JP6354219B2 - Cell culture equipment - Google Patents

Description

  The present invention relates to a cell culture apparatus for perfusion culture of cells on the surface of a scaffold in a culture tank to which a culture solution is continuously supplied.

  In the fields of pharmaceuticals and regenerative medicine, there is a growing need for mass culture of animal cells. Since many animal cells have a strong property of adhering to a solid surface, culture using a scaffold in a petri dish as an attachment surface is generally performed. However, it is difficult to increase the size of the petri dish for mass culture, and mass culture cannot be expected with a general animal cell culture method.

  Therefore, it is aimed to culture a large amount of animal cells by liquid culture. Examples of methods for culturing animal cells by liquid culture include suspension culture, (micro) carrier culture, and perfusion culture.

  Among these, suspension culture is a method in which the property of being easily attached to the solid surface of animal cells is lost by a chemical added to the culture solution so that the animal cells are suspended in the culture solution (for example, Non-patent document 1). Moreover, (micro) carrier culture | cultivation is made to flow in a culture solution with the microcarriers to which the cell was attached (for example, nonpatent literature 2). Further, in perfusion culture, a culture solution is perfused to animal cells attached to the surface of a fixed scaffold (for example, Non-Patent Document 3).

Olmer, R. et al. (2010) Long term expansion of undifferentiated human iPS and ES cells in suspention culture using a defined medium.Stem Cell Research, Vol.5, p.51-64. Phillips, BW, Horne, R., Lay, TS, Rust, WL, Teck, TT, Crook, JM (2008) Attachment and growth of human embryonic stem cells on microcarriers. Journal of Biotechnology, Vol.138, p.24- 32. Janssen, FW, Oostra, J., van Ooschot, A, and van Blitterswijk, CA (2006) A perfusion bioreactor system capable of producing clinically relevant volumes of tissue-engineered bone: In vivo bone formation showing proof of concept.Biomaterials, Vol .27, p.315-323.

  However, suspension culture is not easy to implement because it requires advanced techniques for the composition of the culture solution and the selection of chemicals to be added to the culture solution. In addition, since carrier culture is agitated so that the carrier does not settle in the culture solution, a strong shear force is applied to animal cells that are weak in shear force. Implementation is not easy. In contrast, perfusion culture does not require the addition of chemicals or agitation to the culture medium, so it can be said that the technical obstacles to implementation are low compared to suspension culture and carrier culture.

  Here, when culturing animal cells by perfusion culture, it is necessary to keep the scaffold in a fixed state in the environment in which the suspension is perfused. For that purpose, the scaffold has a strength that can withstand the perfusion of the suspension. It is necessary to make it. In addition, it is important to increase the specific surface area of the scaffold in order to mass-cultivate animal cells. In addition, the task of collecting animal cells cultured from the culture tank must be easy.

  In this way, even in perfusion culture with low technical obstacles to implementation, in practice, the strength of the scaffold and the increase in the ratio of the surface area of the scaffold (specific surface area) on which the animal cells can breed relative to the volume of the culture tank It is difficult to achieve both. For this reason, it was not easy to culture animal cells in large quantities by perfusion culture. Similarly, when culturing cells other than animal cells, it is not easy to culture cells in large quantities by perfusion culture.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a cell culture apparatus capable of culturing a large amount of cells by a perfusion culture method and collecting the cultured cells in a short time.

In order to achieve the above object, the cell culture device of the present invention described in claim 1 comprises:
A cell culture device for perfusion culture of cells,
A culture tank capable of continuously supplying a culture solution;
A scaffold for cell culture provided in the culture tank,
The scaffold is
A plurality of hollow tubes whose passages penetrate the interior;
A support body that supports the plurality of hollow tubes, along the direction of passage of the passage in the flow direction of the culture medium in the culture tank, and arranged in parallel in a direction perpendicular to the direction of penetration.
have,
It is characterized by that.

