JP6594066B2 - Cell culture container, cell culture apparatus and cell culture method - Google Patents

Description

  The present invention relates to a cell culture container, a cell culture device, and a cell culture method.

  For example, the following techniques are known with respect to a cell culture reactor or cell culture substrate constituting a scaffold for cultured cells used for adhesion culture.

  For example, in Patent Document 1, a plurality of cell culture carrier substrates each having a porous structure part with a side length or diameter of 50 μm to 500 μm of a polygonal through hole are stacked in a cell culture tank at intervals. A cell culture reactor constituted by the above is described.

  Further, in Patent Document 2, different types of tissues are arranged in contact with each other, and a tissue contact portion including a solid support having a network structure or a porous structure is fractionated through the tissue contact portion. A two-layer bioreactor having a culture chamber having a medium circulation part and a circulation mechanism part for circulating the medium through the medium circulation part is described. In this two-layer bioreactor, different culture media are supplied to the respective media circulation portions of the culture chamber by the operation of the circulation mechanism, and the tissue having a hierarchical structure is cultured at the tissue contact portion.

  Patent Document 3 describes a cell culture substrate in which a plurality of recesses having a depth of 100 μm to 500 μm and an inner diameter of 100 μm to 1000 μm are provided on the surface of the substrate.

JP 2011-172533 A JP 2007-54026 A JP 2012-249547 A

For example, in order to treat liver disease, heart disease, etc. by cell transplantation, it is considered that transplantation of 1 × 10 ⁹ or more differentiated cells is necessary for one patient. Therefore, it is indispensable to develop a technology for mass-culturing human pluripotent stem cells such as ES cells (Embryonic Stem cells) and iPS cells (induced Pluripotent Stem cells). In order to stably cultivate pluripotent stem cells in large quantities stably while maintaining the optimal environment by conventional adhesion culture methods, it is necessary to enlarge the adhesion area of the cells, which is difficult in terms of structure and cost. is there. Thus, in the conventional adhesion culture method, mass culture becomes inefficient.

  As a technique for solving the above problems in the adhesion culture method, there is a suspension culture method in which cells are cultured while suspended in a culture solution. However, in the conventional suspension culture, there is a problem of fusion of cell clusters (also referred to as spheres or spheroids) that are aggregates of a plurality of cells. That is, in the culturing of pluripotent stem cells, if the size of the cell mass is excessive, the cell masses fuse with each other, and the cells start to differentiate, or the cells at the center of the cell mass are necrotic. Can occur.

  An object of the present invention is to provide a cell culture vessel, a cell culture device, and a cell culture method capable of suppressing fusion of cell masses in suspension culture.

Cell culture chamber according to the present invention has a inner wall surface forming the accommodation space to yield volumes of cells on the inner wall surface, the width gradually narrowed toward the projecting and the distal end side toward the inside of the accommodating space A plurality of tapered pillars are provided.

The plurality of pillars are preferably provided with a size and an interval that inhibit the adhesion of cells to the inner wall surface of the cell culture container. The plurality of pillars are preferably provided in such a size and interval that the adhesion area with the inner wall surface of the cell is reduced as compared with the case where the inner wall surface has a flat structure. The cell culture container may have a plurality of pillars on the entire inner wall surface.

The plurality of pillars are preferably arranged regularly, and may be arranged in a honeycomb shape, for example.

It is preferable coefficient of variation of distance between the centers of the adjacent pillars in a plurality of pillars is 0.1 or less. Moreover, it is preferable that the length of the longest part in the direction parallel to the inner wall surface of the cell culture container at the tip of each of the plurality of pillars is 0.01 μm or more and 10 μm or less.

The inner wall surface of the cell culture container may be formed of a plastic material having a plurality of pillars . The plastic material can include a hydrophobic polymer. It is preferable that the storage space of the cell culture container is sealed. The cell culture container may be configured by bonding a plastic film having a plurality of pillars on the surface.

The cell culture device according to the present invention has a first inflow port and a first outflow port, and an inner wall surface that forms an accommodation space for accommodating cells, and the inner wall surface has a plurality of concave portions and a plurality of convex portions. A cell culture container having a non-flat structure including at least one of the first , the first flow path connecting the first outlet and the first inlet, and the cell culture container provided in the first flow path. A storage container for storing cells to be cultured in the first, having a second inlet connected to the first outlet and a second outlet connected to the first inlet Positioned between the storage container, the first portion in the first flow path located between the second outlet and the first inlet, and between the second inlet and the first outlet. A second channel in the first channel that connects to the second channel, and a second channel that is provided in the second channel, and from the first channel via the first site. A division process is performed to divide the cell mass, which is a cell mass cultured in the cell culture container, and the divided cells are allowed to flow out into the first flow path via the second part. It further includes a division processing unit and a medium supply unit that supplies a medium into the first flow path.

  The cell culture device may further include a pipe that forms the first flow path, and the inner wall surface of the pipe may have a non-flat structure including at least one of a plurality of concave portions and a plurality of convex portions. Good.

The cell culturing method according to the present invention has an inner wall surface that forms an accommodation space for accommodating cells, and projects on the inner wall surface toward the inside of the accommodation space and gradually decreases in width toward the distal end side. The cells are cultured in a state where the cells are suspended in a culture solution in a storage space of a cell culture vessel provided with a plurality of pillars .

  According to the present invention, there are provided a cell culture vessel, a cell culture device, and a cell culture method used for suspension culture, which can suppress the fusion of cell masses.

It is a top view which shows the structure of the cell culture container which concerns on embodiment of this invention. FIG. 2 is a cross-sectional view taken along line 2-2 in FIG. Fig.3 (a) is a top view which shows the structure of the inner wall face of a cell culture container, FIG.3 (b) is sectional drawing along the 3b-3b line | wire in Fig.3 (a). 4 (a) to 4 (d) are cross-sectional views illustrating a method for producing a plastic film having a non-flat structure on the surface including a plurality of recesses arranged in a honeycomb shape. It is the photograph which image | photographed the plastic film which has a some recessed part on the surface using the scanning electron microscope. Fig.6 (a) is a figure which shows typically the state of the cell mass in case the inner wall surface of a cell culture container is flat. FIG. 6B is a diagram schematically illustrating the state of the cell mass when the inner wall surface of the cell culture container has a non-flat structure including a plurality of recesses. Fig.7 (a) is a top view which shows the other example of the non-flat structure formed in the inner wall face of a cell culture container. FIG.7 (b) is sectional drawing along the 7b-7b line | wire in Fig.7 (a). FIG.7 (c) is an enlarged view of the area | region A enclosed with the broken line in FIG.7 (b). It is a figure which shows the manufacturing method of the plastic film which has the non-flat structure containing the some convex part arranged in the honeycomb form on the surface. It is the photograph which image | photographed the plastic film which has a some convex part on the surface using the scanning electron microscope. FIG. 10A to FIG. 10E are photomicrographs showing the state of cells on the fifth day of culture when the cell culture containers according to Experiment 1 to Experiment 5 are used, respectively. FIG. 11A and FIG. 11B are photomicrographs showing the state of cells on the fifth day of culture when the cell culture vessels according to Comparative Experiment 1 and Comparative Experiment 2 are used, respectively. It is a figure which shows the structure of the cell culture apparatus which concerns on embodiment of this invention. It is sectional drawing which shows an example of the internal structure of each piping which concerns on embodiment of this invention. It is a figure which shows the flow of a cell, a culture medium, etc. in case the cell culture apparatus which concerns on embodiment of this invention performs a subculture process. It is a figure which shows the flow of a cell, a culture medium, etc. in case the cell culture apparatus which concerns on embodiment of this invention implements a culture medium exchange process. It is a figure which shows the flow of a cell, a culture medium, etc. in case the cell culture apparatus which concerns on embodiment of this invention performs a division | segmentation process. It is a figure which shows the flow of a cell, a culture medium, etc. in case the cell culture apparatus which concerns on embodiment of this invention performs a division process. It is a figure which shows the flow of a cell, a culture medium, etc. in case the cell culture apparatus which concerns on embodiment of this invention implements a freezing process. It is a flowchart which shows the flow of the process in the cell culture program which the control part which concerns on embodiment of this invention performs.

  Hereinafter, an example of an embodiment of the present invention will be described with reference to the drawings. In the drawings, the same or equivalent components and parts are denoted by the same reference numerals.

(Cell culture vessel)
FIG. 1 is a plan view showing a configuration of a cell culture container 20 according to an embodiment of the present invention. As an example, the cell culture container 20 is formed by bonding two plastic films having flexibility and gas permeability by a technique such as heat sealing (thermocompression bonding). The cell culture container 20 has a seal portion 201 formed along the outer edge thereof, and a storage space 203 for storing cells formed inside the seal portion 201 is sealed. That is, in the present embodiment, the cell culture container 20 has a form of a bag made of plastic, and can be suitably used as a single-use cell culture container. By using the cell culture container 20 as a single use, the risk of cross-contamination is eliminated, and sterilization, washing, and validation thereof are not necessary. The cell culture container 20 may be formed by folding and bonding one plastic film. In addition, the plastic film preferably transmits visible light in order to allow visual observation of cells cultured in the cell culture container 20.

  The plastic film constituting the cell culture vessel 20 is, for example, a vinyl polymer (for example, polyethylene, polypropylene, polystyrene, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl chloride, polyvinylidene chloride, polyvinylidene fluoride, Polyhexafluoropropene, polyvinyl ether, polyvinyl carbazole, polyvinyl acetate, polyvinyl carbazole, polytetrafluoroethylene, etc.), polyester (for example, polyethylene terephthalate, polyethylene naphthalate, polyethylene succinate, polybutylene succinate, polylactic acid, etc.), Polylactone (such as polycaprolactone), polyamide or polyimide (such as nylon or polyamic acid), It is made of materials such as urethane, polyurea, polybutadiene, polycarbonate, polyaromatics, polysulfone, polyethersulfone, polysiloxane derivatives, and cellulose acylate (triacetylcellulose, cellulose acetate propionate, cellulose acetate butyrate). There may be. These polymers may be a homopolymer, or may be in the form of a copolymer or polymer blend, if necessary, from the viewpoints of solubility, optical properties, electrical properties, film strength, elasticity, and the like. Moreover, you may use these polymers as a mixture of 2 or more types of polymers as needed. For use in optical applications, for example, cellulose acylate and cyclic polyolefin are preferred.

