JP2018161115A - Cell culture apparatus and cell culture method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture apparatus and a cell culture method which hardly cause clogging of a filtration membrane due to a cell.SOLUTION: A cell culture apparatus 1 according to the present invention comprises: culture means 2 in which a culture solution CM is encapsulated and a cell C is cultured; cell separation means 3 comprising a filtration membrane 31 which blocks permeation of the cell C contained in the culture solution CM inside the culture means 2, and comprising a discharge part 33 which discharges the culture solution CM passing through the filtration member 31 to the outside of the culture means 2; stirring means 4 for stirring the culture solution CM to generate a stirring flow A; and liquid medium supply means 5 for supplying, into the culture means 2, a liquid medium M for culturing the cell C. The cell separation means 3 is provided in such a manner that the stirring flow A is directed toward a tangential direction relative to the filtration membrane 31.SELECTED DRAWING: Figure 1

Description

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

  For production of biopharmaceuticals, a culture method in which living cells such as microorganisms, cells, and fungi are cultured in a culture tank is used. Living cells and culture solution are accommodated in the culture tank, and the culture solution is stirred by a stirrer. In the culture of aerobic microorganisms and cells, in order to supply oxygen necessary for respiration, a ventilation means for oxygen-containing gas is directly provided on at least one of the upper surface of the culture solution and the culture solution. ing.

  In recent years, it has been desired to cultivate living cells at high density in order to improve the productivity of the target product, and there is a continuous culture method as a culture technique for achieving this. In the continuous culture method, the nutrient period in the liquid medium consumed by the cells is supplemented by supplying a fresh liquid medium in order to increase the cell culture period and improve productivity. In the continuous culture method, in order to maintain the volume of the culture medium in the culture tank to be almost constant, the cells in the culture liquid extracted from the culture tank are separated and returned to the culture tank, and only the liquid components are extracted while a new liquid is extracted. The medium is supplied. For this reason, as a method for separating cells from a culture solution in a continuous culture method, for example, a gravity sedimentation method, a method of sedimenting and separating cells by centrifugation, a porous hollow fiber membrane or a filter medium that prevents passage of cells was used. Cell separation methods such as filtration membrane separation methods have been developed.

  Among these cell separation methods, the filtration membrane separation method has an advantage that the apparatus configuration is simple and the scale-up is easy. A technique suitable for the filtration membrane separation method is described in Patent Document 1, for example. In this Patent Document 1, a filter (filtration membrane) that permeates culture liquid but does not permeate cells is provided in the vicinity of the bottom. A new culture medium supply port is provided in the culture tank, and an old liquid medium discharge port is provided below the filter. And a cell culture tank in which a stirring means having a stirring blade is provided in the vicinity of the upper part of the filter is described. According to Patent Document 1, according to the cell culture tank, a culture solution can be continuously supplied, waste products, metabolic products, purification inhibitors, and the like can be continuously removed, and the filter is not clogged. Therefore, it is described that a large number of cells can be cultured for a long period of time at a high density.

JP 60-259179 A

  However, the method of accommodating the filtration membrane in the culture tank like the cell culture tank described in Patent Document 1 has a problem that clogging of the filtration membrane by cells is substantially inevitable. In addition, since the cell membrane described in Patent Document 1 is provided with a filtration membrane in the vicinity of the bottom, depending on the state of the flow of the culture solution, a cleaning effect may not be obtained, and the filtration membrane may be clogged. . Therefore, when the filtration membrane is clogged and the cells cannot be separated from the culture solution, the culture has to be terminated, and it may be difficult to carry out the culture for a long time. Therefore, the prior art may not fully meet the demand for cell culture at a higher density.

  This invention is made | formed in view of such a condition, and makes it a subject to provide the cell culture apparatus and cell culture method which are hard to generate | occur | produce clogging of the filtration membrane by a cell.

  The cell culture device according to the present invention that has solved the above problems includes a culture means for culturing cells by enclosing a culture solution, a filtration membrane that does not allow cells contained in the culture solution inside the culture means, and the filtration membrane. A cell separation means having a discharge part for discharging the culture solution permeated out of the culture means, a stirring means for stirring the culture solution to generate a stirring flow, and culturing the cells inside the culture means Medium supply means for supplying a liquid medium for the purpose, and the cell separation means is provided so that the stirring flow is tangential to the filtration membrane.

