is automated.

As shown in FIG. 1A, the cell culture apparatus of the present embodiment was separated in a cell separation chamber 1 and a cell separation chamber 1 for separating target cells from a cell-containing solution containing a plurality of different types of cells. It is equipped with a cell proliferation chamber 3 for culturing the cells of interest. In addition, the cell separation chamber 1 and the cell proliferation chamber 3 are connected by a movement conduit 5 for moving the target cells separated in the cell separation chamber 1 from the cell separation chamber 1 to the cell proliferation chamber 3, and move. The cell separation chamber 1 and the cell proliferation chamber 3 are connected to each other by the conduit 5. The moving pipeline 5 is provided with a cock 5a as a moving flow path opening / closing means for opening / closing the flow path in the moving pipeline 5.

The cell separation chamber 1 is a first injection conduit 7 formed of a flexible resin tube for injecting a liquid into the cell separation chamber 1, and a flexible resin for deriving the liquid in the cell separation chamber 1. The first lead-out line 9 formed by the tube of the above is connected. The first injection pipe line 7 has a cock 7a as a means for opening and closing the first injection flow path for opening and closing the flow path in the first injection line line 7, and the first lead-out line line 9 has a first lead-out line line 9. A cock 9a is provided as a means for opening and closing the first lead-out flow path. Similarly, the cell proliferation chamber 3 has a second injection conduit 11 formed of a flexible resin tube for injecting a liquid into the cell proliferation chamber 3, and a flexibility for deriving the liquid in the cell proliferation chamber 3. A second outlet line 13 formed of a resin tube is connected. The second injection pipe 11 has a cock 11a as a means for opening and closing the second injection flow path for opening and closing the flow path in the second injection pipe 11, and the second outlet pipe 13 has a second outlet pipe 13. A cock 13a is provided as a second lead-out flow path opening / closing means for opening / closing.

The cocks 5a, 7a, 9a, 11a, and 13a used as the opening / closing means for each flow path may be any one that can control the flow rate, flow, and cutoff of the liquid in each flow path, and a pinch for sandwiching the tube from the outside. A cock, a roller type cock, a stopcock, etc. can be used as appropriate. Further, as each flow path opening / closing means, in addition to a cock operated manually, an electromagnetic valve or the like that can automatically open / close the flow path by combining with a control means for controlling the operation can also be used.

In the cell culture apparatus as shown in FIG. 1, as shown in FIGS. 1 (a) and 1 (b), the cell separation chamber 1 is a transparent flexible resin sealed in a transparent hard resin box 1a. It is formed by putting a plastic bag 1b. Similarly, the cell proliferation chamber 3 is formed by placing a sealed transparent flexible resin bag 3b in a transparent hard resin box 3a. The moving pipeline 5 is also formed by putting a sealed transparent flexible resin tube 5c in a transparent hard resin tube 5b. One side of the box 1a of the cell separation chamber 1 and the box 3a of the cell proliferation chamber 3 can be attached and detached as appropriate, and the bag 1b and the bag 3b can be taken in and out. Since both ends of the cylinder 5b of the moving pipe line 5 are open, the tube 5c can be taken in and out, and both ends correspond to the box 1a of the cell separation chamber 1 and the box 3a of the cell proliferation chamber 3. It is removable at the position where it is used. The cock 5a is omitted in the tube 5c forming the moving line 5 in FIG. 1 (b).

The bag 1b forming the cell separation chamber 1 is provided with connecting portions 1c, 1d, and 1e for connecting the tube 5c forming the moving pipe 5, the first injection pipe 7, and the first outlet pipe 9. ing. Similarly, the bag 3b forming the cell proliferation chamber 3 is also connected to the connecting portions 3c, 3d, 3e for connecting the tube 5c forming the moving line 5, the second injection line 11 and the second out-line line 13. Is provided. In addition, connecting portions 5d and 5e are also provided at both ends of the tube 5c forming the moving pipeline 5. Each connecting part 1c, 1d, 1e, 3c, 3d, 3e, 5d, 5e is a bag 1b forming a cell separation chamber 1, a bag 3b forming a cell proliferation chamber 3, and a tube forming a moving conduit 5. When 5c is disassembled into the state shown in Fig. 1 (b), each sealed state can be maintained. For example, for connection such as a connector or an injection needle whose flow path opens only when connected. It is made of a resin stopper or the like that can pierce a hollow needle or the like.

Therefore, the first injection line 7 and the first lead-out line 9 connected to the bag 1b forming the cell separation chamber 1, and the second injection line connected to the bag 3b forming the cell proliferation chamber 3. Each end of 11 and the second lead-out pipe 13 also has a structure corresponding to each connecting portion 1c, 1d, 1e, 3c, 3d, 3e.