  According to the cell culture device of the present invention described in claim 1, the penetration direction of the passage of each hollow tube supported by the support is along the flow direction of the culture solution continuously supplied to the culture tank. Therefore, the culture solution flows in the culture tank through the passage inside the hollow tube. For this reason, the inner peripheral surface of each hollow tube can be functioned as a cell scaffold, and the surface area of the scaffold can be increased efficiently. Moreover, since the hollow tube used for the scaffold has a tubular structure, the strength of the scaffold can be maintained high. Therefore, it is possible to achieve both the maintenance of the strength of the scaffold and the increase of the specific surface area of the scaffold.

  Further, by increasing the flow rate of the culture solution, it is possible to detach the cells that are attached to the hollow tube and cultured. Alternatively, the hollow tube can be extracted from the culture tank together with the support, and the hollow tube can be taken out of the culture tank. For this reason, cells can be collected from the culture tank in a short time.

  Therefore, the specific surface area of the scaffold can be increased, and the cells can be cultured in a large amount by the perfusion culture method, and the cultured cells can be collected in a short time.

  Further, the cell culture device of the present invention described in claim 2 is the cell culture device of the present invention described in claim 1, wherein the support is adjacent to the two hollows in a direction orthogonal to the penetration direction. It contains an adhesive that bonds the tubes together.

  According to the cell culture device of the present invention described in claim 2, in the cell culture device of the present invention described in claim 1, a plurality of hollow tubes are bonded by adhering adjacent hollow tubes with an adhesive. It is possible to realize a structure in which the tube body can be integrally extracted from the culture tank.

Further, the cell culture apparatus of the present invention according to claim 1, before Symbol support includes a mesh member which is deployed out movable to and installed in the culture tank in a direction perpendicular to the flow direction, It characterized in that the net-body said in front Symbol to multiple network penetrating the flow direction empty tube body is inserted, respectively.

According to the cell culture device of the present invention described in claim 1, since the space between the hollow tube bodies is secured by the mesh of the mesh body, the hollow tube body is inserted into each mesh, so that The outer peripheral surface of the tubular body can also function as a cell scaffold. This can further increase the specific surface area of the scaffold.

  In addition, by forming the mesh body through which the hollow tube body is inserted into a cage shape, a plurality of hollow tube bodies are integrally extracted from the culture tank together with the mesh body, and the hollow tube body is taken out of the culture tank. be able to. For this reason, cells can be collected from the culture tank in a short time.

The cell culture device of the present invention described in claim 3 is the cell culture device of the present invention described in claim 1 or 2 , wherein the flow direction is a direction from the lower part to the upper part of the culture tank, The plurality of hollow tubes supported by a support are installed in the culture tank with the penetration direction along the vertical direction of the culture tank.

According to the cell culture device of the present invention described in claim 3 , in the cell culture device of the present invention described in claim 1 or 2 , gas bubbles generated during the culture of the cells are formed between the hollow tubes. Or floated in the culture medium inside the hollow tube body and discharged from the upper part of the culture tank without staying in the culture tank.

  According to the present invention, a large amount of cells can be cultured by a perfusion culture method, and the cultured cells can be collected in a short time.

(A) is front sectional drawing which shows schematic structure of the cell culture apparatus which concerns on the reference example of this invention, (b) is the sectional view on the AA line of (a). (A) is a front sectional view showing a schematic configuration of a cell culture device according to an embodiment of the present invention, (b) is a sectional view taken along line BB of (a).

Hereinafter, embodiments of the present invention will be described with reference to the drawings. First, a cell culture device according to a reference example of the present invention will be described with reference to FIG.

FIG. 1A is a front sectional view showing a schematic configuration of a cell culture device according to a reference example of the present invention, and FIG. 1B is a sectional view taken along line AA in FIG.

As shown in FIG. 1 (a), the cell culture device 1 of the present reference example has a plurality of hollow tubes 30, 30,... Inside a cylindrical culture tank 10 to which a culture solution 20 is continuously supplied. It is configured to be housed side by side in the horizontal direction.

  An inlet 11 for introducing the culture solution 20 into the culture tank 10 is connected to the lower end of the culture tank 10 via a funnel-shaped relay pipe 13. In addition, a lid 17 that closes the opening 15 is detachably attached to the upper end of the culture tank 10. Further, an outlet 19 for overflowing the culture solution 20 in the culture tank 10 and discharging it outside the tank is connected to the side wall near the upper end of the culture tank 10 so as not to interfere with the lid body 17.