  The cell culture method according to the present invention includes a culture means for culturing cells by enclosing a culture solution, a filtration membrane that does not permeate cells contained in the culture solution inside the culture means, and the culture that has passed through the filtration membrane. A cell separation means comprising a discharge part for discharging the liquid out of the culture means, a stirring means for stirring the culture liquid to generate a stirring flow, and a liquid medium for culturing the cells inside the culture means A medium supply means for supplying cells, wherein the cell separation means is a method of culturing cells using a cell culture device provided so that the stirring flow is tangential to the filtration membrane. During culture of the cells, the culture solution is stirred by the stirring means to generate the stirring flow, and the culture solution is filtered through a filtration membrane of the cell separation means to remove the culture means from the discharge unit. Discharge process A supply step of supplying substantially the same amount of liquid medium as the culture medium discharged out of the culture means to the inside of the culture means from the culture medium supply means, in any order, and performing these steps continuously .

  In the cell culture device and the cell culture method according to the present invention, clogging of the filtration membrane by cells is difficult to occur.

It is a schematic block diagram explaining the structure of the cell culture apparatus which concerns on this embodiment. It is a schematic sectional drawing which shows the structure of a cell separation means. It is an external view explaining the one aspect | mode of the filtration membrane of a cell separation means. It is an external view explaining the one aspect | mode of the filtration membrane of a cell separation means. It is a schematic sectional drawing explaining the other aspect of a stirring means. It is a schematic sectional drawing explaining a mode that the structure of the cell culture apparatus concerning this embodiment was shown more concretely. It is a schematic sectional drawing which shows the modification of the cell culture apparatus which concerns on this embodiment. It is a schematic sectional drawing which shows the other modification of the cell culture apparatus which concerns on this embodiment. It is the IX section enlarged view of FIG.

  Hereinafter, embodiments (embodiments) for carrying out a cell culture device and a cell culture method according to the present invention will be described in detail with reference to the drawings as appropriate. In addition, the same code | symbol is attached | subjected and represented to the common element in each drawing referred below.

[Cell culture equipment]
FIG. 1 is a schematic configuration diagram illustrating the configuration of the cell culture device according to the present embodiment.
As shown in FIG. 1, the cell culture device 1 according to the present embodiment includes a culture unit 2, a cell separation unit 3, a stirring unit 4, and a medium supply unit 5. These components and each component described later are connected aseptically.

The cell culturing apparatus 1 according to the present embodiment can be applied when culturing microorganisms or animal and plant cells that produce useful substances as main raw materials such as pharmaceuticals and health foods.
Examples of cells that can be used in the present embodiment include Chinese hamster ovary cells (CHO cells), baby hamster kidney cells, and mouse myeloma cells that are commonly used as cells for producing useful substances. It is not limited to these. When the cells are adherent cells, they can be stirred and cultured by attaching them to a carrier such as a microcarrier and allowing them to float. In addition to animal-derived cells, plant cells, photosynthetic bacteria, microalgae, cyanobacteria, insect cells, bacteria, yeast, fungi, algae, Escherichia coli, yeast, and other microbial cells are also applicable.
Examples of useful substances in the present embodiment include physiologically active substances, and in particular, antibody drugs. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies, immunoglobulins and the like. In addition, the physiologically active substance is not limited to antibodies, but also includes tissue-type plasminogen activator used as a thrombolytic agent, biopharmaceuticals such as erythropoietin and interferon, and other industrially useful proteins. In addition, examples include carotenoids such as β-carotene and astaxanthin, pigments such as chlorophyll and bacteriochlorophyll, phycobilin proteins such as phycocyanin used for coloring foods and cosmetics, and physiologically active substances such as fatty acids.

(Culture means)
The culture means 2 is a container for culturing cells by enclosing the culture medium CM. In the present specification, the culture medium CM refers to the liquid and cell culture in the process of seeding and proliferating cells in a liquid medium M for performing unused (fresh) cell culture. A liquid.
The culture means 2 can be a tank made of metal such as stainless steel or aluminum. Moreover, as shown in FIG. 6 as appropriate, the culture means 2 in the present embodiment has flexibility, a culture bag 21 that can enclose the culture medium CM therein, and a shape of the culture bag 21 that encloses the culture medium CM. It is good also as an aspect which comprises the housing 22 which maintains this.

  As the culture bag 21, for example, a bag made of a multilayer film of ethylene / vinyl / acetate or ethyl / vinyl / alcohol can be suitably used. In addition, the culture bag 21 is preferably sterilized in advance by gamma rays, ultraviolet rays, ethylene oxide gas, or the like and maintained in a sterile state. As described above, since the culture bag 21 has flexibility, the contents such as gas and liquid can be almost removed and stored in a folded state or transported. Further, when such a culture bag 21 is used, since it can be single-use (that is, single-use (use only once)), operations such as washing usually performed after the end of culture can be omitted. As a result, the working time can be shortened, the work can be simplified, and the incidental equipment for cleaning can be omitted, so that the cost can be reduced.