Therefore, in the cell culture apparatus as shown in FIG. 1, for example, the bag 1b forming the cell separation chamber 1, the bag 3b forming the cell proliferation chamber 3, and the tube 5c forming the moving conduit 5 are decomposed. After sterilization by autoclave sterilization or γ-ray sterilization, the bag 1b forming the cell separation chamber 1 is placed in the box 1a, the bag 3b forming the cell proliferation chamber 3 is placed in the box 3a, and the moving pipeline 5 is placed. Put the tube 5c to be formed into the cylinder 5b. Then, depending on the structure of the connecting portions 1c, 3c, 5d, and 5e, they are connected using a connector or a hollow needle.

Further, the first injection line 7 and the first lead-out line 9 are placed in the bag 1b forming the cell separation chamber 1, and the second injection line 11 and the second lead-out line 9 are placed in the bag 3b forming the cell proliferation chamber 3. The road 13 is connected by using a connector or a hollow needle according to the structure of the connecting portions 1d, 1e, 3d, and 3e. At this time, the first injection line 7 and the first lead-out line 9, and the second injection line 11 and the second lead-out line 13 form a bag 1b or a cell proliferation room 3 forming the cell separation chamber 1. The ends on the opposite side to the side connected to the bag 3b are appropriately connected to a sterilized cell-containing liquid supply tank, a culture solution tank, a buffer solution tank, a waste liquid tank, etc., which are not shown in FIG. There is.

Here, the cell separation chamber 1 is provided with an attachment portion 15 for attaching cells contained in the cell-containing liquid contained in the cell separation chamber 1. The attachment portion 15 is provided on the bottom surface in the bag 1b forming the cell separation chamber 1, and is a cell adhesion material having cell adhesion and capable of cell detachment according to the adhesion of the cells to be separated. It is formed by providing it on the bottom surface inside the bag 1b, or by performing surface treatment such as coating treatment or plasma treatment of a substance that promotes adhesion of adherent cells. When the adhesion portion 15 is formed by providing a cell-adhesive and peelable material on the bottom surface of the bag 1b, the cell-adhesive material is placed in the bag 1b forming the cell separation chamber 1 each time the culture is performed. The device can be reused by installing.

The basic principle of cell separation in the cell separation chamber 1 of the cell culture device having such a configuration will be described. As described above, as shown in FIG. 2, an attachment portion 15 is provided on the inner surface of the bag 1b forming the cell separation chamber 1. As shown in FIG. 2 (a), a cell-containing solution containing a plurality of types of cells having different adhesiveness is placed in the cell separation chamber 1, and when a predetermined time elapses, as shown in FIG. 2 (b), Adhesive cells 17 indicated by black circles in FIG. 2 adhere to the attachment portion 15, and are weaker or less adherent than other cells indicated by white circles in FIG. 2, that is, adherent cells. The cells 19 are not attached to the attachment part 15 and remain suspended in the liquid. In this state, when a cell-free culture solution or buffer solution is passed through the cell separation chamber 1, the cells 19 suspended in the solution are taken out from the cell separation chamber 1 together with the solution, as shown in FIG. 2 (c). As shown, only the adherent cells 17 attached to the attachment portion 15 are left in the cell separation chamber 1.

In this way, by providing the attachment portion 15 in the cell separation chamber 1, it is possible to separate cells by utilizing the difference in adhesion of the cells. Then, when the cells to be proliferated in the cell proliferation chamber 3, that is, the target cells are the adherent cells 17 attached to the attachment portion 15, the cells 19 suspended in the liquid indicated by the white circles in FIG. 2 are transferred from the cell separation chamber 1. After taking out and removing, as shown in FIG. 2 (d), the cells are detached according to the characteristics of the material forming the attachment portion 15, and the cell-free culture solution is injected into the cell separation chamber 1. Then, the detached cells 17 are moved to the cell proliferation chamber 3 together with the culture solution.

On the other hand, when the cells to be proliferated in the cell proliferation chamber 3, that is, the target cells are floating cells 19 that are not attached to the landing portion 15, the adherent cells 17 are not detached and the cells are contained in the cell separation chamber 1. By injecting a culture solution that does not contain the above, the cells 19 suspended in the solution are moved to the cell proliferation chamber 3 together with the culture solution.

In the cell proliferation chamber 3, when it is necessary to promote the adhesion of the target cells to the inner surface of the bag 3b forming the cell proliferation chamber 3, the cell proliferation chamber is the same as in the case of a generally used culture vessel or the like. Appropriately use a container that has undergone surface treatment such as coating treatment or plasma treatment of a substance that promotes cell adhesion to the inner surface of the bag 3b forming 3. Further, if necessary, the cell adhesion material or the like used for forming the attachment portion 15 of the cell separation chamber 1 can be provided in the cell proliferation chamber 3.

Here, regarding the operation of the cell culture apparatus of the present embodiment when the target cell has adhesiveness or the adhesiveness is stronger than other cells, the adhesion is mainly from the bone marrow fluid of an animal such as a mammal. The case where the cell is a mesenchymal stem cell, that is, a cell targeted by MSC will be described as an example. In this case, as shown in FIG. 3, the attachment portion 15 is formed in the cell separation chamber 1 by covering the bottom surface with a temperature-sensitive cell culture sheet.