  As an example, the cell culture container 20 has an inflow port 21 in the vicinity of the upper end portion in FIG. 1 and an outflow port 22 in the vicinity of the lower end portion in FIG. The cell suspension or culture solution can be injected into the cell culture vessel 20 through the inflow port 21, and the cell suspension or culture solution can be discharged from the cell culture vessel 20 through the outflow port 22. Can be done through. In this embodiment, the inflow port 21 and the outflow port 22 are configured by connectors 21a and 22a inserted into the main body of the cell culture container 20 so as to penetrate the seal portion 201. Thus, the storage space 203 of the cell culture container 20 has a configuration that communicates with the outside only through the inflow port 21 and the outflow port 22, and maintains high sealing performance. Thereby, the contact between the storage space 203 and the atmosphere is limited, and the risk of bacterial contamination is reduced as compared with the case of using a petri dish or a flask. In addition, arrangement | positioning of the inflow port 21 and the outflow port 22 can be changed suitably. Further, a through hole 204 is provided in the seal portion 201 so as to be separated from the accommodation space 203. The through hole 204 can be used to support the cell culture container 20.

  FIG. 2 is a cross-sectional view taken along line 2-2 in FIG. As shown in FIG. 2, the cell culture vessel 20 can be suitably used for floating culture in which cells C are cultured in a suspended state in a culture solution L in an accommodation space 203 formed by an inner wall surface 205. . That is, the cells C that exist in a floating state in the accommodation space 203 during the culture period are surrounded by the inner wall surface 205 of the cell culture container 20. In addition, even if some cells that are not suspended in the culture medium L are present, if a large number of cells are present in a suspended state, they are included in the suspended culture.

  FIG. 3A is a plan view showing the structure of the inner wall surface 205 of the cell culture container 20, and FIG. 3B is a cross-sectional view taken along the line 3b-3b in FIG.

  The inner wall surface 205 of the cell culture container 20 has a non-flat structure including a plurality of recesses 210 that are recessed toward the outer wall surface 206 side of the cell culture container 20. In the present embodiment, the plurality of recesses 210 are formed in the plastic film itself constituting the cell culture container 20. In the present embodiment, the plurality of recesses 210 are formed over the entire inner wall surface 205 of the cell culture container 20. That is, the entire inner wall surface 205 has a non-flat structure, and the entire accommodation space 203 is surrounded by the non-flat structure. Note that “the entire inner wall surface has a non-flat structure” means that most of the inner wall surface 205 (for example, 90% or more) has a non-flat structure, and is accommodated in the accommodation space 203, for example. In some areas where the cells cannot contact, a case where there is no non-flat structure is also included.

  In the present embodiment, the plurality of recesses 210 are regularly arranged. Specifically, the plurality of recesses 210 are arranged in a honeycomb shape (hexagonal close-packed arrangement). That is, each of the recesses 210 arranged in the nth column in FIG. 3A is arranged at a position corresponding to the center in the column direction between the adjacent recesses 210 arranged in the (n + 1) th column. Yes. A plastic film in which a plurality of recesses 210 are regularly arranged in a honeycomb shape can be manufactured by a method described later, and the manufacturing cost can be reduced as compared with the case of using other arrangements. The plurality of recesses 210 may be arranged in a matrix (lattice). In other words, the plurality of recesses 210 may be arranged along the column direction and the row direction orthogonal to the column direction. Although the arrangement of the plurality of recesses 210 can be random, when the density of the recesses 210 formed on the inner wall surface 205 of the cell culture container 20 becomes uneven, the cells to be cultured Since the homogeneity may be adversely affected, the plurality of recesses 210 are preferably arranged regularly.

  The shape of the recess 210 is not particularly limited, but in the present embodiment, each of the recesses 210 has a sphere shape in which a part of a sphere is cut out. That is, each of the recesses 210 forms a circular opening 210 a on the inner wall surface of the cell culture container 20. By making each of the recesses 210 have a spherical shape, a manufacturing method described later can be used, and the manufacturing cost can be reduced as compared with the case of using other shapes. Note that the shape of each of the recesses 210 may be a cylindrical shape or a prismatic shape.

  The plurality of recesses 210 are provided with a size and an interval that inhibit adhesion of cells cultured in the cell culture container 20 to the inner wall surface 205. The plurality of recesses 210 are preferably provided with such a size and interval that the adhesion area of the cells to the inner wall surface 205 is reduced as compared with the case where the inner wall surface 205 has a flat structure. That is, the non-flat structure formed on the inner wall surface 205 of the cell culture container 20 is a flat portion extending between the diameter Dh (hereinafter referred to as the opening diameter Dh) in the opening 210a of the recess 210 and the adjacent recesses 210. Is configured to be smaller than the diameter Ds of the cell mass S formed in the cell culture container 20 during the culture period. By making the opening diameter Dh of the concave portion 210 smaller than the diameter Ds of the cell mass S, it is possible to prevent the cell mass S from entering the concave portion 210, thereby allowing cells to enter the inner wall surface of the concave portion 210. It becomes possible to inhibit the adhesion of the mass S. In addition, by making the width W of the flat portion extending between the concave portions 210 adjacent to each other smaller than the diameter Ds of the cell mass S, adhesion of the cell mass S to the flat portion can be inhibited. The opening diameter Dh is the length of the longest portion in the direction parallel to the inner wall surface 205 of the opening 210a formed in the inner wall surface 205 by the recess 210. The diameter Ds of the cell mass S is the length of the longest part of the cell mass S.

  The assumed range of the diameter Ds of the cell mass S during the culture period in the cell culture container 20 according to the present embodiment is, for example, 100 μm or more and 400 μm or less. The opening diameter Dh of the recess 210 can be set to, for example, 1 μm or more and 80 μm or less for the cell mass S having the above size. By setting the opening diameter Dh of the concave portion 210 to 1 μm or more, the inner wall surface 205 of the cell culture vessel 20 viewed from the cell mass S has a substantially non-flat structure, and the cell mass S to the inner wall surface 205 of the cell culture vessel 20 is obtained. The function of inhibiting the adhesion of is exhibited. On the other hand, by setting the opening diameter Dh of the concave portion 210 to 80 μm or less, the cell mass S can be prevented from entering the concave portion 210, and thereby the cell mass S adheres to the inner wall surface of the concave portion 210. It becomes possible to inhibit.

  Further, the coefficient of variation of the center-to-center distance P1 between the recesses 210 adjacent to each other is preferably 0.1 or less. The variation coefficient is a value obtained by dividing the standard deviation of the population by the arithmetic average, and is an index indicating the degree of relative variation of the population. The smaller the variation coefficient of the center-to-center distance P <b> 1 between the adjacent recesses 210, the smaller the density deviation of the recesses 210 in the inner wall surface 205 of the cell culture container 20. By setting the coefficient of variation of the center-to-center distance P1 between the recesses 210 adjacent to each other to 0.1 or less, the density of the recesses 210 is made uniform, and the homogeneity of cells cultured in the cell culture container 20 is ensured. A center-to-center distance P1 between the recesses 210 adjacent to each other can be set to, for example, 1.5 μm or more and 160 μm or less.

  Below, an example of the manufacturing method of the plastic film which has the non-flat structure on the surface which comprises the cell culture container 20 is demonstrated. FIG. 4A to FIG. 4D are cross-sectional views showing a method for producing a plastic film having a non-flat structure on the surface including a plurality of recesses 210 arranged in a honeycomb shape.

  First, a coating solution is prepared by dissolving a hydrophobic polymer as a material for the plastic film constituting the cell culture container 20 in a solvent. Subsequently, this coating solution is applied onto a support 300 made of glass or a polymer compound to form a coating film 310 on the support 300 (FIG. 4A).

Next, by supplying humid air over the entire surface of the coating film 310, water droplets 320 are formed on the coating film 310 by the dew condensation phenomenon (FIG. 4B). Here, Td and Ts are set so that the difference ΔT (= Td−Ts) between the dew point Td of the humidified air flowing in the vicinity of the coating film 310 and the surface temperature Ts of the coating film 310 satisfies the following expression (1). Control at least one of
3 ° C. ≦ ΔT ≦ 30 ° C. (1)

  Next, the water droplet 320 is grown by continuing supply of humidified air. Each of the water droplets 320 grows to have substantially the same size. Further, the polymer is precipitated by evaporation of the solvent contained in the coating film 310. As the solvent evaporates, the water droplets 320 are regularly arranged so as to form a honeycomb-like arrangement by a capillary force, and enter the coating film 310 (FIG. 4C).

  Next, in parallel with the evaporation of the solvent or after the solvent is evaporated, the water droplet 320 is evaporated. As a result, the portion of the coating film 310 into which the water droplet 320 has entered remains as the recess 210 (FIG. 4D). Thereafter, the coating film 310 is peeled from the support 300 to obtain a plastic film having a non-flat structure including a plurality of recesses 210 on the surface. FIG. 5 is a photograph of a plastic film produced using the above method and having a plurality of recesses on the surface, taken using a scanning electron microscope.

  The opening diameter Dh of the recess 210 can be adjusted, for example, by adjusting the size of the water droplet 320 by changing the supply amount of water vapor contained in the humidified air or the timing at which the water droplet 320 is evaporated. is there. The details of the method for producing a plastic film having a plurality of concave portions arranged on the surface as described above are described in, for example, Japanese Patent Application Laid-Open Nos. 2007-291367 and 2009-256624. According to the above manufacturing method, patterning of the non-flat structure is performed using the water droplets 320. Therefore, the plurality of uniformly sized concave portions 210 arranged in a honeycomb shape are formed on the plastic film without using a mold or a mask. Can be formed. Thereby, manufacturing cost can be suppressed. In addition, it is also possible to manufacture the plastic film which has the non-flat structure which comprises the cell culture container 20 on the surface using other methods, such as an etching, sandblasting, or press molding.

  FIG. 6A schematically shows the state of the cell mass S when the inner wall surface 205 of the cell culture container 20 is flat. When the inner wall surface 205 of the cell culture container 20 is flat, the cell mass S floating in the cell culture container 20 easily adheres to the inner wall surface 205 of the cell culture container 20, and when adhered, It deforms so as to spread along 205 and grows along the inner wall surface 205. Thereby, fusion with other cell mass S adhered to the inner wall surface 205 is induced. When the cell clusters are fused, there may be a problem that the cells start to differentiate or the cells at the center of the cell cluster are necrotized. For this reason, in suspension culture, it is preferable to suppress spontaneous fusion of cell masses.