  Since the culture bag 21 is like a flexible plastic bag, it does not have a fixed shape when the culture solution CM is sealed, and does not have high strength. Therefore, it is preferable to use the housing 22 that maintains the shape of the culture bag 21 in which the culture medium CM is enclosed, and set the inside thereof. The housing 22 contains the culture bag 21 and has only enough strength to maintain the shape even when subjected to the load or pressure, and the shape is not particularly limited. The housing 22 for holding the culture bag 21 is preferably made of metal or hard plastic. The housing 22 may be one in which a part of the culture bag 21 is exposed. The housing 22 may have an open / close lid (not shown) for attaching and detaching the culture bag 21. The opening / closing lid is preferably provided on the side or upper surface of the housing 22 in the form of a double-opening or single-opening door or sliding door.

In the present embodiment, a self-supporting single-use container (not shown) can be used as the culture means 2. When this is used, the housing 22 described above may not be provided. Such a single-use container is preferably made of hard plastic, not stainless steel or aluminum.
The culture means 2 in this embodiment includes a cell separation means 3 to be described later, whether it is a metal tank, a culture bag 21 or a single use container.

(Cell separation means)
FIG. 2 is a schematic cross-sectional view showing the configuration of the cell separation means.
As shown in FIGS. 1 and 2, the cell separation means 3 cultures a filtration membrane 31 that does not allow the cells C contained in the culture medium CM inside the culture means 2 to permeate, and a culture medium CM that has permeated the filtration membrane 31. A discharge portion 33 for discharging outside the means 2. Specifically, the cell separation means 3 is provided with the filtration membrane 31 exposed inside the culture means 2, and discharged to the central portion of the back surface portion 32 that is the opposite surface of the filtration membrane 31 in the cell separation means 3. A portion 33 is provided. In addition, the discharge part 33 can be provided in the arbitrary locations of the cell separation means 3, and is not limited to an above-described position.
The filtration membrane 31 and the back surface portion 32 of the cell separation means 3 are formed integrally with the side wall portion 34, and have a sealed structure that does not communicate with the outside of the cell separation means 3 except for the filtration membrane 31 and the discharge portion 33. As the filtration membrane 31 used in the present embodiment, a known ultrafiltration membrane having a pore size that allows only unnecessary substances U such as a solution of culture solution CM and wastes to pass through without allowing cells C to permeate can be used.

When the culture means 2 is the metal tank described above (for example, see FIGS. 1 and 7), the cell separation means 3 may be attached at a position where the filtration membrane 31 is parallel to the stirring flow A on the wall surface of the tank.
When the culture means 2 is the culture bag 21 described above (see, for example, FIG. 5, FIG. 6, FIG. 8), the position where the filtration membrane 31 is parallel to the stirring flow A on the wall surface of the culture bag 21, that is, the stirring flow The cell separation means 3 may be welded at a position where A is tangential to the filtration membrane 31. In addition, “parallel” in the present embodiment is not strict, and may be tilted within a range in which the cells attached to the filtration membrane 31 can be removed by stirring shear stress. Further, the “tangential direction” may be a direction substantially parallel to the filtration membrane 31 and may be inclined.

3 and 4 are external views for explaining one embodiment of the filtration membrane of the cell separation means.
As shown in FIG. 3, the filtration membrane 31 of the cell separation means 3 can be planar. In this case, since the filtration membrane 31 is planar, if the stirring flow A of the culture solution CM is parallel to the filtration membrane 31, it is filtered regardless of the direction in which the culture solution CM flows. Can do. That is, the degree of freedom of installation of the filtration membrane 31 is high and suitable.
Moreover, as shown in FIG. 4, the filtration membrane 31 of the cell separation means 3 can be made into a corrugated shape having a plurality of peaks and valleys. In this case, since the filtration membrane 31 is corrugated and has a wide contact area with the culture solution CM, the culture solution CM can be efficiently filtered. In this case, in order to perform efficient filtration, the cell separation means 3 is provided so that the direction of the stirring flow A of the culture medium CM is along the peaks and valleys of the filtration membrane 31, that is, in parallel. preferable.