The temperature-sensitive cell culture sheet forming the adhesion portion 15 exhibits hydrophobicity at a temperature inside the cell separation chamber 1, that is, when the environmental temperature around the temperature-sensitive cell culture sheet is relatively high, for example, about 37 ° C. It is a known high molecular weight cell adhesion material in which MSC is attached to the cell culture and becomes hydrophilic at a relatively low temperature, for example, about 30 ° C., so that MSC can be peeled off. Such cell-adhesive materials include temperature-responsive polymer polymers such as N-isopropylacrylamide, which is known by the abbreviation of N-PAM.

By forming the attachment part 15 in the cell separation chamber 1 with such a temperature-sensitive cell culture sheet, trypsin and the like which may reduce the ability of the cell such as the proliferative ability and the differentiation ability of the cell, etc. The cells attached to the attachment portion 15 can be detached without using the proteolytic enzyme of. That is, by forming the attachment portion 15 with the temperature-sensitive cell culture sheet, it is possible to suppress a decrease in the ability of the cells such as proliferation ability and differentiation ability, whereby the cells can be cultured in the cell growth chamber thereafter. The MSC can be exfoliated while suppressing the deterioration of the quality of the cells.

In such a cell separation chamber 1, as shown in FIGS. 3 (a)-(b) and 4, a plurality of different species of bone marrow fluid collected from an animal, either directly or in a state of being diluted with an appropriate culture medium. As a cell-containing liquid containing the cells of the above, the cock 7a is opened to inject the cells from the first injection conduit 7 (step 101). At this time, the cock 9a of the first lead-out pipe 9 and the cock 5a of the moving pipe 5 in FIG. 1 are closed, and the cell-containing liquid is injected only into the cell separation chamber 1.

In FIG. 3, the moving pipe 5, the first injection pipe 7, the first outlet pipe 9, the second injection pipe 11, and the second outlet pipe 13 are cocks 5a provided in the respective pipes. , 7a, 9a, 11a, 13a are shown in black when closed, and cocks 5a, 7a, 9a, 11a, 13a are shown in white when closed. Further, in FIG. 3, the target cell MSC21 is indicated by a black circle, and the other cells 23 are indicated by a white circle.

After that, as shown in FIG. 3 (b), the cock 7a of the first injection line 7 was closed, and the cells were placed in the cell separation chamber 1 under the same conditions as the culture conditions for growing MSC21, that is, at a temperature of about 37 ° C. Incubate while maintaining the temperature, that is, the ambient temperature around the cells. In the case of general adherent cells, they adhere to the attachment portion 15 in the cell separation chamber 1 after several hours to about 24 hours, but cells other than the adherent cells do not adhere. Therefore, as shown in FIG. 4, it is determined whether or not MSC21 has adhered to the attachment portion 15 in the cell separation chamber 1 by photographing the passage of time or the culture state and performing image analysis (step 102).

When it is determined in step 102 that MSC21 has adhered to the attachment portion 15 in the cell separation chamber 1, cock 7a and the first derivation of the first injection line 7 as shown in FIGS. 3 (c) and 4 show. The cock 9a of the line 9 is opened, and the inside of the cell separation chamber 1 in which the culture solution or the buffer solution is injected from the first injection line 7 and discharged from the first out-line line 9 to be perfused is washed (step 103). As a result, the floating cells that did not adhere to the attachment part 15 in the cell separation chamber 1 can be discharged from the cell separation chamber 1, and the target cells attached to the attachment part 15 in the cell separation chamber 1 can be used. Some MSC21 and other cells 23 can be separated.

After cleaning the inside of the cell separation chamber 1 in step 103, the cock 7a of the first injection line 7 and the cock 9a of the first lead-out line 9 are closed. In this state, the ambient temperature around the cells is lowered to a temperature lower than the temperature under the culture conditions when MSC21 is grown, for example, about 30 ° C (step 104). At this time, for example, when the cell separation chamber 1 is placed in the incubator together with the cell proliferation chamber 3, the environment temperature around the cells can be lowered by adjusting the temperature of the incubator to lower the environment temperature around the cells. In addition, the ambient temperature around the cells can be lowered by injecting a culture solution or a buffer solution at about 30 ° C from the first injection conduit 7 into the cell separation chamber 1. As a result, the phase transition of the temperature-sensitive cell culture sheet forming the attachment portion 15 makes the temperature-sensitive cell culture sheet hydrophilic, and the MSC21 is detached from the attachment portion 15.