  FIG. 6B is a diagram schematically showing the state of the cell mass S when the inner wall surface 205 of the cell culture container 20 has a non-flat structure including a plurality of recesses 210. When the inner wall surface 205 of the cell culture container 20 has a non-flat structure, the contact area between the cell cluster S and the inner wall surface 205 becomes small, and there is no region where the cell cluster S can stably adhere. Adhesion of S to the inner wall surface 205 is hindered. Thereby, during the culture period, the shape of the cell mass S is maintained, and fusion of the cell masses is suppressed. By suppressing the fusion of cell masses, it is possible to suppress a decrease in cell culture efficiency, and it is possible to suppress cell differentiation in the culture of pluripotent stem cells. In the cell culture container 20 according to the present embodiment, since the entire inner wall surface 205 has a non-flat structure and the entire storage space 203 is surrounded by the non-flat structure, the cells stored in the storage space 203 The effect of suppressing the fusion of lumps is promoted.

  FIG. 7A is a plan view showing another example of the non-flat structure formed on the inner wall surface 205 of the cell culture container 20. FIG.7 (b) is sectional drawing along the 7b-7b line | wire in Fig.7 (a). FIG.7 (c) is an enlarged view of the area | region A enclosed with the broken line in FIG.7 (b).

  The non-flat structure formed on the inner wall surface 205 of the cell culture container 20 includes a plurality of protrusions (pillars) 220 protruding toward the inside of the cell culture container 20 as shown in FIG. Also good. In the present embodiment, the plurality of convex portions 220 are formed on the plastic film itself constituting the cell culture container 20. In the present embodiment, the convex portion 220 is formed over the entire inner wall surface 205 of the cell culture container 20. That is, the entire inner wall surface 205 has a non-flat structure, and the entire accommodation space 203 is surrounded by the non-flat structure.

  In the present embodiment, the plurality of convex portions 220 are regularly arranged. Specifically, the plurality of convex portions 220 are arranged in a honeycomb shape. A plastic film in which a plurality of convex portions 220 are regularly arranged in a honeycomb shape can be manufactured by a method described later, and the manufacturing cost can be reduced as compared with a case where other arrangements are used. Note that the plurality of convex portions 220 may be arranged in a matrix (lattice). That is, the plurality of convex portions 220 may be arranged along the column direction and the row direction orthogonal to the column direction. In addition, the arrangement of the plurality of convex portions 220 can be made random, but when the density of the convex portions 220 formed on the inner wall surface 205 of the cell culture container 20 becomes uneven, the cells are cultured. Since the cell homogeneity may be adversely affected, the plurality of convex portions 220 are preferably arranged regularly.

  The shape of the convex portion 220 is not particularly limited, but in the present embodiment, each of the convex portions 220 has a tapered shape whose width gradually decreases toward the tip side of the convex portion 220. . Each of the convex portions 220 may have a cylindrical shape or a prismatic shape.

  The plurality of convex portions 220 are provided with a size and an interval that inhibit adhesion of cells cultured in the cell culture container 20 to the inner wall surface 205. It is preferable that the plurality of convex portions 210 be provided with a size and an interval that reduce the adhesion area of the cell to the inner wall surface 205 as compared with the case where the inner wall surface 205 has a flat structure. That is, the diameter Dp (hereinafter referred to as the tip diameter Dp) at the distal end of the convex portion 220 and the distance P2 between the convex portions 220 adjacent to each other are determined by the cell mass S formed in the cell culture container 20 during the culture period. It is smaller than the diameter Ds. By making the tip diameter Dp of the convex portion 220 smaller than the diameter Ds of the cell mass S, adhesion of the cell mass S to the tip portion of the convex portion 220 can be inhibited. Further, by making the distance P2 between the convex portions 220 adjacent to each other smaller than the diameter Ds of the cell mass S, the adhesion of the cell mass S to the region between the convex portions 220 adjacent to each other can be inhibited. The tip diameter Dp of the convex portion 220 is the length of the tip portion of the convex portion 220 in the direction parallel to the inner wall surface 205 of the cell culture container 20.

  As described above, the assumed range of the diameter Ds of the cell mass S during the culture period is, for example, 100 μm or more and 400 μm or less. For the cell mass S having the above size, the tip diameter Dp of the convex portion 220 can be set to be 0.01 μm or more and 10 μm or less, for example. By setting the tip diameter Dp of the convex part 220 to 0.01 μm or more, the inner wall surface 205 of the cell culture container 20 viewed from the cell mass S has a substantially non-flat structure, and A function of inhibiting the adhesion of the cell mass S is exhibited. On the other hand, the adhesion of the cell mass S to the tip of the convex part 220 can be inhibited by setting the tip diameter Dp of the convex part 220 to 10 μm or less. Moreover, it becomes possible to form a non-flat structure on the surface of a plastic film by the method mentioned later by making the magnitude | size of the front-end | tip diameter Dp of the convex part 220 into said range.

  On the other hand, the center-to-center distance P2 between the convex portions 220 adjacent to each other can be set to 2 μm or more and 90 μm or less, for example. By setting the distance P2 between the convex portions 220 adjacent to each other to be 2 μm or more, the inner wall surface 205 of the cell culture container 20 viewed from the cell mass S has a substantially non-flat structure, and the inner wall surface 205 of the cell culture container 20 The function of inhibiting the adhesion of the cell mass S to is exerted. On the other hand, by setting the center-to-center distance P2 between the convex portions 220 adjacent to each other to 90 μm or less, the adhesion of the cell mass S to the region between the convex portions 220 adjacent to each other can be inhibited.

  Moreover, it is preferable that the variation coefficient of the center-to-center distance P2 of the convex portions 220 adjacent to each other is 0.1 or less. Decreasing the coefficient of variation of the center-to-center distance P2 between the convex portions 220 adjacent to each other means that the unevenness of the density of the convex portions 220 on the inner wall surface 205 of the cell culture container 20 is reduced. By setting the coefficient of variation of the center-to-center distance P2 between the convex portions 220 adjacent to each other to be 0.1 or less, the density of the convex portions 220 on the inner wall surface 205 of the cell culture container 20 is made uniform, and is cultured in the cell culture container 20. Cell homogeneity.

  FIG. 8 is a diagram showing a method of manufacturing a plastic film having a non-flat structure including a plurality of convex portions 220 arranged in a honeycomb shape on the surface. A plastic film having a plurality of convex portions 220 on the surface can be manufactured using the above-described plastic film having a plurality of concave portions 210 on the surface. That is, as shown in FIG. 8, the adhesive tape 330 is peeled off after the adhesive tape 330 is applied to the coating film 310 that forms the plastic film formed on the support 300 and has a plurality of recesses 210. . Thereby, the upper layer part of the coating film 310 is removed, and the site | part between the mutually adjacent recessed parts 210 remains as the convex part 220. FIG. FIG. 9 is a photograph of a plastic film produced using the above method and having a plurality of convex portions on the surface, taken using a scanning electron microscope. According to the above manufacturing method, it is possible to form the plurality of uniform-sized convex portions 220 arranged in a honeycomb shape on the plastic film without using a mold or a mask, and it is possible to reduce the manufacturing cost. .

  Even when the non-flat structure of the inner wall surface 205 of the cell culture container 20 is configured to include a plurality of convex portions 220, the cell mass S and the non-flat structure are configured to include the plurality of concave portions 210. The contact area with the inner wall surface 205 is reduced. Thereby, since there is no region where the cell mass S can stably adhere, the adhesion of the cell mass S to the inner wall surface 205 is inhibited. Thereby, during the culture period, the shape of the cell mass S is maintained, and fusion of the cell masses is suppressed. By suppressing the fusion of cell masses, it is possible to suppress a decrease in cell culture efficiency, and it is possible to suppress cell differentiation in the culture of pluripotent stem cells. In the cell culture container 20 according to the present embodiment, since the entire inner wall surface 205 has a non-flat structure and the entire storage space 203 is surrounded by the non-flat structure, the cells stored in the storage space 203 The effect of suppressing the fusion of lumps is promoted.

  The cell culture container 20 is an example of the cell culture container in the present invention. The inner wall surface 205 is an example of the inner wall surface in the present invention. The accommodation space 203 is an example of the accommodation space in the present invention. The recess 210 is an example of a recess in the present invention. The convex part 220 is an example of the convex part in this invention.

(Cell culture experiment)
Cell culture experiments were conducted to demonstrate the effect of inhibiting the adhesion of cells to the inner wall surface and the effect of suppressing the fusion of cell clumps obtained by making the inner wall surface of the cell culture container non-flat. . A plurality of types of plastic films having the above-described non-flat structure on the surface were prepared, and the cells were cultured using those which were respectively arranged on the bottom surface of a commercially available well plate as a cell culture container. As a comparative experiment, cells were cultured using a well plate in which the plastic film having the above-described non-flat structure was not disposed on the bottom surface. That is, in the comparative experiment, cells were cultured on the bottom surface of a flat well plate. Table 1 shows the details of the configuration of the cell culture vessel used in each experiment and the evaluation results regarding the cell state on the fifth day of culture.

  The cell culture container according to Experiment 1 to Experiment 4 is configured by arranging a plastic film having a plurality of concave portions on the surface as shown in FIG. 5 on the bottom surface of a commercially available well plate. That is, in Experiment 1 to Experiment 4, cells were cultured on a plastic film having a plurality of recesses on the surface. The plastic films according to Experiments 1 to 4 have different opening diameters Dh of the recesses. That is, the opening diameters Dh of the recesses formed in the plastic films according to Experiments 1 to 4 are 0.8 μm, 1.5 μm, 5 μm, and 35 μm, respectively.

  On the other hand, the cell culture container according to Experiment 5 is configured by arranging a plastic film having a plurality of convex portions on the surface as shown in FIG. 9 on the bottom surface of a commercially available well plate. That is, in Experiment 5, cells were cultured on a plastic film having a plurality of convex portions on the surface. The tip diameter Dp of the convex portion formed on the plastic film according to Experiment 5 is 0.1 μm. In addition, the material of the plastic film concerning Experiment 1 to Experiment 5 is polybutadiene.