  The flow state in the culture means 2 varies depending on the shape of the culture means 2 and the mode of the stirring means 4 (for example, the shape of the stirring blade 41 (see FIG. 1) and the mounting position of the stirring blade 41). It also varies depending on the mounting position of the second wall. Therefore, in determining the mounting position of the cell separation means 3, it is preferable to confirm the direction of the stirring flow A of the culture medium CM by a water flow test using the actual culture means 2. It is also preferable to determine a suitable mounting position of the cell separation means 3 by analyzing the flow state in the culture means 2 on a computer. In this case, since the flow state in the peripheral part of the cell separation means 3 can be calculated by incorporating the shape of the cell separation means 3, a more desirable attachment position can be determined.

The discharge unit 33 (see FIG. 2) is connected to the collection tank 6 by a discharge pipe 62 provided with a discharge pump 61. The culture liquid CM filtered by the filtration membrane 31 of the cell separation means 3 is discharged from the discharge part 33 by driving the discharge pump 61 and is recovered in the recovery tank 6 through the discharge pipe 62.
As the collection tank 6, any container can be used as long as it can store the culture liquid CM filtered by the filtration membrane 31 of the cell separation means 3.
As the discharge pump 61, any pump can be used as long as it can send the culture solution CM in the discharge pipe 62.
The discharge pipe 62 is preferably a flexible pipe made of any synthetic resin.

  In the present embodiment, the number of cell separation means 3 provided in the culture means 2 is not limited, and one or two or more can be provided. When two or more cell separation means 3 are provided, the filtration ability can be enhanced by operating them simultaneously and filtering the culture medium CM. Further, when two or more cell separation means 3 are provided, even if one of the plurality of cell separation means 3 is clogged, and the culture medium CM cannot be sufficiently filtered, other cell separation means By operating 3, the culture medium CM can be filtered. Therefore, the cell culture period can be extended, and the yield of useful substances produced by the cells can be increased.

(Stirring means)
The agitating means 4 agitates the culture medium CM to generate a stirring flow A. In this embodiment, various methods can be applied as the stirring means 4.
For example, FIG. 1 shows an example in which a magnetic coupling 43 is used as the stirring means 4. The stirring means 4 shown in FIG. 1 includes a stirring blade 41 having a magnet (driven magnet) provided inside the culturing means 2 and a stirring drive device having a magnet (driving magnet) that rotates the driven magnet in synchronization with the driven magnet (drive magnet). Magnet pump) 42. Although depending on the shape of the stirring blade 41, when the stirring drive device 42 is driven to rotate the stirring blade 41, as shown in FIG. 1, the stirring flow A in which the culture medium CM flows along the wall surface from the bottom of the culture means 2. Occurs. In FIG. 1, since the stirring flow A of the culture medium CM is parallel to the wall surface from the middle of the wall surface of the culture means 2, the filtration membrane 31 is separated from the stirring flow A at a position higher than the middle wall surface of the culture means 2. The cell separation means 3 is provided so as to be parallel. By doing in this way, the cell separation means 3 can make it hard to generate | occur | produce clogging of the filtration membrane 31 by the cell C (refer FIG. 2).

Various methods other than the above-described method can be applied to the stirring means 4.
FIG. 5 is a schematic cross-sectional view illustrating another embodiment of the stirring means 4. As shown in FIG. 5, as another aspect of the stirring means 4, when the cell culture device 1 has a housing 22, the housing 22 can be provided with the stirring means 4. The stirring means 4 in this embodiment includes a central shaft member 4C provided vertically downward at a central position outside the bottom of the housing 22, and an actuator (not shown) that rotates the central shaft member 4C in a horizontal plane. ). Since such a stirring means 4 is provided, the housing 22 can rotate in the horizontal plane together with the central shaft member 4C. The stirring means 4 in the present embodiment can suitably employ such a system, that is, a system that revolves the housing 22 but does not rotate (hereinafter referred to as a shaking stirring system). In this way, since the culture medium CM can be shaken and stirred by rocking, it is not necessary to provide the stirring blade 41 (see FIG. 1) or the like inside the culture means 2 and the structure of the stirring means 4 is simplified. Can be Further, it is possible to prevent the culture means 2 (particularly, the culture bag 21 described above) from being damaged by the rotation of the stirring blade 41. Furthermore, damage due to the stirring shear stress on the cells can be reduced.

(Medium supply means)
The medium supply unit 5 supplies a liquid medium M for culturing cells inside the culture unit 2. The medium supply means 5 includes a medium storage tank 51 for storing an unused fresh liquid medium M, a supply pipe 53 provided with a supply pump 52, which connects the medium storage tank 51 and the culture means 2, It has. In the present embodiment, as described above, the culture medium CM is discharged out of the culture means 2 by the cell separation means 3. At the time of culture, the medium supply means 5 supplies the culture medium 2 through the supply pipe 53 by driving the supply pump 52 with the fresh liquid medium M having approximately the same amount as the culture liquid CM discharged outside the culture means 2. Supply in. The medium supply means 5 also supplies the liquid medium M to the culture means 2 when culturing is started.