After lowering the ambient temperature around the cells in step 104 and confirming that MSC21 has detached from the attachment 15 (step 105), as shown in FIGS. 3 (d), (e) and 4. With the cocks 11a and 13a of the second injection line 11 and the second outlet line 13 closed, the cock 5a of the moving line 5 and the cock 7a of the first injection line 7 are opened, and the first injection line 7 is opened. By injecting the culture solution into the cell separation chamber 1 from the cell, the culture solution is perfused from the cell separation chamber 1 toward the cell proliferation chamber 3 to move the target cell MSC21 to the cell proliferation chamber 3. Then, the cock 5a of the moving tube 5 is closed, and the temperature in the cell proliferation chamber 3 is set to a cell culture condition, for example, about 37 ° C., so that MSC21 is proliferated (step 106). At this time, if necessary, the cock 11a of the second injection line 11 is temporarily opened, the culture solution is added from the second injection line 11 into the cell growth chamber 3, and the cells are cultured under appropriate conditions.

Further, during the culture in which the MSC21 is propagated in step 106, after the MSC21 adheres to the culture bed of the cell proliferation chamber 3, as necessary, as shown in FIG. 3 (e), the second injection conduit 11 and After opening the cocks 11a and 13a of the second outlet pipe 13 and discharging the culture medium from the second outlet pipe 13, the cock 13a of the second outlet pipe 13 is closed and the culture solution is discharged from the second injection pipe 11. The medium is exchanged by injecting and closing the cock 11a.

After the MSC21 has grown in step 106, the grown MSC21 is recovered from the cell growth chamber 3 as shown in FIG. 4 (step 107). In step 107, the cocks 11a and 13a of the second injection pipe 11 and the second outlet pipe 13 are opened, and the buffer solution is put in from the second injection pipe 11 in the same manner as in the operation shown in FIG. 3 (e). 2 Wash the MSC21 in the cell proliferation chamber 3 by draining it from the outlet line 13. After washing, the buffer solution is removed from the cell proliferation chamber 3, and as shown in FIG. 3 (f), the cock 13a of the second lead-out line 13 is closed, and the second infusion line 11 is used to decompose proteins such as trypsin. Inject a cell release agent containing the enzyme.

Then, as shown in FIG. 3 (g), the cock 11a of the second injection conduit 11 is closed and a cell exfoliation reaction is performed. After the cell exfoliation reaction, the cock 11a of the second injection pipe 11 was opened and the neutralizing agent was added, and then the cock 11a of the second injection pipe 11 was closed again in the same manner as in the operation shown in FIG. 3 (f). , The cell exfoliation reaction is stopped by stirring. After the cell detachment reaction is stopped, as shown in FIG. 3 (h), the cock of the cock 13a of the second lead-out pipe 13 is opened and the MSC21 is collected.

By the way, in the above description, the case where the attachment portion 15 is formed by the temperature-sensitive cell culture sheet provided in the cell separation chamber 1 is shown. However, as the attachment part 15 that can detach cells without using a cell release agent containing a proteolytic enzyme such as trypsin, in addition to the temperature-sensitive cell culture sheet, a substance that animal cells do not have, that is, derived from plant cells. It is also possible to form the attachment portion 15 using the substance of.

For example, the adhesion portion 15 can be formed by cellulose, which is a substance derived from plant cells, and a protein or peptide having cell adhesion. When the attachment portion 15 formed of cellulose and a protein or peptide having cell adhesion is provided in the cell separation chamber 1 in this way, the inside of the cell separation chamber 1 in step 104 shown in FIG. 4 described above is set as a cell detachment condition. Instead of the changing operation, the cock 7a of the first injection line 7 is temporarily opened, and in order to decompose cellulose from the first injection line 7, a cell detaching agent containing cellulase is injected to form the adhesion part 15. Perform an operation to decompose the cellulose. By this operation, the attachment portion 15 is decomposed and the target cells can be detached.

Since cellulase does not easily affect animal cells, it is possible to detach the target cells from the attachment portion 15 of the cell separation chamber 1 while suppressing the deterioration of the quality of the cells obtained by culturing in the cell proliferation chamber. .. Further, as the plant cell-derived substance forming the attachment portion 15, various substances peculiar to the plant cell such as alginic acid can be used in addition to cellulose.

On the other hand, regarding the operation of the cell culture apparatus of the present embodiment when the target cell does not have adhesiveness or the adhesiveness is weaker than other cells, the embryonic stem cell after co-culturing with the feeder cell A case where feeder cells are separated from such cells, that is, ES cells, and only ES cells are proliferated will be described as an example. In this case, the attachment portion 15 on the inner bottom surface of the bag 1b forming the cell separation chamber 1 promotes the attachment of cells to the inner bottom surface of the bag 1b, as in the case of a generally used culture container or the like. It is formed by performing surface treatment such as coating treatment of a substance that promotes cell adhesion and plasma treatment. Further, in order to promote the adhesion of cells to the inner surface of the bag 3b forming the cell proliferation chamber 3, the adhesion of cells to the inner surface of the bag 3b is promoted as in the case of a generally used culture vessel or the like. Those that have undergone surface treatment such as material coating treatment or plasma treatment are appropriately used.

