JP2018038339A - Cell separation device and culture apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell separation apparatus and a culture apparatus capable of carrying out a separation operation of separating cultured cells in a culture solution from the solution for an extended period.SOLUTION: The cell separation apparatus 1 comprises: a vessel 2 provided with a first communication part 6 having an opening at one end part 5 communicating with the outside and a second communication part 8 having an opening at another end part 7 communicating with the outside; a plurality of filters 3 arranged in parallel in the vessel 2, and connected to the first communication part 6 and the second communication part 8; and a switching mechanism 4 which switches any of the filters 3 and which allows the first communication part 6, the filter 3, and the second communication part 8 to communicate.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell separation device and a culture device having the same.

Animal cells can be cultured to produce useful substances such as antibody proteins, or microorganisms and plant cells can be cultured to produce useful substances such as antibiotics and physiologically active substances. Culture methods for culturing living cells such as animal cells and microorganisms include batch culture, fed-batch culture, fed-batch culture, high-density fed-batch culture,
Continuous culture (Perfusion Culture), high density continuous culture (Concentrated Perfusion Culture)
There is.

  In general, since the cost of a culture medium is high, there is an intention to increase the density of living cells per unit volume of the culture medium in order to improve productivity. Examples of the culture method capable of increasing the living cell density include the above-described high-density fed-batch culture and high-density continuous culture. In order to maintain a high density of living cells, it is essential to separate the cultured cells and the solution in the culture solution, extract only the solution from the culture tank, and supply a new medium. Various cell separation means for performing operations have been developed.

  Examples of the cell separation means include a gravity sedimentation method, a centrifugal separation method, an ultrasonic agglutination method, and a filtration separation method. Among these, the filtration separation method has an advantage that the apparatus configuration is simple, and when production is performed on an industrial scale, it is relatively easy to scale up.

  An invention using a filtration separation method is described in Patent Document 1, for example. This Patent Document 1 illustrates and explains that a container containing a bundle of hollow fiber separation membranes is connected to a container containing a cell suspension, a waste liquid container, and a collection container. . A container containing a bundle of hollow fiber type separation membranes described in Patent Document 1 is sometimes referred to as a hollow fiber module.

Japanese Patent Laying-Open No. 2015-12837

  However, the filtration separation method including the invention described in Patent Document 1 is substantially inevitable for clogging of the filtration membrane, and the separation operation for separating the cultured cells and the solution in the culture solution is performed for a long time. There is a problem that it is difficult to implement.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a cell separation device and a culture device capable of performing a separation operation for separating a cultured cell and a solution in a culture solution for a long period of time. .

  The cell separation device according to the present invention that has solved the above problems includes a first communication portion having an opening communicating with the outside at one end, and a second communication having an opening communicating with the outside at the other end. A container provided with a portion, a plurality of filters arranged in parallel in the container, and connected to the first communication portion and the second communication portion, and arbitrarily switching the filter, the first communication portion, A switching mechanism for communicating the filter and the second communication portion.

  Moreover, the culture apparatus according to the present invention includes a first communication portion having an opening communicating with the outside at one end, and a second communication portion having an opening communicating with the outside at the other end. A plurality of containers, a filter arranged in parallel in the container, and connected to the first communication part and the second communication part; and the filter is arbitrarily switched, and the first communication part, the filter and the first A cell separation device having a switching mechanism for communicating two communicating portions, a culture vessel for storing a culture solution, a pressure generating device for generating pressure in the cell separation device, and one open end portion of the culture vessel A first flow path in which the other open end is connected to the first communicating portion, one open end is connected to the second communicating portion, and the other open end is the pressure. And a second flow path connected to the generator.

  The cell separation device and the culture device according to the present invention can perform a separation operation for separating a cultured cell and a solution in a culture solution for a long period of time.