  A net 40 is disposed at the boundary between the culture tank 10 and the relay pipe 13. The mesh body 40 has a mesh 41 having an opening smaller than the outer shape of the hollow tube body 30. The culture solution 20 introduced from the inlet 11 passes through the mesh 41 of the mesh body 40 and is introduced into the culture tank 10.

  The culture solution 20 is a liquid medium for animal cells, plant cells, and aerobic microbial cells that require oxygen (hereinafter collectively referred to as “cells”). Then, the culture solution 20 in the culture tank 10 can be replaced by continuing the introduction of the culture solution 20 from the inflow port 11 and collecting the overflowed culture solution 20 from the outflow port 19. Moreover, the culture solution 20 can be recovered from the culture vessel 10 by discharging the culture solution 20 from the inlet 11 to the outside of the vessel.

  Each hollow tube 30 has a hollow cylindrical shape with a passage 31 passing through the inside thereof, and the flow direction of the culture solution 20 in the culture tank 10 from the inlet 11 to the outlet 19 is determined as the through direction. It is accommodated in the culture tank 10 along (vertical direction). The lower end of each hollow tube 30 is supported by a net 40.

  As shown in FIG. 1B, the hollow tube bodies 30 are arranged side by side in, for example, a lattice shape in the horizontal direction. The two adjacent hollow tube bodies 30 are bonded with an adhesive 50. Therefore, in the present embodiment, the adhesive 50 corresponds to the support in the claims.

  As the hollow tube body 30, for example, a commercially available plastic straw can be used. Moreover, you may give uneven | corrugated processes, such as a satin finish, to the outer peripheral surface 33 and the inner peripheral surface 35 of the hollow tube 30 so that a cell may adhere easily.

  Examples of the adhesive 50 include glue based on collagen and gelatin extracted from skin and bones of animals and fish, and fibrin glue (fibrin glue) based on fibrinogen which is a coagulation component in blood. It is desirable to use a material having excellent biocompatibility.

  Alternatively, a polymer obtained by crosslinking a polymer such as collagen as an adhesive component with a curing component activated with citric acid to increase the adhesive strength may be used as the adhesive 50. Since citric acid is one of the reaction systems of the citric acid cycle in biological energy metabolism, there is no problem in terms of biocompatibility even if it is used as a crosslinking agent.

  It should be noted that the inner peripheral wall of the culture tank 10 and the hollow tube body 30 facing the inner wall may be adhered with an adhesive 50 as shown in FIG. The outer peripheral surface of the hollow tube 30 may be brought into contact with the inner peripheral wall of the culture tank 10. If the hollow tube body 30 is bonded to the inner peripheral wall of the culture tank 10, the hollow tube bodies 30, 30 can be firmly fixed in the culture tank 10. On the other hand, if the hollow tube 30 is brought into contact with the inner peripheral wall of the culture vessel 10 without being bonded, the plurality of hollow tubes 30, 30,. Can be put in and out.

In the cell culturing apparatus 1 of this reference example configured as described above, the culture solution 20 is continuously supplied from the inlet 11 to the culture tank 10, and between the hollow tubes 30 or inside the hollow tubes 30. The culture solution 20 flows through the culture tank 10 from below to above. A symbol X in FIG. 1A indicates the flow direction of the culture solution 20 in the culture tank 10. The overflowed culture solution 20 in the culture tank 10 is discharged out of the culture tank 10 through the outlet 19.

  The culture tank 10 (including the lid body 17) described above is preferably composed of, for example, glass, metal, silicon rubber, or the like that does not change under normal steam sterilization conditions of 121 ° C. and 2 atmospheres. Or when using sterilization means other than steam sterilization, it is preferable to comprise with the material which does not change by irradiation of the sterilization means, such as a gamma ray, an electron beam, peracetic acid, ozone. In addition, if the principal part of the culture tank 10 is comprised with a transparent material, since the mode in a tank can be confirmed from the outside of a tank, it is still more preferable.