  In Comparative Experiment 1 and Comparative Experiment 2, cells were cultured on the bottom surface of a well plate having a flat structure without using a plastic film having a non-flat structure. In Comparative Experiment 2, a well plate having a bottom surface subjected to low adhesion treatment was used.

iPS cells (strain: 207B7) were suspended in serum-free medium TeSR (registered trademark) -E8 (registered trademark: ST-05940) at an initial cell density of 2 × 10 ⁵ cells / ml. It became cloudy, 3 ml was seeded in each cell culture vessel, and culture was started under conditions of 5% CO ₂ and 37 ° C. During the culture period, each cell culture vessel was kept stationary, and the culture solution was changed every day.

The evaluation criteria shown in Table 1 relating to the adhesion of the cell mass to the bottom surface of the cell culture container are as follows.
A: Most of the cell mass is not adhered to the bottom of the container.
B: A part of the cell mass is adhered to the bottom surface of the container.
C: Many cell clusters are adhered to the bottom of the container.

Evaluation criteria for the fusion of cell masses shown in Table 1 are as follows.
A: Most cell clusters are not fused with other cell clusters.
B: A part of the cell mass is fused with another cell mass.
C: Many cell clusters are fused with other cell clusters.

  FIG. 10A to FIG. 10E are photomicrographs showing the state of cells on the fifth day of culture when the cell culture containers according to Experiment 1 to Experiment 5 are used, respectively. FIG. 11A and FIG. 11B are photomicrographs showing the state of cells on the fifth day of culture when the cell culture containers according to Comparative Experiment 1 and Comparative Experiment 2 are used, respectively.

  When the cell culture container according to Experiment 1 to Experiment 5 was used, adhesion of the cell mass to the bottom surface of the container was not confirmed. In addition, as shown in FIGS. 10 (b) to 10 (d), when the cell culture container according to Experiment 2 to Experiment 5 is used, deformation of the cell mass and fusion of the cell masses are hardly confirmed. It was. On the other hand, as shown in FIG. 10A, when the cell culture container according to Experiment 1 was used, deformation of the cell mass and fusion of the cell masses were confirmed in some cell masses.

  On the other hand, when the cell culture containers according to Comparative Experiment 1 and Comparative Experiment 2 were used, adhesion to the bottom surface of the culture container was confirmed in some cell clusters. In addition, as shown in FIGS. 11A and 11B, when the cell culture containers according to Comparative Experiment 1 and Comparative Experiment 2 are used, in many cell masses, deformation of the cell mass and cell masses The fusion between them was confirmed.

  Thus, the inner wall surface of the cell culture container has a non-flat structure including a concave portion having an opening diameter Dh smaller than the diameter of the cell mass or a convex portion having a tip diameter Dp smaller than the diameter of the cell mass. It is confirmed that adhesion of the cell mass to the inner wall surface of the cell culture container can be inhibited, and deformation of the cell mass that spreads along the inner wall surface of the cell culture container and spontaneous fusion of the cell masses are suppressed. It was confirmed. Furthermore, as shown in FIGS. 10 (b) to 10 (d), the inner wall surface of the cell culture container has a non-flat structure, so that even when the cell masses are close to each other, It became clear that no fusion was induced. This suggests that the non-flat structure formed on the inner wall surface of the cell culture vessel acts directly on the cells so as to stop or suppress the fusion reaction between the cell masses. .

  In order to suppress the fusion of cell masses, a suspension culture method has been proposed in which a water-soluble polymer that brings about a thickening action such as methylcellulose is added to a culture solution (WO2013 / 077423A1). According to the cell culture container 20 according to the embodiment of the present invention, it becomes possible to suppress the fusion of cell masses without adding a water-soluble polymer that brings about a thickening action to the culture solution. The complexity of the medium exchange process and the like carried out in the above can be alleviated, and cell culture can be performed at a lower cost than in the past.

  As is clear from the above description, according to the cell culture container 20 of the embodiment of the present invention, it is possible to suppress the fusion of cell masses floating in the accommodation space 203. As a result, cell necrosis can be suppressed, and in the culture of pluripotent stem cells, cell differentiation can be suppressed, and the culture efficiency can be improved as compared with the conventional case.

  In the above embodiment, the case where the non-flat structure formed on the inner wall surface 205 of the cell culture container 20 includes any one of the plurality of concave portions 210 and the plurality of convex portions 220 is exemplified. It is not limited to. The non-flat structure formed on the inner wall surface 205 of the cell culture container 20 may be configured to include both the plurality of concave portions 210 and the plurality of convex portions 220.

  Further, when the cell culture container 20 is configured by bonding plastic films by thermocompression bonding, the bonding surface of the seal portion 201 (see FIG. 1) has a non-flat structure from the viewpoint of adhesion between the plastic films. It is preferable not to have.

  In the above embodiment, the case where the plastic film itself having a non-flat structure on the surface is used as the cell culture container is exemplified, but the present invention is not limited to this mode. For example, the cell culture container 20 can be configured by attaching a plastic film having a non-flat structure on the surface to another support. The support may be made of, for example, plastic, glass, metal, or the like.

  In the above embodiment, the case where the inner wall surface of the cell culture container is made of a plastic material has been exemplified. However, the inner wall surface of the cell culture container may be made of metal, glass, ceramic, or the like, for example. In this case, the non-flat structure can be formed by, for example, thermal spraying, sandblasting, or etching. Moreover, the whole cell culture container may be comprised with the metal, glass, or ceramics. From the viewpoint of enhancing the effect of inhibiting cell adhesion, it is preferable that the inner wall surface of the cell culture container is made of a plastic material. Moreover, by using a plastic film having a non-flat structure on the surface as the base material of the cell culture container as in the cell culture container 20 according to the present embodiment, it can be used as an inexpensive single-use cell culture bag. It is.

(Cell culture device)
FIG. 12 is a diagram showing a configuration of the cell culture device 10 according to the embodiment of the present invention. The cell culture apparatus 10 includes a cell supply unit 100, a medium supply unit 110, a diluent supply unit 120, and a frozen solution supply unit 130. The cell culture device 10 includes a cell culture container 20, a storage container 30, a division processing unit 40, a waste liquid collection container 16, and a freezing unit 17.

  The cell culture device 10 stores the cells supplied from the cell supply unit 100 in the cell culture container 20 together with the medium (culture solution) supplied from the medium supply unit 110, and the cells are contained in the medium in the cell culture container 20. For example, the cells are cultured in a suspended state.

<Cell supply unit>
The cell supply unit 100 includes a cell storage unit 101 that stores cells to be cultured by the cell culture apparatus 10 in a frozen state, and a cell that is stored in the cell storage unit 101, including the pipe c1. And a pump P1 fed to the flow path F3. Moreover, the cell supply part 110 has the opening-and-closing valve V1 provided in the downstream of the pump P1 of the piping which connects the cell accommodating part 101 and the piping c1. The cells stored in the cell storage unit 101 are sent to the flow path F3 when the pump P1 is driven and the open / close valve V1 is opened.

  Cells to be cultured by the cell culture device 10 according to the present embodiment are not particularly limited, and are animal cells, plant cells, fungal cells, bacterial cells, protoplasts, established cell lines, artificially genetically modified. Any cell can be targeted, such as a treated cell.

  An example of a cell to be cultured is a stem cell. The stem cells are not particularly limited as long as they have self-replicating ability and differentiation ability, and may be pluripotent stem cells or somatic stem cells.

  A pluripotent stem cell is a cell having self-renewal ability and pluripotency capable of differentiating into any of ectoderm, mesoderm and endoderm. As pluripotent stem cells, embryonic stem cells (Embryonic Stem cells; ES cells), induced pluripotent stem cells (iPS cells), embryonic germ cells (Embryonic Germ cells; EG cells), embryonic cancers Cells (Embryonal Carcinoma cells; EC cells), pluripotent adult progenitor cells (MAP cells), adult pluripotent stem cells (Adult Pluripotent Stem cells; APS cells), Muse cells (Multi-lineage differentiating Stress Enduring) cell).

  Examples of somatic stem cells include mesenchymal stem cells, hematopoietic stem cells, neural stem cells and the like.

  Other examples of cells to be cultured are somatic cells constituting the living body and precursor cells thereof. Specifically, lymphocytes, neutrophils, monocytes, megakaryocytes, macrophages, fibroblasts, basal cells, keratinocytes, epithelial progenitor cells, pericytes, endothelial cells, adipose precursor cells, myoblasts, osteoblasts Cells, chondrocytes, hepatocytes, pancreatic β cells, glial cells, and the like.

  Other examples of cells to be cultured include animal cells such as CHO (chinese hamster ovary), COS, HeLa, HepG2, C127, 3T3, BHK, HEK293, Bowes melanoma cells; insect cells such as Drosophila S2 and Spodoptera Sf9; Fungal cells such as yeast and Aspergillus; bacterial cells such as Escherichia coli and Bacillus subtilis; plant cells and callus; These cells may be cells into which a protein expression vector has been introduced for the purpose of mass expression of proteins.

  Note that the cell supply unit 100 can be omitted from the cell culture apparatus 10 when cell culture is started in a state where cells and a medium are previously stored in the cell culture container 20.

<Medium supply unit>
The medium supply unit 110 includes medium storage units 111 and 114 that store a medium (culture solution) used for cell culture, and a pump P2 that sends the medium stored in the medium storage units 111 and 114 to the flow path F3. And P3, and filters 113 and 116 for sterilizing the media sent from the pumps P2 and P3, respectively. Further, the culture medium supply unit 110 includes an opening / closing valve V2 provided on the downstream side of the filter 113, a pipe connecting the culture medium storage unit 111 and the pipe c1, and a pipe connecting the culture medium storage unit 114 and the pipe c1. And an open / close valve V3 provided on the downstream side of the filter 116. Thus, the culture medium supply unit 110 according to this embodiment includes the first system including the culture medium storage unit 111, the pump P2, the filter 113, and the open / close valve V2, the medium storage unit 114, the pump P3, the filter 116, and the open / close valve. It has a two-line medium supply function consisting of a second line containing V3 and can supply two different kinds of mediums. Note that the number of lines in the medium supply unit 110 can be appropriately increased or decreased according to a cell culture protocol or the like. That is, the culture medium supply unit 110 may be configured to be able to supply three or more types of culture media, or may be configured to be capable of supplying one type of culture medium. The medium accommodated in the medium accommodating part 111 is sent to the flow path F3 when the pump P2 is driven and the open / close valve V2 is opened. The medium stored in the medium storage unit 114 is sent to the flow path F3 when the pump P3 is driven and the open / close valve V3 is opened.