In such a cell separation chamber 1, as shown in FIGS. 5 (a)-(b) and 6, a culture medium containing ES cells 25 and feeder cells 27 after co-culture is directly or appropriately cultured. As a cell-containing liquid containing a plurality of different types of cells in a diluted state with the liquid, the cock 7a is opened to inject the cells from the first injection line 7 (step 201). At this time, the cock 9a of the first lead-out pipe 9 and the cock 5a of the moving pipe 5 in FIG. 1 are closed, and the cell-containing liquid is injected only into the cell separation chamber 1.

In addition, in FIG. 5, the moving pipe line 5, the first injection pipe line 7, the first lead-out line line 9, the second injection line line 11 and the second lead-out line line 13 are each as in the case of FIG. When the cocks 5a, 7a, 9a, 11a, 13a provided in the pipeline are closed, they are shown in black, and when the cocks 5a, 7a, 9a, 11a, 13a are closed, they are shown in white. There is. Further, in FIG. 5, ES cells 25, which are the target cells, are indicated by white circles, feeder cells 27 are indicated by black circles, and the attachment portion 15 in the cell separation chamber 1 is omitted.

Then, as shown in FIG. 5 (b), the cock 7a of the first injection conduit 7 is closed, and the cells are cultured under the same conditions as those for growing the ES cells 25. In the case of general feeder cells 27, they adhere to the attachment part 15 in the cell separation chamber 1 after several hours, but ES cells, which are weaker in adhesion than the feeder cells, do not attach. Therefore, as shown in FIG. 4, it is determined whether or not only the feeder cells 27 have adhered to the attachment portion 15 in the cell separation chamber 1 by photographing the passage of time or the culture state and analyzing the image (step). 202).

When it is determined in step 202 that the feeder cells 27 have adhered to the attachment portion 15 in the cell separation chamber 1, as shown in FIGS. 5C and 6, the second injection line 11 and the second lead-out tube are used. With the cocks 11a and 13a of the passage 13 closed, the cock 5a of the moving conduit 5 and the cock 7a of the first injection conduit 7 are opened, and the culture solution is injected into the cell separation chamber 1 from the first injection conduit 7. By perfusing the culture solution from the cell separation chamber 1 toward the cell proliferation chamber 3, ES cells 25, which are the target cells, are moved to the cell proliferation chamber 3. After the movement of the ES cells 25, the cock 5a of the movement tube 5 is closed, the temperature in the cell growth chamber 3 is set to the culture temperature for growing the ES cells, and the culture for growing the ES cells 25 is performed (step). 203). At this time, if necessary, the cock 11a of the second injection line 11 is temporarily opened, and the culture solution is added into the cell growth chamber 3 from the second injection line 11 to obtain appropriate conditions.

In this way, by injecting the culture solution into the cell separation chamber 1 from the first injection conduit 7 in step 203, the culture solution is perfused from the cell separation chamber 1 toward the cell proliferation chamber 3 to cause the cell proliferation chamber. By moving the ES cell 25, which is the target cell, to 3, the ES cell 25, which is the target cell that was floating without adhering to the attachment part 15 in the cell separation chamber 1, and the ES cell 25, which is the target cell, and the cell separation chamber 1 The feeder cells 27 attached to the attachment portion 15 can be separated.

Further, during the culture in which the ES cells 25 are proliferated in step 203, after the ES cells 25 adhere to the culture bed of the cell proliferation chamber 3, the second injection is performed as required as shown in FIG. 5 (d). After opening the cocks 11a and 13a of the pipeline 11 and the second outlet pipeline 13 and discharging the culture medium from the second outlet pipeline 13, the cock 13a of the second outlet pipeline 13 is closed and the second injection pipeline 11 The culture medium is exchanged by injecting the culture solution from the tube and closing the cock 11a.

After the ES cells 25 proliferate in step 203, the ES cells 25 proliferated from the cell proliferation chamber 3 are recovered as shown in FIG. 6 (step 204). In step 204, as shown in FIG. 5 (e), the cocks 11a and 13a of the second injection pipe 11 and the second outlet pipe 13 are opened, the buffer solution is put in from the second injection pipe 11 and the second lead out. The ES cells 25 in the cell proliferation chamber 3 are washed by draining from the conduit 13. After washing, the buffer solution is removed from the cell proliferation chamber 3, and as shown in FIG. 5 (f), the cock 13a of the second lead-out line 13 is closed, and the second infusion line 11 is used to decompose proteins such as trypsin. Inject a cell release agent containing the enzyme.

Then, as shown in FIG. 5 (g), the cock 11a of the second injection conduit 11 is closed and a cell exfoliation reaction is performed. After the cell exfoliation reaction, the cock 11a of the second injection pipe 11 was opened and the neutralizing agent was added, and then the cock 11a of the second injection pipe 11 was closed again in the same manner as in the operation shown in FIG. 5 (f). , The cell exfoliation reaction is stopped by stirring. After the cell detachment reaction is stopped, as shown in FIG. 5 (h), the cock of the cock 13a of the second lead-out conduit 13 is opened and the ES cells 25 are collected.