Any medium can be used as the medium storage tank 51 as long as it is a container that can store an unused fresh liquid medium M.
Any supply pump 52 can be used as long as it can feed the liquid medium M in the supply pipe 53.
The supply pipe 53 is preferably a flexible pipe formed of an arbitrary synthetic resin, like the discharge pipe 62.
The liquid medium M used for the culture is not particularly limited, and any conventional liquid medium can be used.
The supply of the liquid medium M from the medium supply means 5 to the culture means 2 can be performed continuously, intermittently or at an arbitrary timing.

(Function and effect)
The cell culture apparatus 1 according to the present embodiment described above discharges the culture liquid CM separated from the cells by the cell separation means 3 to the outside of the culture means 2 and is roughly the same as the culture liquid CM discharged to the outside of the culture means 2. By supplying the same amount of the liquid medium M from the medium supply means 5, it is possible to supply nutrient components consumed by the cells. In this way, the amount of the culture medium CM in the culture means 2 can be maintained almost constant. Thus, the cell culture period can be extended, and the yield of useful substances produced by the cells can be increased.

The cell separation means 3 in this embodiment is provided so that the stirring flow A generated by the stirring means 4 described above is tangential to the filtration membrane 31. That is, the membrane surface of the filtration membrane 31 of the cell separation means 3 is provided so as to be parallel to the flow direction of the culture medium CM (the cell separation means 3 has a stirring flow A in a direction perpendicular to the filtration membrane 31. Not provided).
Therefore, as shown in FIG. 2, the cells C that are substances that are impermeable to the filtration membrane 31 flow along the surface of the filtration membrane 31. That is, the cell C flows in a direction perpendicular to the filtration direction of the filtration membrane 31. Therefore, it is difficult for the cells C to adhere to the filtration membrane 31. In addition to this, even when the cells C are attached to the filtration membrane 31, they are removed by the stirring shear stress caused by the stirring flow A of the culture medium CM. Clogging can be made difficult to occur.
Therefore, the cell culture device 1 according to the present embodiment can perform long-term culture and can sufficiently meet the demand for cell culture at a higher density.

(Specific example of cell culture device)
The cell culture device 1 according to the present embodiment will be described with reference to an example in which the configuration is more specifically shown.
FIG. 6 is a schematic cross-sectional view for explaining the state of the configuration of the cell culture device according to this embodiment more specifically.
As shown in FIG. 6, the cell culture apparatus 1 includes a culture bag 21 and a housing 22 as the culturing means 2, and employs the shaking and stirring method shown in FIG. 5 as the stirring means 4. The culture means 2 is supported by the stirring means 4, and the stirring means 4 is fixed to the support frame 7. Although not shown in FIG. 6, the cell culture device 1 includes a hot water / cold water supply facility and a water supply / drainage facility that are indispensable for culturing cells.

  As shown in FIG. 6, the culture bag 21 includes a gas supply pipe 8 for submerged aeration gas, a gas supply pipe 9 for gas phase, a supply pipe 53, a culture liquid discharge pipe 11, and a vent gas discharge pipe 12. Is provided. The liquid supply gas supply pipe 8 and the gas-phase gas supply pipe 9 are connected to gas supply facilities 17 and 18 for supplying air, oxygen, nitrogen, carbon dioxide gas and the like, respectively. A diffuser 13 for generating bubbles is connected to the tip of the submerged aeration gas supply pipe 8 provided in the culture bag 21. The submerged gas supply pipe 8, gas phase gas supply pipe 9, and gas discharge pipe 12 are provided with a gas filter 14 for preventing invasion of microorganisms from the outside.

  The cell separating means 3 is fixed to the inner wall surface of the culture bag 21 by means such as adhesion or attachment. By driving the discharge pump 61, the culture solution CM (see FIG. 2) separated from the cells C by the filtration membrane 31 of the cell separation means 3 is collected in the collection tank 6 through the discharge pipe 62.

The cell culture device 1 is provided with a pipe for sampling and injection of a drug for adjusting the pH other than those described above, but the description in FIG. 6 is omitted. Further, as a means for measuring the culture state, a device for measuring pH, temperature, dissolved oxygen concentration (DO), dissolved carbon dioxide concentration (DCO ₂ ), and the like, and respective sensors are provided. Is simply shown as the measuring means 15.
The culture bag 21 is fixed by fixtures 16 a and 16 b of the housing 22.
In addition, the cell culture device 1 has a control device 20. Based on the pH, temperature, DO, and DCO ₂ information of the culture medium CM obtained from the measuring means 15, the control device 20 controls the stirring means 4, the supply pump 52, the discharge pump 61, and the gas supply facilities 17 and 18. Perform output control.