  The medium that can be applied in the cell culture by the cell culture apparatus 10 according to the present embodiment is not particularly limited, and any medium can be applied. Specifically, basic medium for mammalian cells (for example, DMEM (Dulbecco's Modified Eagle's Medium), DMEM / F-12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), EMEM (Eagle's minimal essential medium), BME (Basal Medium Eagle), RPMI 1640 Medium (Roswell Park Memorial Institute 1640 Medium), E8 base medium, SkBM (Skeletal Muscle Cell Basal Medium), MCDB104, MCDB153, 199, L15), commercially available stem cell maintenance medium, insect It is a liquid medium such as a basic medium for cells, a medium for yeast, a medium for bacteria, and the like.

  The medium that can be applied in cell culture by the cell culture apparatus 10 according to the present embodiment is a polymer that does not have cytotoxicity for the purpose of continuously floating cells and / or preventing excessive adhesion between cells. A compound may be added. Examples of the polymer compound added to the medium for the above purpose include a polymer compound that adjusts the specific gravity of the medium, a polymer compound that adjusts the viscosity of the medium, and a polymer compound that forms a three-dimensional network structure in the medium. . Examples of such high molecular compounds include cellulose, methylcellulose, carboxymethylcellulose, gellan gum, deacylated gellan gum, hyaluronic acid, alginic acid, carrageenan, xanthan gum, starch, pectin and other polysaccharides; collagen, gelatin and other proteins Synthetic polymers such as polyethylene glycol and polyvinyl pyrrolidone; and the like.

  Medium that can be applied to cell culture by the cell culture device 10 according to the present embodiment includes various components that can be generally added, for example, antibiotics such as penicillin and streptomycin; vitamins or vitamin derivatives such as ascorbic acid and retinoic acid; glucose Sugar sources such as amino acids; inorganic salts; serum, serum substitutes; proteins such as transferrin; hormones such as insulin; growth factors; differentiation inhibitors; antioxidants such as 2-mercaptoethanol and dithiothreitol; Metal ions such as magnesium ions, zinc ions, iron ions, copper ions, etc. may be added.

<Diluent supply unit>
The diluent supply unit 120 includes a diluent storage unit 121 that stores a diluent used in a dilution process that is appropriately performed in the cell culture process. Further, the diluent supply unit 120 includes a pump P4 that sends the diluent stored in the diluent storage unit 121 to the flow path F3, and a filter 123 for sterilizing the diluent sent from the pump P4. . In addition, the diluent supply unit 120 includes an open / close valve V4 provided on the downstream side of the filter 123 in a pipe connecting the diluent storage part 121 and the pipe c1. The diluent stored in the diluent storage unit 121 is sent to the flow path F3 when the pump P4 is driven and the open / close valve V4 is opened.

  The diluent that can be used for cell culture by the cell culture device 10 according to the present embodiment is not particularly limited, and for example, a basic medium for mammalian cells (for example, DMEM, DMEM / F-12, MEM, DME, RPMI1640). MCDB104, 199, MCDB153, L15, SkBM, Basal Medium, E8 base medium) can be used as a diluent. In addition, when the dilution process is unnecessary in the cell culture process, the diluent supply unit 120 can be omitted.

<Frozen liquid supply unit>
The frozen solution supply unit 130 includes a frozen solution storage unit 131 that stores a frozen solution used when the cultured cells are cryopreserved in the freezing unit 17. In addition, the frozen liquid supply unit 130 includes a pump P5 for sending the frozen liquid stored in the frozen liquid storage part 131 to the flow path F3, and a filter 133 for sterilizing the frozen liquid sent from the pump P5. Have. In addition, the frozen liquid supply unit 130 includes an opening / closing valve V5 provided on the downstream side of the filter 133 in a pipe connecting the frozen liquid storage unit 131, the pump P5, and the filter 133. The frozen liquid stored in the frozen liquid storage part 131 is sent to the flow path F3 when the pump P5 is driven and the open / close valve V5 is opened. In addition, when it is not necessary to cryopreserve the cultured cells, it is possible to omit the frozen liquid supply unit 130 in the cell culture device 10.

<Cell culture container>
The cell culture container 20 is a container for storing the cells supplied from the cell supply unit 100 together with the medium supplied from the medium supply unit 110 and culturing the stored cells. As described above, the cell culture container 20 has a non-flat structure in which the inner wall surface 205 surrounding the accommodation space 203 for containing cells includes at least one of a plurality of concave portions and a plurality of convex portions (FIGS. 2, 3 and (See FIG. 7). The cell culture container 20 has an inlet 21 for allowing cells and culture medium to flow into the cell culture container 20 and an outlet for allowing cells and culture medium contained in the cell culture container 20 to flow out of the cell culture container 20. 22.

The cell culture vessel 20 can be accommodated in an incubator 24 that is controlled and sealed at, for example, a temperature of 30 ° C. to 40 ° C. (preferably 37 ° C.) and a CO ₂ concentration of 2% to 10% (preferably 5%). The incubator 24 includes a gas supply mechanism 25 for supplying oxygen (O ₂ ) and carbon dioxide (CO ₂ ) to cells accommodated in the cell culture container 20 together with the medium. The incubator 24 includes a pressure adjustment mechanism 26 that adjusts the pressure in the cell culture container 20. The pressure adjusting mechanism 26 pressurizes the atmosphere in the cell culture container 20 by introducing air into the cell culture container 20 or discharges the atmosphere in the cell culture container 20 to the outside. Free the atmosphere to the atmosphere. The pressure adjusting mechanism 26 increases the pressure in the cell culture vessel 20 to be higher than the pressure in the circulation channel F1, which will be described later, so that the cells and the medium stored in the cell culture vessel 20 flow out into the circulation channel F1. Let The cell culture container 20 has a distribution port for receiving carbon dioxide supplied from the gas supply mechanism 25, and a distribution port for performing intake and exhaust of air in the cell culture container 20, with the inlet 21 and the outlet 22. May be provided separately.

<Circulation channel>
The cell culture device 10 has a circulation flow path F <b> 1 including pipes a <b> 1 to a <b> 7 that connect the outlet 22 and the inlet 21 of the cell culture container 20. The cells and the medium accommodated in the cell culture container 20 circulate in the circulation channel F1 during the culture process. The cells and the medium flowing in the circulation channel F1 flow into the cell culture container 20 via the inflow port 21, and the cells and medium accommodated in the cell culture container 20 pass through the outflow port 22. It flows out into the circulation channel F1.

  An opening / closing valve V11 is provided in the piping a7 that constitutes the circulation channel F1 connected to the inlet 21 of the cell culture vessel 20, and the piping that constitutes the circulation channel F1 connected to the outlet 22 of the cell culture vessel 20 An opening / closing valve V12 is provided at a1. The on-off valve V11 is opened when cells and culture medium are allowed to flow into the cell culture container 20 from the circulation flow path F1, and is otherwise closed. The on-off valve V12 is opened when the cells and the medium are allowed to flow out from the cell culture container 20 into the circulation channel F1, and is otherwise closed.

  A flow path F3 constituted by the pipe c1 connected to the cell supply section 100, the culture medium supply section 110, the diluent supply section 120, and the frozen liquid supply section 130 is connected to the circulation flow path F1 at the connection site X3. That is, the cells stored in the cell storage unit 101, the culture medium stored in the culture medium storage units 111 and 114, the diluent stored in the diluent storage unit 121, and the frozen solution stored in the freezing solution storage unit 131, It is supplied into the circulation flow path F1 via the flow path F3 and the connection part X3.

  An opening / closing valve V6 is provided in the vicinity of the connection site X3 in the pipe c1 constituting the flow path F3. The on-off valve V6 is opened when cells, a medium, a diluent, and a frozen solution are supplied from the cell supply unit 100, the medium supply unit 110, the diluent supply unit 120, and the frozen solution supply unit 130, respectively, into the circulation channel F1. In other cases, it is closed.

<Storage container>
The storage container 30 is provided in the circulation channel F1, that is, in the middle of the circulation channel F1. The storage container 30 is a container for temporarily storing cells, medium, diluent and frozen liquid flowing in the circulation flow path F1, and will be described later during subculture, medium exchange, Used in splitting and freezing processes. The form of the storage container 30 is not particularly limited, and for example, a glass or stainless steel container or a container having a plastic bag form can be used.

  The storage container 30 includes an inlet 31 for allowing cells, medium, diluent and frozen liquid flowing in the circulation channel F1 to flow into the storage container 30, and cells, medium, diluent and An outlet 32 is provided for allowing the frozen liquid to flow into the circulation flow path F1. The inflow port 31 of the storage container 30 is connected to the outflow port 22 of the cell culture container 20 by pipes a1, a2, and a3 that constitute the circulation flow path F1. The outlet 32 of the storage container 30 is connected to the inlet 21 of the cell culture container 20 by pipes a4, a5, a6, and a7 that constitute the circulation flow path F1. Moreover, in this embodiment, although the connection part X3 to which the circulation flow path F1 and the flow path F3 are connected is arrange | positioned in the vicinity of the inflow port 31 of the storage container 30, the circulation flow path F1, the flow path F3, and These connection positions can be arranged at any position in the circulation flow path F1.

  An opening / closing valve V13 is provided in the vicinity of the inlet 31 of the storage container 30 in the pipe a2 constituting the circulation flow path F1. The on-off valve V13 is in an open state when cells, a medium, and the like are allowed to flow into the storage container 30 from the circulation channel F1, and is closed in other cases. An opening / closing valve V14 is provided in the vicinity of the outlet 32 of the storage container 30 in the pipe a5 that constitutes the circulation flow path F1. The on-off valve V14 is opened when cells, a medium, and the like are transferred from the storage container 30 to the circulation channel F1 to the cell culture container 20, the division processing unit 40, and the freezing unit 17, and otherwise. Closed.

  The storage container 30 includes a pressure adjustment mechanism 33 that adjusts the pressure in the storage container 30. The pressure adjustment mechanism 33 pressurizes the atmosphere in the storage container 30 by introducing air into the storage container 30 or discharges the atmosphere in the storage container 30 to the outside to discharge the atmosphere in the storage container 30 to the atmosphere. To release. The pressure adjustment mechanism 33 circulates the cells, the culture medium, the diluted solution, and the frozen solution stored in the storage container 30 from the outflow port 32 by increasing the pressure in the storage container 30 to be higher than the pressure in the circulation channel F1. It flows out into the flow path F1.