In this example, the case where the ES cells from which the feeder cells have been separated is proliferated and collected is illustrated, but in the culture in the cell proliferation chamber 3, some differentiation and growth factors are added to differentiate the ES cells into other cells. In some cases, cells are collected from the cell growth chamber 3. In this case, the operations up to step 202 are the same, but in step 203, after culturing by adding appropriate differentiation and growth factors to the culture solution, depending on the type and properties of the differentiated cells, the cell proliferation chamber is placed from the incubator or the like. 3 or the entire cell culture apparatus is taken out and the cells are manually collected from the cell growth chamber 3, or the cells are collected by the same operation as in step 204.

Next, a modified example of the configuration of the cell culture device of the present embodiment will be described. The cell culture device having the configuration shown in Fig. 1 is premised on repeated use, but in order to eliminate washing and sterilization work for reducing cross-contamination in repeated use, cell separation chamber 1, cells The growth chamber 3, the mobile line 5, the first injection line 7, the first out-line line 9, the second infusion line 11, the second out-line line 13, and the like connected to each of them shall be disposable. May be desirable. In this case, for example, the cell separation chamber 1 and the cell proliferation chamber 3 are each formed of a transparent resin container similar to a flat square blood bag, as shown in FIG. Therefore, the cell separation chamber 1 and the cell proliferation chamber 3 are formed by welding two resin sheets in a frame shape. At this time, at least one part of the inner surface of the container that becomes the cell separation chamber 1 is provided with an appropriate attachment part 15.

Connecting portions 1c, 1d, and 1e are provided side by side on one side surface of the cell separation chamber 1. Similarly, connecting portions 3c, 3d, and 3e are provided side by side on one side surface of the cell proliferation chamber 3. Then, resin tubes such as silicon tubes are placed in the connecting portions 1c, 1d, 1e of the cell separation chamber 1 and the connecting portions 3c, 3d, 3e of the cell proliferation chamber 3, respectively, and the moving pipe line 5, the first injection pipe, respectively. It is connected as a road 7, a first lead-out line 9, a second injection line 11 or a second lead-out line 13. In these moving pipes 5, the first injection pipe 7, the first outlet pipe 9, the second injection pipe 11, and the second outlet pipe 13, cocks 5a, 7a, 9a made of a resin stopcock or the like are used. , 11a and 13a are provided respectively. In addition, the ends of the first injection line 7, the first out-line line 9, the second infusion line 11 and the second out-line line 13 on the side opposite to the side connected to the cell separation chamber 1 or the cell proliferation chamber 3. The portions are made of resin and have tubular connecting portions 7b, 9b, 11b, and 13b, which can be connected by inserting the tip of the injection tube, for example.

The cell separation chamber 1 and the cell proliferation chamber 3 as shown in FIG. 7 are dedicated to the cell separation chamber 1 and the cell proliferation chamber 3 of the cell culture apparatus having the configuration of the present embodiment by using the same material and manufacturing method as the blood bag. The cell separation chamber 1 and the cell proliferation chamber 3 can also be formed by using the blood bag itself, as shown in FIG. In this case, for example, two blood bags are used as the cell separation chamber 1 and the cell proliferation chamber 3, respectively. At this time, at least one portion of the inner surface of the blood bag forming the cell separation chamber 1 on the bottom surface side is provided with an appropriate attachment portion 15.

Further, in the configuration shown in FIG. 8, when the cell separation chamber 1 and the cell proliferation chamber 3 are formed by the blood bag, the cells are used to support the blood bag in a laid state during culturing and to facilitate handling. A frame 29 capable of supporting a blood bag serving as a separation chamber 1 and a cell proliferation chamber 3 is used. The frame 29 has support frame portions 29a and 29b for supporting the blood bag forming the cell separation chamber 1 and the blood bag forming the cell proliferation chamber 3, respectively. In the support frame portions 29a and 29b, the cell separation chamber 1 and the cell proliferation chamber are sandwiched between three rim portions 1f and 3f formed on the periphery of each of the blood bags serving as the cell separation chamber 1 and the cell proliferation chamber 3. A holding member 29c for attaching the blood bag of 3 to the frame 29 is provided.

In FIG. 8, the conduit formed integrally with the blood bag serving as the cell separation chamber 1 is referred to as the first outlet conduit 9, and the two connecting portions 1c and 1d formed in the blood bag serving as the cell separation chamber 1 The moving pipe line 5 and the first injection pipe line 7 are connected to each of them. Similarly, the conduit formed integrally with the blood bag serving as the cell proliferation chamber 3 is designated as the second outlet conduit 13, and moves to the two connecting portions 3c and 3d formed in the blood bag serving as the cell proliferation chamber 3, respectively. The pipeline 5 and the second injection pipeline 11 are connected. Further, FIG. 8 shows a case where two cocks 7a and 11a are provided in the first injection pipe line 7 and the second injection pipe line 11, respectively, and the cocks provided with the second outlet line 13 are provided. 13a is omitted.