(Operating procedure)
Next, although the operation procedure of the cell culture apparatus 1 etc. are demonstrated with reference to FIG. 6, the outline | summary of operation is demonstrated and it does not necessarily restrain with the following procedure.
The new culture bag 21 in a folded state is installed at a predetermined position using the fixtures 16 a and 16 b of the housing 22.
The supply pump 52 of the medium supply means 5 is driven to supply the liquid medium M in the medium storage tank 51 to the culture bag 21 through the supply pipe 53. Then, target cells are supplied into the culture bag 21. The culture bag 21 is inflated by the supplied liquid medium M, and is crimped to the inner wall surface of the housing 22 to maintain a predetermined shape.
The liquid medium M to be supplied to the culture bag 21 may be one in which target cells are suspended in advance and cultured to a predetermined cell concentration. In this case, cell culture may be performed in a storage tank for an initial medium (not shown) so as to have a predetermined cell concentration, and supplied to the culture bag 21 (culture container 2) using a pipe (not shown).

Next, the gas supply facilities 17 and 18 are driven, and an oxygen mixed gas of a predetermined concentration is vented from the submerged gas supply pipe 8 and the gas phase gas supply pipe 9 connected to the gas supply facilities 17 and 18, respectively. The culture is started by stirring the liquid medium M (culture medium CM) containing the cells. After the culture starts, as the cells grow and the cell density increases, the oxygen consumption rate increases and the carbon dioxide concentration increases. In order to maintain the oxygen concentration of the culture medium CM within a predetermined range, DO, DCO _{2 and the} like are measured by the measuring means 15 and the amount of aeration in the liquid from the aeration means 13 is controlled by the control device 20 based on the information. Increase. By the aeration in the liquid from the air diffuser 13 and the agitation by the agitation means 4, the oxygen concentration in the culture medium CM is kept substantially uniform.

  Further, in order to avoid that nutrient components consumed by the cells are reduced and cell culture cannot be continued efficiently, the fresh liquid medium M in the medium reservoir 51 is cultured in the culture bag 21 from the supply pipe 53. Supply to liquid CM. In order to determine whether or not it is necessary to supply the liquid medium M, the consumption amount of the components of the culture medium CM is calculated from the increase amount of the cell concentration by a corresponding sensor representatively described as the measuring means 15. In addition, by incorporating a sensor for measuring the concentration of nutrient components such as glucose and glutamine in the culture medium CM, the nutrient component concentration may be directly measured to calculate the required supply amount of the liquid medium M. When an appropriate sensor cannot be used, a necessary supply amount of the liquid medium M may be calculated by sampling a part of the culture medium CM and measuring the cell concentration or the nutrient concentration.

  Subsequent to the supply of the fresh liquid medium M, the discharge pump 61 is driven, and the culture medium CM of approximately the same amount as the supplied fresh liquid medium M is filtered and discharged by the cell separation means 3, and is discharged through the discharge pipe 62. Store in the collection tank 6. The supply amount of the fresh liquid medium M and the discharge amount of the culture medium CM filtered by the cell separation means 3 are respectively calculated from the drive time of the supply pump 52 and the discharge pump 61 and the flow rate measured in advance. It can be calculated. Although not clearly shown in the figure, the weight of the tank may be measured by mass measuring means provided in each of the medium storage tank 51 and the collection tank 6 and calculated from the variation amount. At this time, the product can be continuously obtained by performing a series of post-process treatments such as separation and recovery and purification of the target culture product from the culture solution CM collected in the collection tank 6.

  In addition, when the foaming of the culture medium CM becomes intense as the aeration amount in the liquid increases and a foam layer is formed on the upper surface of the culture medium CM, the fresh liquid medium M is supplied from the supply pipe 53. In addition, the antifoaming effect can be enhanced by adding chemicals such as silicone antifoaming agents, polyether antifoaming agents, and surfactants.

As described above, the pH, temperature, DO, and DCO ₂ of the culture solution CM are measured by the measuring unit 15 and the information is input to the control device 20. Based on the input information, the control device 20 controls the output of the agitating means 4, the supply pump 52, the discharge pump 61, and the gas supply facilities 17 and 18, and adjusts the air flow rates of the mixed gas of air, oxygen and nitrogen. predetermined pH, for example, by adjusting, DO, and controls to maintain the DCO _2.