  The cell culture device 10 includes a connection part X1 located between the outlet 32 of the storage container 30 and the inlet 21 of the cell culture container 20 in the circulation channel F1, and the storage container 30 in the circulation channel F1. A flow path F2 including pipes b1 and b2 that connect the connection portion X2 located between the inflow port 31 and the outflow port 22 of the cell culture container 20. Cells, a medium, and the like flowing in the circulation flow path F1 can flow into the flow path F2 via the connection site X1. In addition, cells, culture medium, and the like flowing in the flow path F2 can flow into the circulation flow path F1 via the connection portion X2.

<Division processing unit>
The division processing unit 40 is provided in the flow path F2, that is, in the middle of the flow path F2. The division processing unit 40 includes a processing container 42 for performing a division process for dividing a cell mass generated by culturing cells in the cell culture container 20. The dividing process performed in the processing container 42 may be a mechanical dividing process or an enzyme process using a cell dissociating enzyme. When mechanical division processing is applied, a mesh filter (not shown) may be disposed in the processing container 42. By passing the cell mass through the mesh filter, the cell mass is divided into sizes according to the mesh size of the mesh filter. On the other hand, when a division treatment by an enzyme treatment is applied, a cell dissociation enzyme such as trypsin-EDTA (ethylenediaminetetraacetic acid) can be accommodated in the treatment container 42. The cell mass is divided by immersing the cell mass in a cell dissociation enzyme for a certain time.

  The division processing unit 40 divides the cell mass flowing into the flow path F2 from the circulation flow path F1 via the connection portion X1 in the processing container 42. The cells subjected to the division process flow out into the circulation channel F1 via the connection part X2.

  The division processing unit 40 includes a pressure container 41 that communicates with the processing container 42, and a pressure adjustment mechanism 43 that adjusts the pressure in the pressure container 41 and the processing container 42. The pressure adjustment mechanism 43 pressurizes the atmosphere in the pressure vessel 41 and the processing vessel 42 by introducing air into the pressure vessel 41, or discharges the atmosphere in the pressure vessel 41 to the outside. The atmosphere in the processing container 42 is released to the atmosphere. The pressure adjusting mechanism 43 causes the cells subjected to the division process to flow out into the circulation channel F1 by increasing the pressure in the pressure vessel 41 and the processing vessel 42 to be higher than the pressure in the circulation channel F1.

  An open / close valve V21 is provided in the vicinity of the connection portion X1 in the pipe b1 that is disposed on the upstream side of the division processing unit 40 and that constitutes the flow path F2. The on-off valve V21 is opened when cells or the like are transferred from the storage container 30 to the division processing unit 40, and is closed otherwise. On the other hand, an opening / closing valve V22 is provided in the vicinity of the connection site X2 in the pipe b2 that is disposed on the downstream side of the division processing unit 40 and that constitutes the flow path F2. The on-off valve V22 is opened when the cells subjected to the division process by the division processing unit 40 are allowed to flow out into the circulation flow path F1, and is otherwise closed.

  On the pipe a1 constituting the circulation flow path F1, an opening / closing valve V15 is provided in the vicinity of the connection site X2 and upstream of the connection site X2. The on-off valve V15 is opened when cells and culture medium are transferred from the cell culture container 20 to the storage container 30, and is otherwise closed.

<Waste liquid collection container>
The cell culture device 10 is between the outflow port 32 of the storage container 30 and the inflow port 21 of the cell culture container 20 in the circulation flow path F1 and on the upstream side (near the storage container 30) from the connection site X1. In the connection part X4 located, it has the flow path F4 comprised including the piping d1 connected to the circulation flow path F1. A waste liquid collection container 16 is provided at the end of the flow path F4. The waste liquid recovery container 16 is a container for recovering the waste liquid flowing into the flow path F4 from the circulation flow path F1 via the connection part X4. The waste liquid collected in the waste liquid collection container 16 includes a used medium, a used diluent, a frozen liquid accompanying the cells supplied in a frozen state from the cell supply unit 100, and the like. The form of the waste liquid collection container 16 is not particularly limited. For example, a glass or stainless steel container or a container having a plastic bag form can be used.

  An opening / closing valve V31 is provided in the vicinity of the connection site X4 in the pipe d1 constituting the flow path F4. The on-off valve V31 is opened when the waste liquid flowing out from the storage container 30 is collected in the waste liquid collection container 16, and is closed otherwise.

<Frozen part>
The cell culture device 10 has a flow path F5 configured to include a pipe e1 connected to the circulation flow path F1 at the connection site X1. The freezing part 17 is provided in the edge part of the flow path F5. The freezing unit 17 includes a storage container 17a that stores the cells flowing into the flow path F5 from the circulation flow path F1 via the connection portion X1 together with the freezing liquid supplied from the freezing liquid supply unit 130. The storage container 17a may have a form of, for example, a vial, a cryotube, or a bag. The freezing unit 17 may be configured to include a freezer that freezes cells stored in the storage container 17a and a frozen solution. Moreover, the freezing part 17 may be provided with the tank filled with liquid nitrogen, and may be comprised so that the storage container 17a can be accommodated in a tank. Moreover, the freezing part 17 may be comprised including the cryo-library (trademark) system made from Taiyo Nippon Sanso, for example. An open / close valve V41 is provided in the vicinity of the connection site X1 in the pipe e1 constituting the flow path F5. The on-off valve V41 is opened when the cells and the frozen liquid are transferred from the storage container 30 to the freezing unit 17, and is otherwise closed. The connection position between the flow path F5 and the circulation flow path F1 may be any position as long as it is between the outlet 32 of the storage container 30 and the inlet 21 of the cell culture container 20. Further, when it is not necessary to cryopreserve the cells, the freezing unit 17 can be omitted.

<Control unit>
The control unit 18 comprehensively controls the operations of the pumps P1 to P5, the open / close valves V1 to V5, V11 to V16, V21, V22, V31 and V41, the gas supply mechanism 25, and the pressure adjustment mechanisms 26, 33 and 43. As a result, cell culture in accordance with a predetermined cell culture protocol is automatically performed without human intervention. In FIG. 12, the electrical connection wiring between the control unit 18 and each of the above-described components controlled by the control unit 18 is omitted from the viewpoint of avoiding the complexity of the drawing.

<Piping>
FIG. 13 is a cross-sectional view showing an example of the internal structure of each of the pipes a1 to a7, b1, b2, c1, d1 and e1 constituting the circulation flow path F1 and the flow paths F2 to F5. As shown in FIG. 13, the inner wall surface 400 of the pipes a1 to a7, b1, b2, c1, d1, and e1 includes a plurality of concave portions 210 or a plurality of convex portions 220 formed on the inner wall surface 205 of the cell culture vessel 20. It may have a non-flat structure similar to the non-flat structure included. That is, on the inner wall surface 400 of each of the pipes a1 to a7, b1, b2, c1, d1, and e1, at least one of a plurality of concave portions or a plurality of convex portions is an inner wall surface of a cell cultured in the cell culture vessel 20 It may be provided with a size and an interval that inhibit adhesion to 400. Thereby, adhesion to the inner wall surface 400 of the cell which distribute | circulates the inside of piping a1-a7, b1, b2, c1, d1, and e1 is inhibited, and fusion of the cell clusters which distribute | circulate the inside of each piping is suppressed. Moreover, it can prevent that a cell remains inside piping a1-a7, b1, b2, c1, d1, and e1. The materials of the pipes a1 to a7, b1, b2, c1, d1 and e1 are not particularly limited. However, when the entire cell culture apparatus 10 is used as a single use, the pipes a1 to a7, b1, b2, c1, d1 and e1 are preferably made of an inexpensive material such as plastic.

  The inlet 21 of the cell culture container 20 is an example of the first inlet in the present invention. The outlet 22 of the cell culture container 20 is an example of a first outlet in the present invention. The storage container 30 is an example of the storage container in the present invention. The inlet 31 of the storage container 30 is an example of a second inlet in the present invention. The outlet 32 of the storage container 30 is an example of a second outlet in the present invention. The circulation channel F1 is an example of the first channel in the present invention. The flow path F2 is an example of the second flow path in the present invention. Connection part X1 is an example of the 1st part in the present invention. The connection site X2 is an example of a second site in the present invention. The division processing unit 40 is an example of a division processing unit in the present invention. The culture medium supply unit 110 is an example of a culture medium supply unit in the present invention.

  Below, an example of the process which can be implemented in the cell culture apparatus 10 which concerns on this embodiment is shown. The cell culture device 10 performs, for example, a passage process, a medium exchange process, a division process, and a freezing process exemplified below. In the subculture process, the medium exchange process, the division process, and the freezing process described below, the control unit 18 uses the open / close valves V1 to V5, V11 to V16, V21, V22, V31 and V41, the pumps P1 to P5, and the pressure. This is realized by controlling the operations of the adjusting mechanisms 26, 33 and 43.

<Passage processing>
The cell culturing apparatus 10 carries out the subculture process in which the cells accommodated in the cell accommodating part 101 are accommodated in the cell culture container 20 together with the medium accommodated in the medium accommodating parts 111 and 114 and cell culture is started as follows. To implement. In the following description, a case where the division process in the division processing unit 40 is a mechanical division process will be exemplified. FIG. 14 is a diagram illustrating the flow of cells, culture medium, and the like when the cell culture device 10 performs the passage process. FIG. 14 shows the correspondence between the flow of cells and culture medium, etc., and the following processing steps.

  In step S1, the on-off valves V1, V4 and V6 are opened, and the pumps P1 and P4 are driven. As a result, the cells stored in the cell storage unit 101 in a frozen state and the diluent stored in the diluent storage unit 121 flow into the storage container 30 via the flow path F3 and the circulation flow path F1.

  In step S <b> 2, a concentration process is performed to remove the frozen solution and the diluted solution from the mixture stored in the storage container 30, the frozen solution and the diluted solution accompanying the cells. The concentration process is performed, for example, by causing the cells stored in the storage container 30 together with the diluent to settle in the storage container 30 (natural sedimentation), and removing the supernatant liquid containing the diluent and the frozen liquid. . Specifically, after the cells are settled in the storage container 30, the open / close valve V14 is opened for a short period of time. As a result, while leaving the supernatant liquid in the storage container 30, the cells are allowed to flow out of the storage container 30 and stay in the pipe a5. Thereafter, the open / close valve V14 is closed and the open / close valve V31 is opened. As a result, the waste liquid containing the diluent and the frozen liquid remaining in the storage container 30 flows out from the outlet 32 of the storage container 30 and is recovered in the waste liquid recovery container 16 via the flow path F4. Note that the transfer of cells from the storage container 30 to the pipe a5 and the transfer of the diluent and the frozen liquid from the storage container 30 to the waste liquid collection container 16 are performed by pressurizing the atmosphere in the storage container 30 by the pressure adjustment mechanism 33. Is called.