Further, FIGS. 1, 7 and 8 show the case where the cell separation chamber 1 and the cell proliferation chamber 3 formed separately are used, but as shown in FIG. 9, the cell separation chamber 1 and the cell proliferation are shown. The chamber 3 can also be formed as one isolation culture unit 31. In this case, for example, as shown in FIG. 9, a cell separation chamber 1 and a cell proliferation chamber 3 are formed in one transparent resin container 31a, and further, a moving pipe line 5 and a first injection pipe line 7 are formed. , The first lead-out line 9, the second injection line 11, and the second lead-out line 13 are provided. Further, cocks 5a, 7a, 9a, 11a and 13a are provided in the moving pipe 5, the first injection pipe 7, the first outlet pipe 9, the second injection pipe 11 and the second outlet pipe 13.

At this time, at least one part of the inner surface of the cell separation chamber 1 that is on the bottom surface side is subjected to a process of providing an appropriate attachment portion 15. Further, in the separation culture unit 31, the first injection line 7 and the second injection line 11 are connected to one injection port 31b formed on the side wall of the container 31a, and the first lead-out line 9 and the first lead-out line 9 and the first are connected. 2 The outlet line 13 is connected to one outlet 31c formed on the side wall of the container 31a. The separation and culture unit 31 is made of a cell separation chamber 1, a cell proliferation chamber 3, a moving pipe 5, a first injection pipe 7, a first outlet pipe 9, and a second injection pipe by resin molding using a mold or the like. It is possible to integrally form 11 and the second lead-out pipe 13.

If such a separation culture unit 31 is used and the operations of the cocks 5a, 7a, 9a, 11a, and 13a provided in the separation culture unit 31 can be controlled mechanically or electrically, FIGS. 3 to 6 It becomes easy to automate the separation culture operation as shown in. When automating the separation culture operation using the separation culture unit 31, the cell culture device accommodates the separation culture unit 31 in addition to the separation culture unit 31 to create a cell culture environment, as shown in FIG. Controllable incubator 33, control unit 35 that controls the operation of cocks 5a, 7a, 9a, 11a, 13a, etc. provided in the isolation culture unit 31, control of the operation of the incubator 33, commands to the control unit 35, and various settings. It is equipped with a computer 37 for inputting values, a plurality of reagent bottles 39 each containing a culture solution, a buffer solution, a cell stripping agent, etc., and a waste liquid tank 41 containing the waste liquid from the separation culture unit 31.

A supply line 43 is connected to each of the plurality of reagent bottles 39, and the supply line 43 connected to each reagent bottle 39 joins one line in the middle and is an injection port of the separation culture unit 31. Connected to 31b. A solenoid valve 45 for controlling the supply and stop of the liquid in each reagent bottle 39 to the separation culture unit 31 is provided in each of the branched portions of the supply pipe 43. The solenoid valve 45 is electrically connected to the control unit 35 via wiring 47, and its operation is controlled by the control unit 35. Although not shown, the control unit 35 is electrically connected to a drive mechanism such as cocks 5a, 7a, 9a, 11a, and 13a provided in the separation culture unit 31 via wiring. The computer 37 is electrically connected to the control unit 35 and the incubator 33 via the wiring 47. The waste liquid tank 41 is connected to the outlet 31c of the separation culture unit 31 via the waste liquid pipeline 49.

By adopting such a cell culture device having the configuration shown in FIG. 10, the computer 37 and the control unit 35 can perform cocks 5a, 7a, 9a, according to the separation culture operation as shown in FIGS. 3 to 6. By controlling the operation of 11a, 13a, incubator 33, electromagnetic valve 45, etc., it is possible to automatically perform a culture in which a target cell is separated from a plurality of different types of cells and proliferated.

As described above, in the cell culture apparatus of the present embodiment, in the cell separation chamber 1, cells contained in the cell-containing solution that have adhesiveness or are stronger than other cells are attached to the attachment portion 15. The cells attached to the attachment portion 15 and the cells not attached to the attachment portion 15 can be separated by adhering to the attachment portion 15. Furthermore, different types of cells into the cell separation chamber 1 by the first injection line 7 and the first lead-out line 9, the second injection line 11 and the second lead-out line 13, and the moving line 5 Injection of cell-containing liquid or buffer solution containing cells, derivation of unnecessary cells or liquid from the cell separation chamber 1, movement of cells separated in the cell separation chamber 1 to the cell proliferation chamber 3, inside the cell proliferation chamber 1 It is possible to inject a culture solution or a buffer solution into the cells, or collect cells from the cell proliferation chamber 1. Therefore, a consistent space isolated from the outside without the need for the operator to manually perform the operation of separating the target cells and the operation of planting the separated target cells in a medium for proliferation using a pipette or the like. Can be done in. Therefore, it is possible to suppress the occurrence of contamination in the cell culture for separating and proliferating the target cells from the cell-containing solution containing a plurality of different types of cells.

Further, since it is not necessary to use an expensive device such as a cell sorter to separate the target cells, the cost of the cell culture device capable of separating and proliferating the target cells can be reduced.