When the control device 20 satisfies a preset condition or receives a signal indicating that the culture is stopped by an operator's operation, the control device 20 stops the stirring means 4 and the gas supply equipments 17 and 18 to perform the culture. finish.
Thereafter, the culture solution CM is discharged through the culture solution discharge pipe 11 provided in the lower part of the culture bag 21. A post-process such as collection and purification of the target culture product is performed from the discharged culture medium CM, and a series of culture production processes is completed. After the culture medium CM is discharged, the culture bag 21 is removed from the housing 22 and discarded.
In order to start the next culture, the new culture bag 21 folded as described above is installed at a predetermined position using the fixtures 16a and 16b of the housing 22, and the culture is started in the same manner as described above. .

(Cell culture method)
The cell culture method according to the present embodiment is a method for culturing cells using the cell culture apparatus described above. Since the cell culture device has already been described in detail, the description of the device is omitted.
The cell culture method according to the present embodiment includes a discharging step and a supplying step in no particular order, and these steps are continuously performed. Any of these steps may be performed first. Moreover, you may advance these processes simultaneously.

(Discharge process)
In the discharging step, the culture medium CM is stirred by the stirring means 4 to generate the stirring flow A, while the culture liquid CM is filtered by the filtration membrane 31 of the cell separating means 3 and discharged from the discharge section 33 to the outside of the culture means 2. It is a process to do. If it does in this way, since it removes by the stirring shear stress by the stirring flow A of the culture solution CM, it can make it hard to produce clogging of the filtration membrane 31 by the cell C adhering.

(Supply process)
The supplying step is a step of supplying approximately the same amount of the liquid medium M as the culture medium CM discharged outside the culture means 2 from the medium supply means 5 to the inside of the culture means 2. In this way, since the same amount of fresh liquid medium M as the discharged culture medium CM is continuously supplied, long-term culture can be performed, and it is sufficient for the desire to perform cell culture at a higher density. I can respond. Further, since the volume of the culture medium CM is always kept substantially constant, cell culture can be stably performed.

  As mentioned above, although the cell culture apparatus and the cell culture method which concern on one Embodiment of this invention were demonstrated in detail, the summary of this invention is not limited to this, Various modifications are included. For example, the above-described embodiment has been described in detail for easy understanding of the present invention, and is not necessarily limited to one having all the configurations described. Further, a part of the configuration of an embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of an embodiment. Moreover, it is possible to add / delete / replace other configurations for a part of the configurations of the embodiments.

In addition, the following are mentioned as an above-described modification.
FIG. 7 is a schematic cross-sectional view showing a modification of the cell culture device according to the present embodiment.
As shown in FIG. 7, the cell culture device 1 according to the present modification has a pressure sensor 24 that detects the drawing pressure of the discharged filtrate (culture solution CM) in the path of the discharge pipe 62. . Further, the cell culture device 1 according to this modification has a branch pipe 54 branched from the supply pipe 53. This branch pipe 54 is connected to the back surface portion 32 of the cell separation means 3. The supply pipe 53 is provided with an opening / closing valve V1 in the path leading to the culture means 2. Similarly, the branch pipe 54 is provided with an opening / closing valve V2 in the path leading to the cell separation means 3.

  In the cell culture device 1 according to this modification, when the filtrate drawing pressure detected by the pressure sensor 24 becomes excessively high, the control device 20 (not shown in FIG. 7) performs cell separation means 3 based on the pressure information. It is determined that the filter membrane 31 is clogged.

  The control device 20 that has made the determination normally sets the open / close valve V1 in the “open” state to the “closed” state and the open / close valve V2 in the “closed” state to the “open” state. In this way, the liquid medium M that has been directly supplied to the culture means 2 from the medium storage tank 51 until then is cultured from the discharge part 33 (not shown in FIG. 7) side of the cell separation means 3 through the branch pipe 54. The liquid medium M is supplied toward the inside 2. In this way, since the liquid medium M flows from the back surface of the filtration membrane 31 into the culture means 2, so-called backwashing can be performed, and the cells adhering to the surface of the filtration membrane 31 are peeled off and clogged. Can be eliminated. Therefore, it is possible to carry out culture for a longer period.

  In the example shown in FIG. 7, the liquid medium M is flowed from the back surface of the filtration membrane 31 into the culture means 2, and the cells adhering to the surface of the filtration membrane 31 are peeled off to eliminate clogging. However, the present invention is not limited to this. For example, when the culture means 2 has two or more cell separation means 3, if the pressure sensor 24 determines that one cell separation means 3 is clogged, switch to another cell separation means 3. You may make it continue filtration. Even in this way, it is possible to carry out culture for a longer period of time. This modification and the modification described with reference to FIG. 7 can be used together.