  In addition, since the mesh body 40 is left in the culture solution 20 in the culture tank 10, it is preferable that the mesh body 40 is made of a material having a specific gravity (weight) on which gravity exceeding buoyancy acts in the culture solution 20. When the hollow tube body 30 is brought into contact with the inner peripheral wall of the culture tank 10 without being bonded, a plurality of hollow tube bodies 30, 30,... It is preferable to make the whole of the medium have a specific gravity (weight) at which gravity exceeding buoyancy acts in the culture solution 20.

  When cell culture is performed with the cell culture apparatus 1 having the above-described configuration, the plurality of hollow tubes 30, 30,... The culture solution 20 is introduced from the inflow port 11 into the culture tank 10 which is accommodated in the culture vessel 10 and the opening 15 is closed by the lid 17. When the culture solution 10 is filled in the culture vessel 10, the lid 17 is removed and the cells cultured in the culture vessel 10 together with the culture solution 20 continuously supplied to the culture vessel 10 from the opening 15 or from the inlet 11. Inoculate.

  Thereafter, the culture solution 20 is continuously supplied from the inlet 11 to the culture tank 10, and the overflowed culture solution 20 is recovered from the outlet 19. The collected culture solution 20 may be introduced again into the culture tank 10 from the inlet 11. Thereby, the inner peripheral surface 35 of the hollow tube 30 that is a passage of the culture solution 20 functions as a cell scaffold, and the inoculated cells adhere to the inner peripheral surface 35 and are cultured.

  The cultured cells are separated from the inner peripheral surface 35 of the hollow tube body 30 by increasing the flow rate of the culture solution 20 in the culture tank 10 from the inlet 11 toward the outlet 19, and are discharged together with the overflowed culture solution 20. 19 may be recovered. Alternatively, in a sterile environment, by using an instrument (not shown) such as sterile tweezers, the plurality of hollow tubes 30, 30,... Are taken out from the culture tank 10 through the opening 15 from which the lid 17 is removed, The cells may be recovered by peeling from the inner peripheral surface 35 of each hollow tube 30 outside the culture tank 10.

As described above, according to the cell culture device 1 of the present reference example , the penetration direction of each hollow tube body 30 adjacent to each other by the adhesive 50 is supplied to the culture tank 10 continuously. A plurality of hollow tubes 30, 30,... Are installed in the culture tank 10 as the vertical direction along the flow direction X. For this reason, the culture solution 20 flows in the culture tank 10 through the inside of each hollow tube 30. Therefore, the inner peripheral surface 35 of each hollow tube 30 can be functioned as a cell scaffold, and the surface area of the scaffold can be efficiently increased. Moreover, since the hollow tube 30 used for the scaffold has a tubular structure, the strength of the scaffold can be maintained high. Therefore, it is possible to achieve both the maintenance of the strength of the scaffold and the increase of the specific surface area of the scaffold.

  Further, the inner peripheral surface 35 of the hollow tube 30 is increased by increasing the flow rate of the culture solution 20 in the culture tank 10 from the inlet 11 toward the outlet 19 or by removing each hollow tube 30 from the culture tank 10. Since the cultured cells can be peeled off, the cultured cells can be collected from the culture tank 10 in a short time.

Therefore, according to the cell culture device 1 of the present reference example , it is possible to increase the specific surface area of the scaffold and to culture a large amount of cells by the perfusion culture method, and it is possible to collect the cultured cells in a short time.

Moreover, according to the cell culture apparatus 1 of this reference example , since each hollow tube 30 was installed in the culture tank 10 with the penetrating direction along the vertical direction, as shown in FIG. The gas bubbles 60 generated during the culture float up in the culture solution 20 between the hollow tube bodies 30 and 30 or inside each hollow tube body 30 and do not stay in the culture bath 10. The gas can be discharged out of the culture tank 10 together with the overflowed culture solution 20 from the upper outlet 19.

Next, a cell culture device according to an embodiment of the present invention will be described with reference to FIG.

FIG. 2A is a front sectional view showing a schematic configuration of a cell culture device according to an embodiment of the present invention, and FIG. 2B is a sectional view taken along line BB in FIG.

In the cell culture apparatus 1A of the present embodiment shown in FIG. 2A, a plurality of hollow tube bodies 30, 30,... Are arranged in the horizontal direction in the culture tank 10 using the net bodies 70 , 80. Unlike the cell culture device 1 of the first embodiment shown in FIG. 1 (a), the other configurations are the same as those of the cell culture device 1 of the first embodiment.