  In step S3, the on-off valves V2, V3, and V6 are opened, and the pumps P2 and P3 are driven. Thereby, the culture medium A and the culture medium B accommodated in the culture medium accommodating parts 111 and 114 flow in in the storage container 30 via the flow path F3 and the circulation flow path F1. Thereafter, the open / close valve V14 is opened, and the mixed medium composed of the medium A and the medium B flows out of the storage container 30 and joins the cells staying in the pipe a5.

  In step S4, the open / close valves V14, V16 and V21 are opened. Further, the atmosphere in the storage container 30 is pressurized by the pressure adjusting mechanism 33. As a result, the cells and the medium that stay in the pipe a <b> 5 flow into the flow path F <b> 2 via the connection part X <b> 1 and flow into the division processing unit 40. The cells that have flowed into the division processing unit 40 are subjected to division processing in the processing container 42. The division process here is performed for the purpose of crushing the frozen cells.

  In step S5, the on-off valves V22 and V13 are opened. The atmosphere in the pressure vessel 41 and the processing vessel 42 is pressurized by the pressure adjustment mechanism 43. As a result, the cells that have undergone the division process flow out into the circulation flow path F1 through the connection portion X2 together with the culture medium, and are transferred into the storage container 30.

  In step S6, the opening / closing valves V14, V16 and V11 are opened. Further, the atmosphere in the storage container 30 is pressurized by the pressure adjusting mechanism 33. Thereby, the cells and culture medium stored in the storage container 30 flow into the cell culture container 20 via the circulation channel F1. The passaging process is completed through the above steps S1 to S6.

  In the above example, the concentration process and the supply of a new medium are performed before the division process. However, the concentration process and the supply of a new medium may be performed after the division process.

<Medium medium replacement process>
In cell culture, the medium is altered by metabolites secreted from the cells. Therefore, a medium replacement process is required to replace the used medium in the cell culture container 20 with a new medium at an appropriate time within the culture period. The cell culture device 10 according to the present embodiment performs the above-described medium exchange process as follows. FIG. 15 is a diagram illustrating a flow of cells, a medium, and the like when the cell culture device 10 performs a medium exchange process. Note that FIG. 15 shows the correspondence between the flow of cells and culture medium and the following processing steps.

  In step S11, the on-off valves V12, V15, and V13 are opened. Further, the atmosphere in the cell culture container 20 is pressurized by the pressure adjusting mechanism 26. Thereby, the cells accommodated in the cell culture container 20 and the used medium flow out into the circulation channel F1 and are transferred into the storage container 30.

  In step S12, a concentration process is performed to remove the used medium from the mixture containing the cells stored in the storage container 30 and the used medium. The concentration process is performed in the same procedure as the process in step S2 of the passage process described above. By the concentration treatment, the used medium is collected in the waste liquid collection container 16, and the cells stay in the pipe a5. In order to promote cell sedimentation, a diluent may be introduced into the storage container 30.

  In step S13, the on-off valves V2, V3 and V6 are opened, and the pumps P2 and P3 are driven. Thereby, the new culture medium A and the new culture medium B accommodated in the culture medium accommodating parts 111 and 114 flow in in the storage container 30 via the flow path F3 and the circulation flow path F1. Thereafter, the open / close valve V14 is opened, and a new medium including the medium A and the medium B flows out of the storage container 30 and merges with the cells staying in the pipe a5.

  In step S14, the open / close valves V14, V16 and V11 are opened. Further, the atmosphere in the storage container 30 is pressurized by the pressure adjusting mechanism 33. Thereby, the cells staying in the pipe a <b> 5 and the new medium flow into the cell culture container 20. The cells accommodated in the cell culture container 20 are cultured with a new medium. The medium replacement process is completed through the processes in steps S11 to S14 described above.

<Division processing (mechanical division processing)>
For example, in culturing pluripotent stem cells, if the size of the cell mass produced by culturing the cells becomes excessive, the cell masses adhere and fuse together, and the cells start to differentiate, or the cells in the center of the cell mass Can cause problems such as necrosis. Therefore, in order to prevent the size of the cell mass from becoming excessive, a division process for dividing the cell mass may be required at an appropriate time during the culture period. The cell culture device 10 according to the present embodiment performs the above division process as follows. In the following description, a case where the division process in the division processing unit 40 is a mechanical division process will be exemplified. FIG. 16 is a diagram illustrating a flow of cells, a medium, and the like when the cell culture device 10 performs the division process. In FIG. 16, the correspondence between the flow of cells, culture medium, and the like and each processing step shown below is shown.

  In step S21, the on-off valves V12, V15, and V13 are opened. Further, the atmosphere in the cell culture container 20 is pressurized by the pressure adjusting mechanism 26, and the cell mass and the used medium generated in the cell culture container 20 flow out into the circulation flow path F1 and enter the storage container 30. Be transported

  In step S <b> 22, a concentration process is performed to remove the used medium from the mixture containing the cell mass and the used medium stored in the storage container 30. The concentration process is performed in the same procedure as the process in step S2 of the above-described passaging process. By the concentration treatment, the used medium is recovered in the waste liquid recovery container 16, and the cell mass stays in the pipe a5. In order to promote cell sedimentation, a diluent may be introduced into the storage container 30.

  In step S23, the on-off valves V2, V3, and V6 are opened, and the pumps P2 and P3 are driven. Thereby, the new culture medium A and the new culture medium B accommodated in the culture medium accommodating parts 111 and 114 flow in in the storage container 30 via the flow path F3 and the circulation flow path F1. Thereafter, the open / close valve V14 is opened, and a new medium including the medium A and the medium B flows out of the storage container 30 and merges with the cell mass remaining in the pipe a5.

  In step S24, the open / close valves V14, V16 and V21 are opened. Further, the atmosphere in the storage container 30 is pressurized by the pressure adjusting mechanism 33. Thereby, the cell mass and culture medium which remain | survive in the piping a5 flow in in the flow path F2 via the connection part X1, and flow in in the division | segmentation process part 40. FIG. The cells that have flowed into the division processing unit 40 are subjected to division processing in the processing container 42.

  In step S25, the on-off valves V22 and V13 are opened. The atmosphere in the pressure vessel 41 and the processing vessel 42 is pressurized by the pressure adjusting mechanism 43. As a result, the cells that have undergone the division process flow out into the circulation flow path F1 through the connection portion X2 together with the culture medium, and are transferred into the storage container 30.

  In step S26, the open / close valves V14, V16 and V11 are opened. Further, the atmosphere in the storage container 30 is pressurized by the pressure adjusting mechanism 33. As a result, the cells that have been divided and stored in the storage container 30 and the new medium flow into the cell culture container 20. In the cell culture container 20, the culture of the cells subjected to the division process is continued. The division process is completed through the processes in steps S21 to S26 described above.

  In the above example, the concentration process and the supply of a new medium are performed before the division process. However, the concentration process and the supply of a new medium may be performed after the division process.

<Division treatment (enzyme treatment)>
FIG. 17 is a diagram illustrating a flow of cells, a medium, and the like when the cell culture device 10 performs the division process when the division process in the division processing unit 40 includes the enzyme process using the cell dissociation enzyme described above. is there. Note that FIG. 17 shows the correspondence between the flow of cells and culture medium, etc., and the following processing steps.

  Since the processing of steps S31 to S33 in FIG. 17 is the same as the processing of steps S21 to S23 described above, a duplicate description is omitted.

  In step S34, the open / close valves V14, V16 and V21 are opened. Further, the atmosphere in the storage container 30 is pressurized by the pressure adjusting mechanism 33. Thereby, the cell mass and culture medium which remain | survive in the piping a5 flow in in the flow path F2 via the connection part X1, and flow in in the division | segmentation process part 40. FIG. The cells that have flowed into the division processing unit 40 are subjected to a division process using a cell dissociating enzyme in the processing container 42.

  In step S35, the open / close valves V22 and V13 are opened. The atmosphere in the pressure vessel 41 and the processing vessel 42 is pressurized by the pressure adjustment mechanism 43. As a result, the cells that have been subjected to the division treatment flow out into the circulation channel F1 through the connection site X2 together with the medium containing the cell dissociation enzyme, and are transferred into the storage container 30.

  In step S36, the on-off valves V4 and V6 are opened, and the pump P4 is driven. Thereby, the diluent stored in the diluent storage part 121 flows into the storage container 30 via the flow path F3 and the circulation flow path F1. The cell dissociation action by the cell dissociation enzyme is stopped by adding the diluent to the medium containing the cells and cell dissociation enzyme stored in the storage container 30.

  In step S <b> 37, a concentration process is performed in which the medium stored in the storage container 30, the medium containing the cell dissociation enzyme and the mixture containing the diluent are removed from the mixture containing the cell dissociation enzyme and the diluent. The concentration process is performed in the same procedure as the process in step S2 of the above-described passaging process. By the concentration treatment, the medium containing the cell dissociation enzyme and the diluted solution are collected in the waste liquid collection container 16, and the cells stay in the pipe a5.

  In step S38, the on-off valves V2, V3, and V6 are opened, and the pumps P2 and P3 are driven. Thereby, the new culture medium A and the new culture medium B accommodated in the culture medium accommodating parts 111 and 114 flow in in the storage container 30 via the flow path F3 and the circulation flow path F1. Thereafter, the open / close valve V14 is opened, and a new medium including the medium A and the medium B flows out of the storage container 30 and merges with the cells staying in the pipe a5.

  In step S39, the on-off valves V14, V16 and V11 are opened. Further, the atmosphere in the storage container 30 is pressurized by the pressure adjusting mechanism 33. Thereby, the cell and the new culture medium in which the division process staying in the pipe a <b> 5 is performed flow into the cell culture container 20. In the cell culture container 20, the culture of the cells subjected to the division process is continued. The division process is completed through the processes in steps S31 to S39.

  In the above example, the concentration process and the supply of a new medium are performed before the division process. However, the concentration process and the supply of a new medium may be performed after the division process.