Further, in the cell culture apparatus of the present embodiment, the first injection pipe line 7 has a cock 7a serving as a first injection flow path opening / closing means, and the first lead-out pipe line 9 has a first lead-out flow path opening / closing means. The cock 9a is provided with a cock 11a serving as a second injection flow path opening / closing means in the second injection pipe line 11, and a cock 13a serving as a second lead-out flow path opening / closing means in the second lead-out pipe line 13. ing. Therefore, all the channels for flowing in and out of the cell separation chamber 1 and the cell proliferation chamber 3 can be blocked by the cocks 5a, 7a, 9a, 11a, and 13a, and only the necessary conduits can be opened to allow the liquid to flow in and out. Therefore, it becomes easy to maintain the sterilized state of the cell separation chamber 1 and the cell proliferation chamber 3, and the occurrence of contamination can be suppressed more reliably. In addition, it becomes easy to control the movement of liquids and cells to and from the cell separation chamber 1 and the cell proliferation chamber 3 and the movement of cells from the cell separation chamber 1 to the cell proliferation chamber 3.

Further, cocks 5a, 7a, 9a, 11a, 13a are provided in the mobile line 5, the first injection line 7, the first lead line 9, the second injection line 11, and the second lead line 13. By making it possible to mechanically or electrically control the operation of each flow path opening / closing means such as cocks 5a, 7a, 9a, 11a, and 13a, the operation of separating the target cells and the medium of the separated cells can be obtained. A series of culture operations for separating and proliferating target cells, such as a planting operation and a recovery operation of proliferated cells, can be easily automated.

Further, in the present embodiment, the attachment portion 15 provided in the cell separation chamber 1 is a cell adhesive material or a component peculiar to plant cells, which is a component that can be enzymatically decomposed and a component that has adhesion to cells. Is formed of. Therefore, when the cells attached to the attachment portion 15 are the target cells, it is not necessary to detach the cells using a proteolytic enzyme derived from animal cells such as trypsin, and the cells have the ability to proliferate and differentiate. It is possible to suppress the deterioration of the ability to do. Therefore, it becomes easy to obtain cells of the quality required in the cell proliferation chamber 3.

In addition, in the present embodiment, when the cells not attached to the attachment portion 15 of the cell separation chamber 1 are the target cells, the attachment portion 15 in the cell separation chamber 1 is a surface for promoting cell attachment. It is formed by applying treatment. Therefore, the adhesion of cells other than the target cells to the attachment portion 15 is promoted, and the separation ability of the target cells in the cell separation chamber 1 can be improved.

Further, the present invention is not limited to the cell culture apparatus having the configuration of the present embodiment, and may be various as long as it includes a cell separation chamber having an attachment portion, a cell proliferation chamber communicating with the cell culture chamber via a moving flow path, and the like. Can be configured.

<figref num="1">It is a figure which shows schematic structure of one Embodiment of the cell culture apparatus to which this invention is applied, (a) is the whole structure, (b) is a cell separation chamber, a moving tube and a cell proliferation chamber. It is a perspective view which shows the state which took out and disassembled the bag and tube which make up.</figref><figref num="2">It is a figure which schematically explains the principle of cell separation in the cell separation chamber of the cell culture apparatus to which the invention is applied.</figref><figref num="3">In one embodiment of the cell culture apparatus to which the present invention is applied, the operation when the target cell is an adherent cell is schematically shown in the order of (a) to (h).</figref><figref num="4">It is a flow chart which shows the operation when the target cell is an adherent cell in one Embodiment of the cell culture apparatus to which this invention is applied.</figref><figref num="5">In one embodiment of the cell culture apparatus to which the present invention is applied, the operation when the target cell is a cell not attached to the attachment portion is schematically shown in the order of (a) to (h). is there.</figref><figref num="6">It is a flow chart which shows the operation when the target cell is a cell which is not attached to the attachment part in one embodiment of the cell culture apparatus to which this invention is applied.</figref><figref num="7">It is a figure which shows another schematic structure of another embodiment of the cell culture apparatus to which this invention is applied, (a) is the whole plan view, (b) is the side view of the cell separation chamber.</figref><figref num="8">It is a figure which shows the other schematic structure of another embodiment of the cell culture apparatus to which this invention is applied, (a) is a plan view of the whole, (b) is a plan view of only a frame, (c). ) Is a side view of the frame only.</figref><figref num="9">It is a top view which shows still another schematic structure of one Embodiment of the cell culture apparatus to which this invention is applied.</figref><figref num="10">It is a perspective view which shows typically the schematic structure of one Embodiment in the case of automating the culture operation in the cell culture apparatus to which this invention is applied.</figref>

Code description

1 Cell separation chamber 3 Cell proliferation chamber 5 Mobile pipeline 7 1st injection pipeline 9 1st outlet pipeline 11 2nd injection pipeline 13 2nd outlet pipeline 15 Attachment 5a, 7a, 9a, 11a, 13a Cock