  As shown in FIG. 2 (a), the mesh bodies 70 and 80 are orthogonal to the flow direction of the culture solution 20 at two locations spaced vertically between the mesh body 40 and the outlet 19 of the culture tank 10. They are deployed in the horizontal direction. The vertical spacing of the nets 40, 70, 80 is held constant by spacers 75, 85 interposed between the nets 40, 70, 80.

  As shown in FIG. 2B, the upper mesh body 70 has a mesh 71 having an opening that is slightly larger than the outer shape of the hollow tube body 30. As shown in FIG. 2A, the lower mesh body 80 formed to be the same size as the upper mesh body 70 also has an open mesh 81 that is slightly larger than the outer shape of the hollow tube 30. . The nets 70 and 80 are respectively arranged in the culture tank 10 so that the positions of the respective nets 71 and 81 are aligned in the horizontal direction.

  .. Are inserted through meshes 71 and 81 having the same horizontal position of the mesh bodies 70 and 80, and the lower ends of the hollow tube bodies 30 are formed by the mesh body 40. It is supported.

  Therefore, in the cell culture apparatus 1A of the present embodiment, the mesh bodies 70 and 80 support the hollow tube bodies 30 arranged in a grid in the horizontal direction as shown in FIG. In addition, a gap corresponding to the difference between the pitch of the meshes 71 and 81 of the mesh bodies 70 and 80 and the tube diameter of the hollow tube body 30 is generated between two adjacent hollow tube bodies 30 and 30. The culture solution 20 also flows through the gap. In the present embodiment, the nets 70 and 80 correspond to the supports in the claims.

  In addition, since the nets 70 and 80 are left in the culture solution 20 in the culture tank 10, similarly to the net 40, the nets 70 and 80 are made of a material having a specific gravity (weight) on which gravity exceeding buoyancy acts. It is preferable to do this. Moreover, in this embodiment, since the hollow tube 30 is supported by the nets 70 and 80, the hollow tube 30 itself has a specific gravity (weight) at which gravity exceeding buoyancy acts in the culture solution 20. It is preferable to do so. Alternatively, a frictional force exceeding the buoyancy acting on the hollow tube 30 in the culture solution 20 acts between the meshes 71 and 81 of the mesh bodies 70 and 80 and the outer peripheral surface 33 of the hollow tube 30. It is preferable that the meshes 71 and 81 have a mesh size that matches the outer diameter of the hollow tube 30.

  In the cell culture apparatus 1A configured as described above, since the hollow tube bodies 30 inserted through the meshes 71 and 81 of the mesh bodies 70 and 80 are separated from each other, in addition to the inner peripheral surface 35 of the hollow tube body 30 The outer peripheral surface 33 also serves as a passage for the culture solution 20 and functions as a cell scaffold. For this reason, in the cell culture apparatus 1A of the present embodiment, the inoculated cells are attached to the outer peripheral surface 33 and the inner peripheral surface 35 and cultured.

  The cultured cells were separated from the outer peripheral surface 33 and the inner peripheral surface 35 of the hollow tube 30 by increasing the flow rate of the culture solution 20 in the culture tank 10 from the inlet 11 to the outlet 19 and overflowed. You may collect | recover from the outflow port 19 with the culture solution 20. FIG. Alternatively, in an aseptic environment, each hollow together with the mesh bodies 70, 80 (or the mesh bodies 40, 70, 80) from the culture tank 10 through the opening 15 from which the lid body 17 is removed, using an instrument or the like (not shown). The tube body 30 may be taken out and separated from the outer peripheral surface 33 and the inner peripheral surface 35 of each hollow tube body 30 outside the culture tank 10 to collect cells.

According to the cell culture apparatus 1A of the present embodiment configured as described above, the outer peripheral surface 33 and the inner peripheral surface 35 of each hollow tube body 30 function as cell scaffolds, respectively, and the specific surface area of the scaffold is used as a reference example. The cell culture device 1 can be further increased. Further, by increasing the flow rate of the culture solution 20 and taking out the hollow tube 30 from the culture tank 10, the cells cultured from the outer peripheral surface 33 and the inner peripheral surface 35 of the hollow tube 30 are separated and collected in a short time. can do. Furthermore, gas bubbles 60 generated during cell culture can float in the culture solution 20 and can be discharged out of the culture tank 10 through the outlet 19 without staying in the culture tank 10.