<Freezing process>
When the cultured cells are collected and stored, the cells are generally collected in a storage container and stored frozen. The cell culture device 10 according to the present embodiment performs a freezing process for collecting and freezing the cultured cells as follows. FIG. 18 is a diagram illustrating a flow of cells, a medium, and the like when the cell culture device 10 performs a freezing process. In FIG. 18, the correspondence between the flow of cells and culture medium and the following processing steps is shown.

  In step S41, the on-off valves V12, V15, and V13 are opened. Further, the atmosphere in the cell culture container 20 is pressurized by the pressure adjusting mechanism 26. Thereby, the cells accommodated in the cell culture container 20 and the used medium flow out into the circulation flow path F1 and are transferred into the storage container 30.

  In step S42, a concentration process for removing the used medium from the mixture containing the cells stored in the storage container 30 and the used medium is performed. The concentration process is performed in the same procedure as the process in step S2 of the above-described passaging process. By the concentration treatment, the used medium is collected in the waste liquid collection container 16, and the cells stay in the pipe a5. In order to promote cell sedimentation, a diluent may be introduced into the storage container 30.

  In step S43, the on-off valves V5 and V6 are opened, and the pump P5 is driven. Thereby, the frozen liquid stored in the frozen liquid storage part 131 flows into the storage container 30 via the flow path F3 and the circulation flow path F1. Thereafter, the open / close valve V14 is opened, and the frozen liquid flows out of the storage container 30 and merges with the cells staying in the pipe a5.

  In step S44, the open / close valves V14, V16 and V41 are opened. Further, the atmosphere in the storage container 30 is pressurized by the pressure adjusting mechanism 33. Thereby, the cells and the frozen liquid staying in the pipe a5 are accommodated in the storage container 17a of the freezing part 17 via the flow path F5. The freezing unit 17 freezes the cells accommodated in the storage container 17a together with the freezing solution. The division process is completed through the processes in steps S41 to S44 described above.

  As a method for cryopreserving cells, for example, the method described in Japanese Patent No. 4705473 may be applied. This method includes the step of rapidly freezing the cells in a medium containing a predetermined amount of dimethyl sulfoxide (DMSO), propylene glycol, acetamide and a medium. Further, the method described in Japanese Patent No. 4223961 may be applied. This method is a method for cryopreserving cells using a cryopreservation solution containing at least one selected from the group consisting of dimethyl sulfoxide, glycerin, ethylene glycol, propylene glycol, and polyvinylpyrrolidone as a cryoprotectant at a predetermined concentration. A step of suspending cells in a cryopreservation solution, a cooling step of cooling the cryopreservation solution to −80 ° C. or less at a predetermined cooling rate, and a preservation step of storing the cryopreservation solution at −80 ° C. or less. including.

  In addition, although the case where two types of medium A and medium B are supplied from the medium supply unit 110 is illustrated in the above passage process, medium exchange process, and division process, the type of the medium used is the cell culture It can be appropriately changed according to the protocol. That is, the medium to be used may be one type or three or more types according to the culture protocol.

<Cell culture treatment>
The cell culture device 10 can automatically perform cell culture without human intervention by the control unit 18 executing a cell culture processing program exemplified below. FIG. 19 is a flowchart showing the flow of processing in the cell culture program executed by the control unit 18.

  In step S <b> 101, the control unit 18 stores the cells supplied from the cell supply unit 100 and the medium supplied from the medium supply unit 110 in the cell culture container 20 by executing the above-described passage process. Start culture.

  In step S102, the control unit 18 stores the used medium in the cell culture container 20 by storing the medium in the cell culture container 20 by performing the medium replacement process (first time) after a lapse of a predetermined period from the start of cell culture. It replaces | exchanges for the new culture medium accommodated in the parts 111 and 114, and a cell culture is continued.

  In step S <b> 103, the control unit 18 performs the medium replacement process (second time) after a predetermined period has elapsed since the first medium replacement process, thereby performing the used medium in the cell culture container 20. Is replaced with a new medium accommodated in the medium accommodating part 111 and 114, and the cell culture is continued.

  In step S104, the control unit 18 continues the cell culture by dividing the cell mass by performing the above-described division processing after a predetermined period of time has elapsed since the second medium exchange processing.

  In step S105, the control unit 18 determines whether or not the number of culture cycles in which the processing in steps S102 to S104 described above is one cycle has reached a predetermined number. When it is determined that the number of culture cycles has not reached the predetermined number, the control unit 18 returns the process to step S102. On the other hand, if the control unit 18 determines that the number of culture cycles has reached a predetermined number, the process proceeds to step S106. As the culture cycle progresses, the cell culture scale increases.

  In step S106, the control unit 18 stores the cells cultured by performing the above-described freezing process in the storage container 17a of the freezing unit 17 and cryopreserves them.

  In the above example, the medium exchange process is performed twice in one cycle of the culture cycle. However, the number of times of performing the medium exchange process in one cycle of the culture cycle can be changed as appropriate. .

  As is clear from the above description, the cell culture device 10 according to the present embodiment has the circulation flow path F1 that connects the inlet 21 and the outlet 22 of the cell culture container 20. Moreover, the cell culture apparatus 10 has the storage container 30 provided in the circulation flow path F1. The storage container 30 has an inlet 31 connected to the outlet 22 of the cell culture container 20 and an outlet 32 connected to the inlet 21 of the cell culture container 20. The storage container 30 is used for the concentration process performed in the above passage process, medium exchange process, division process and freezing process. In addition, the cell culture device 10 includes the connection part X1 in the circulation channel F1 positioned between the outlet 32 of the storage container 30 and the inlet 21 of the cell culture container 20, the inlet 31 of the storage container 30, and the cells. It has the flow path F2 which connects the connection site | part X2 in the circulation flow path F1 located between the outflow port 22 of the culture container 20. FIG. The cell culture device 10 is provided in the flow path F2, divides the cell mass flowing from the circulation flow path F1 via the connection part X1, and divides the divided cell mass via the connection part X2. It has the division | segmentation process part 40 made to flow out in F1. The cell culture device 10 also has a medium supply unit 110 that supplies a medium into the circulation flow path F1.

  By configuring the cell culture device 10 as described above, it is possible to continuously perform a series of processes required for cell culture such as a medium exchange process and a division process in a closed system. This enables mass production of homogeneous cells. According to the cell culture device 10 according to the present embodiment, it is possible to perform a series of processes necessary for cell culture from the passaging process to the freezing process without intervention of human hands.

  Moreover, according to the cell culture apparatus 10 which concerns on this embodiment, the transfer of the cell from the cell culture container 20 to the storage container 30, the transfer of the cell from the storage container 30 to the division | segmentation process part 40 and the cell culture container 20, and a division | segmentation process Transfer of cells from the unit 40 to the storage container 30 is performed by pressure feeding by the pressure adjusting mechanisms 26, 33 and 43. As a result, damage to cells can be reduced compared to the case of transferring cells using a wet pump such as a diaphragm pump, tube pump, peristaltic pump, and Mono pump, and the viability of the cells can be reduced. It becomes possible to suppress. In the cell culture device 10 according to the present embodiment, the control unit 18 includes the open / close valves V1 to V5, V11 to V16, V21, V22, V31 and V41, the pumps P1 to P5, and the pressure adjusting mechanisms 26, 33 and 43. Although the case where the process from the passaging process to the freezing process is automated by controlling the operation is illustrated, the present invention is not limited to this mode. The user may manually operate the on-off valves V1 to V5, V11 to V16, V21, V22, V31 and V41, the pumps P1 to P5, and the pressure adjusting mechanisms 26, 33 and 43.

  Moreover, according to the cell culture device 10 according to the present embodiment, the inner wall surface 205 of the cell culture container 20 has a non-flat structure including at least one of a plurality of concave portions and a plurality of convex portions. It becomes possible to suppress the fusion of the cell masses floating in the accommodation space 203. As a result, cell necrosis can be suppressed, and in the culture of pluripotent stem cells, cell differentiation can be suppressed, and the culture efficiency can be improved as compared with the conventional case. In addition, the inner wall surface 400 of each of the pipes a1 to a7, b1, b2, c1, d1, and e1 constituting the circulation channel F1 and the channels F2 to F5 is a non-flat surface formed on the inner wall surface 205 of the cell culture container 20. Since it has a non-flat structure similar to the structure, adhesion of cells flowing through the pipes a1 to a7, b1, b2, c1, d1 and e1 to the inner wall surface 400 is inhibited, and the cell mass flowing through each pipe Fusion between each other is suppressed. Moreover, it can prevent that a cell remains inside piping a1-a7, b1, b2, c1, d1, and e1. Note that a non-flat structure similar to the non-flat structure formed on the inner wall surface 205 of the cell culture container 20 may also be formed on the inner wall surface of the storage container 30. Thereby, the adhesion of the cells to the inner wall surface of the storage container 30 is inhibited, and the fusion of the cell clusters stored in the storage container 30 is suppressed.

DESCRIPTION OF SYMBOLS 10 Cell culture apparatus 17 Freezing part 20 Cell culture container 21 Inlet 22 Outlet 30 Reservoir 31 Inlet 32 Outlet 33, 43 Pressure adjustment mechanism 40 Division processing part 42 Processing container 100 Cell supply part 110 Medium supply part 203 Storage space 205 Inner wall surface 210 Concave portion 220 Convex portion F1 Circulating flow path F1
F2-F7 flow path X1-X4 connection part

Claims (2)

A non-flat structure having a first inflow port, a first outflow port, and an inner wall surface that forms an accommodation space for accommodating cells, wherein the inner wall surface includes at least one of a plurality of concave portions and a plurality of convex portions. A cell culture vessel having
A first flow path connecting the first outlet and the first inlet;
A storage container that is provided in the first flow path and stores cells to be cultured in the cell culture container, the second inlet connected to the first outlet, and the first A first storage container having a second outlet connected to the inlet of
A first portion in the first flow path located between the second outlet and the first inlet; and between the second inlet and the first outlet. A second channel connecting the second part in the first channel positioned,
Division process for dividing a cell mass that is provided in the second flow channel and flows from the first flow channel via the first part and that is a cell mass cultured in the cell culture container A dividing unit that causes the cells subjected to the dividing process to flow out into the first flow path via the second part;
A medium supply unit for supplying a medium into the first flow path;
Further comprising a cell culture device. A pipe that forms the first flow path;
The cell culture device according to claim 1, wherein an inner wall surface of the pipe has a non-flat structure including at least one of a plurality of concave portions and a plurality of convex portions.