  Furthermore, as shown in FIG. 7, a plurality of stirring blades 41 may be provided. In this way, the ability to stir the culture medium CM is improved, which makes it easy to keep the oxygen concentration in the culture medium CM uniform, which is preferable. The stirring blade 41 in FIG. 7 is connected to a stirring drive device 45 such as a motor provided above the culture means 2 via a rotating shaft 44. When the rotating shaft 44 transmits the rotational force of the stirring drive device 45 to the stirring blade 41, the stirring blade 41 rotates. The rotary shaft 44 and the culture means 2 are mechanically sealed with a known mechanical seal (shaft seal device) to prevent contamination such as germs.

FIG. 8 is a schematic cross-sectional view showing another modification of the cell culture device according to the present embodiment. FIG. 9 is an enlarged view of the IX portion of FIG.
As shown in FIG. 8, the cell culturing apparatus 1 according to another modified example is agitated in which a stirring blade 41 is provided above the culturing means 2 via a rotating shaft 44 as in the modified example shown in FIG. A drive device 45 is connected. The rotating shaft 44 and the culture means 2 are mechanically sealed with a known mechanical seal (shaft seal device). And the cell culture apparatus 1 which concerns on another modification is provided with the hollow fiber-shaped cell separation means 3 (filtration membrane 31) wound around the stirring blade 41 helically. In this modification, the culture solution CM filtered by the cell separation means 3 is present inside the hollow fiber. A discharge pump, a discharge pipe and a recovery tank (not shown in FIG. 8) are connected to the base end of the hollow fiber-shaped cell separation means 3, and the culture solution CM filtered by the cell separation means 3 is collected in the recovery tank (FIG. 8 and not shown in FIG. 9).

In the example shown in FIG. 8, the stirring flow A flows between the spirally wound hollow fiber filtration membrane 31 and the hollow fiber filtration membrane 31. Therefore, in this case, as shown in FIG. 9, the stirring flow A is tangential to the filtration membrane 31 on the opposed surfaces of the adjacent hollow fiber filtration membrane 31 and the hollow fiber filtration membrane 31. Therefore, the cells C (not shown in FIG. 9) are removed by the shear stress, and therefore the clogging of the filtration membrane 31 due to the attachment of the cells C hardly occurs.
Therefore, also in the cell culture device 1 according to this modification, it is possible to perform long-term culture, and it is possible to sufficiently meet the demand for cell culture at a higher density.

DESCRIPTION OF SYMBOLS 1 Cell culture apparatus 2 Culture means 21 Culture bag 22 Housing 3 Cell separation means 31 Filtration membrane 33 Discharge part 4 Stirring means 41 Stirring blade 5 Medium supply means A Stirring flow CM Culture liquid M Liquid medium

Claims (4)

A culture means for culturing cells by enclosing the culture solution;
A cell separation means comprising a filtration membrane that does not permeate cells contained in the culture medium inside the culture means, and a discharge part that discharges the culture liquid that has passed through the filtration membrane to the outside of the culture means;
Stirring means for stirring the culture solution to generate a stirring flow;
Medium supply means for supplying a liquid medium for culturing the cells inside the culture means,
The cell culture device, wherein the cell separation means is provided so that the stirring flow is in a tangential direction with respect to the filtration membrane. In claim 1,
The culture means includes a culture bag having flexibility and capable of enclosing the culture solution therein, and a housing for maintaining the shape of the culture bag in which the culture solution is enclosed. Cell culture device. In claim 2,
The cell culturing apparatus, wherein the agitating means causes the housing to rotate in a horizontal plane or is agitated by an agitating blade disposed in the culture bag. A culture means for culturing cells by enclosing the culture solution, a filtration membrane that does not permeate cells contained in the culture solution inside the culture means, and the culture solution that has permeated the filtration membrane is discharged out of the culture means A cell separation means comprising a discharge section, a stirring means for stirring the culture solution to generate a stirring flow, and a medium supply means for supplying a liquid medium for culturing the cells inside the culture means, The cell separation means is a method of culturing cells using a cell culture device provided so that the stirring flow is tangential to the filtration membrane,
When culturing the cells,
A discharging step of stirring the culture solution with the stirring means to generate the stirring flow, filtering the culture solution with a filtration membrane of the cell separating means and discharging the culture solution out of the culture means;
A supply step of supplying a liquid medium of approximately the same amount as the culture solution discharged out of the culture means from the culture medium supply means to the inside of the culture means;
In a random order, and these steps are carried out continuously.

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