For this reason, the same effect as that of the cell culture device 1 of the reference example can be obtained even in the cell culture device 1A of the present embodiment.

In the cell culture apparatuses 1 and 1A of the reference examples and embodiments described above, the culture solution 20 is supplied from the outlet 19 to the culture tank 10, and the supplied culture solution 20 is discharged from the inlet 11 to the outside of the culture tank 10. May be. In this case, the outflow port 19 may be provided in the lid body 17 to serve as a supply port for the culture solution 20.

  In this way, since the culture solution 20 is always filled in the inlet 11 for discharging the culture solution 20, the culture solution 20 can be removed from the culture tank 10 without sucking air even if the volume of the culture solution 20 is reduced by evaporation. It can be discharged and recirculated from the outlet 19 to the culture vessel 10.

  In addition, by supplying the culture solution 20 from the outlet 19 to the culture vessel 10, cells are inoculated near the upper liquid surface of the culture solution 20, and the culture solution 20 in the culture vessel 10 heading from the outlet 19 toward the inlet 11. The cells can be diffused in each hollow tube 30 by being placed on the descending flow of.

Furthermore, in the cell culture apparatuses 1 and 1A of the reference example and the embodiment described above, the flow direction X of the culture solution 20 in the culture tank 10 is the vertical direction, and in accordance with this, a plurality of hollow tube bodies 30, 30, ... has been described for the case where each penetrating direction is installed along the vertical direction.

  However, for example, the flow direction of the culture solution 20 in the culture tank 10 is set to the horizontal direction, and in accordance with this, the plurality of hollow tubes 30, 30,. It may be installed side by side in the vertical direction. That is, the installation direction of the hollow tube 30 in the culture tank 10 is not limited to the vertical direction as long as it is along the flow direction of the culture solution 20 in the culture tank 10.

  Further, as a method of supporting a plurality of hollow tube bodies 30, 30,... In parallel in a direction orthogonal to the penetrating direction, the hollow tube bodies 30 using the adhesive 50 shown in FIGS. And the support of the hollow tube body 30 by the mesh bodies 70 and 80 shown in FIGS. 2A and 2B may be used in combination.

  Further, the shape of the hollow tube body 30 is not limited to a cylindrical shape, and may be a rectangular tube shape or the like as long as the hollow tube 30 has a cylindrical shape with a passage therethrough.

DESCRIPTION OF SYMBOLS 1,1A Cell culture apparatus 10 Culture tank 11 Inflow port 13 Relay pipe 15 Opening 17 Lid body 19 Outlet 20 Culture solution 30 Hollow tube body 31 Passage 33 Hollow tube outer peripheral surface 35 Hollow tube body outer peripheral surface 40 Network body 41, 71, 81 Mesh 50 Adhesive (support)
60 bubbles 70,80 mesh (support)
75,85 Spacer X Culture fluid flow direction

Claims (3)

A cell culture device for perfusion culture of cells,
A culture tank capable of continuously supplying a culture solution;
A scaffold for cell culture provided in the culture tank,
The scaffold is
A plurality of hollow tubes whose passages penetrate the interior;
A support body that supports the plurality of hollow tubes, along the direction of passage of the passage in the flow direction of the culture medium in the culture tank, and arranged in parallel in a direction perpendicular to the direction of penetration .
The support includes a mesh body that is detachably installed in the culture tank and is developed in a direction orthogonal to the flow direction, and the hollow is formed in a plurality of meshes that penetrate the mesh body in the flow direction. Each tube is inserted,
A cell culture device.   The cell culture device according to claim 1, wherein the support includes an adhesive that bonds two hollow tubes adjacent to each other in a direction orthogonal to the penetration direction. The flow direction is a direction from the lower part to the upper part of the culture tank, and the plurality of hollow tubes supported by the support body have the penetration direction along the vertical direction of the culture tank. cell culture equipment according to claim 1 or 2, characterized in that it is installed in.