It is a schematic side view explaining the structure of the cell separation apparatus which concerns on this embodiment. It is a partially cutaway view showing a configuration example of a first communication part, a filter, and a second communication part in the present embodiment. It is a top view of the cell separation apparatus which shows the arrangement position of a filter. It is a schematic block diagram explaining the structure of the culture apparatus which concerns on this embodiment. It is a schematic block diagram explaining the other aspect of the switching mechanism of the cell separation apparatus and culture apparatus which concern on this embodiment. It is a schematic block diagram explaining the other aspect of the switching mechanism of the cell separation apparatus and culture apparatus which concern on this embodiment. It is a schematic block diagram explaining the other aspect of the switching mechanism of the cell separation apparatus and culture apparatus which concern on this embodiment. It is a schematic block diagram explaining the other aspect of the switching mechanism of the cell separation apparatus and culture apparatus which concern on this embodiment. It is a schematic block diagram explaining the other aspect of the switching mechanism of the cell separation apparatus and culture apparatus which concern on this embodiment.

Hereinafter, an embodiment of a cell separation device and a culture device according to the present invention will be described in detail with reference to the drawings as appropriate.
[Cell separator]
FIG. 1 is a schematic side view illustrating the configuration of the cell separation device 1 according to the present embodiment.
FIG. 2 is a partially cutaway view showing a configuration example of the first communication part, the filter, and the second communication part in the present embodiment.
As shown in FIG. 1, the cell separation device 1 mainly includes a container 2, a filter 3, and a switching mechanism 4.
The cell separation device 1 forms part of a culture device 100 (see FIG. 4) to which a filtration separation method is applied, and serves to separate the cultured cells and the solution in the culture medium M and extract only the solution. . Here, in the present specification, the cultured cell refers to a cell cultured in the culture medium M. The culture solution M refers to a solution for culturing cultured cells. As the culture solution M, one appropriately prepared according to the cell to be cultured or one that is commercially available can be used, and is not limited to a specific one. Examples of cells to be cultured include animal cells, plant cells, insect cells, bacteria, yeasts, fungi, and algae, but any cells that can produce useful substances may be used. However, it is not limited to these. Examples of useful substances include proteins such as antibodies and enzymes, and physiologically active substances such as low molecular compounds and high molecular compounds, but are not limited thereto. The solution separated by the cell separation device 1 includes nutrient components for growing the cells and waste products such as secreted ammonia and lactic acid produced when the cells are grown. Hereinafter, the solution separated by the cell separation device 1 may be referred to as “filtrate”. Further, the useful substance may be secreted into the cell, but may be secreted outside the cell and contained in the filtrate F. In this embodiment, the case where a useful substance is secreted extracellularly and is contained in the filtrate F is mainly demonstrated.

(container)
The container 2 includes a first communication portion 6 having an opening portion that communicates with the outside at one end portion 5, and a second communication portion 8 that has an opening portion that communicates with the outside at the other end portion 7. That is, in the example shown in FIG. 1, the container 2 includes one set (a pair) of the first communication portion 6 and the second communication portion 8. As will be described later, the first communication section 6 is connected to the first flow path 40 to communicate with the culture tank 20 (see FIG. 4), and the second communication section 8 is connected to the second flow path 50. And communicates with the pressure generator 30. In addition, the 1st communication part 6 and the 2nd communication part 8 can be made into the pipe joint 6a and the pipe joint 8a which have a shape like what is called a hose nipple provided with the above-mentioned opening part (refer FIG. 2). That is, the first flow path 40 is inserted and connected to the first communication part 6, and the second flow path 50 is connected to the second communication part 8. As for the pipe joint 6a and the pipe joint 8a, the unevenness | corrugation which prevents that these fall | off | omit in the surface which contact | connects the 1st flow path 40 or the 2nd flow path 50 may be provided. It is preferable that the apertures of the first communication part 6 and the second communication part 8 have the same diameter as the inner diameter of the first flow path 40 and the second flow path 50. The first channel 40, the second channel 50, the culture tank 20, and the pressure generator 30 will be described later.

  One end portion 5 and the other end portion 7 may be lid members that can be attached to and detached from the main body 21 of the container 2. However, the container 2 is configured so that the inside and outside of the container 2 cannot be communicated except for the first communication part 6 and the second communication part 8 in order to prevent contamination of the culture solution M during culture. The container 2 can be made of, for example, a metal such as stainless steel or aluminum, a plastic such as polypropylene or polystyrene, or a glass. As for the container 2, it is preferable that the main body 21 is being fixed to the wall part or support | pillar of a plant | facility by the support member which is not illustrated. Moreover, it is preferable that the lower part of the container 2 has a discharge port 9 for discharging the filtrate F out of the container 2. The outlet 9 is preferably provided at the bottom of the container 2, that is, directly above the other end 7 so that the filtrate F does not remain in the container 2.

(Filter and switching mechanism)
The filter 3 in the present embodiment is a structure using an ultrafiltration membrane having a pore size that allows only a solution of the culture medium M to pass through without allowing cultured cells to permeate. Therefore, the filter 3 allows only the solution of the culture solution M to permeate and separates the cultured cells and the filtrate F in the culture solution M.
In the present embodiment, a plurality of filters 3 are arranged in parallel in the container 2, and are sequentially connected to the first communication part 6 and the second communication part 8 of the container 2.

The switching mechanism 4 plays a role of switching the filter 3 arbitrarily to allow the first communication unit 6, the filter 3, and the second communication unit 8 to communicate with each other. Since the switching mechanism 4 connects the first communication unit 6, the filter 3, and the second communication unit 8, the switching unit 4 joins the first communication unit 6 and the filter 3, and the second communication unit 8 and the filter 3. A sealing material 11 such as an O-ring is provided at the joint portion. The sealing material 11 may be provided in the filter 3, or may be provided in the first communication part 6 or the second communication part 8. Since the switching mechanism 4 is provided with the sealing material 11, the filter 3 is arbitrarily switched by the switching mechanism 4, and then the first communication portion 6 and the second communication portion 8 of the container 2 are moved by an actuator (not shown). Airtightness is obtained by pressing against the filter 3. In addition, as an actuator used when pressing the first communication portion 6 and the second communication portion 8 to the filter 3 and releasing the above-described pressing when the filter 3 is switched, for example, an electric motor or hydraulic pressure is used. A pump, an air cylinder, etc. are mentioned. However, the actuator is not limited to these, and any actuator can be used as long as it can be connected to each of the first communication portion 6 and the second communication portion 8 and can apply a pressing force thereto. it can.

(One aspect of switching mechanism)
The switching mechanism 4 described above has several modes, which will be described later, and one mode among them will be described below.
Since the switching mechanism 4 arbitrarily switches a plurality of filters 3 arranged in parallel in the container 2, it is preferable that the filter 3 can be easily switched. From such a viewpoint, the filter 3 is preferably a hollow fiber membrane module 3a as shown in FIG.

  As shown in FIG. 2, the hollow fiber membrane module 3a has a hollow fiber membrane 3b through which the culture medium M flows, and a hollow fiber opening end that holds the openings 3c and 3c of the hollow fiber membrane 3b open. Parts 3d and 3d. The hollow fiber opening end 3d may be obtained by collecting the hollow fiber membranes 3b in a bundle, applying a resin or the like to the peripheral surface in the vicinity of the opening 3c, and bonding and solidifying the resin. Bottom outer cylindrical members 3h and 3i are attached to the outer end surfaces of the hollow fiber opening end portions 3d and 3d, respectively, so that predetermined spaces 3j and 3k can be secured between the hollow fiber opening end portions 3d. It has become. In addition, an opening 3n for communicating the space 3j and the first communication portion 6 is provided in the central portion of the bottom of the outer member 3h. Similarly, an opening 3p for communicating the space 3k and the second communication portion 8 is provided in the central portion of the bottom of the outer member 3i. Further, a sealing material 11 such as an O-ring is provided on the outer surfaces of the outer members 3h and 3i in order to obtain airtightness. The hollow fiber membrane module 3a has a cover 3f. The cover 3f will be described later.

  Since it is set as such a structure, the culture solution M introduce | transduced from the 1st communication part 6 can be introduce | transduced uniformly to each opening part 3c of the hollow fiber membrane 3b once it introduce | transduces into the space 3j. . Then, when the culture solution M is introduced into the hollow fiber membrane 3b from the opening 3c, only the solution of the culture solution M permeates out of the hollow fiber membrane 3b without allowing the cultured cells to permeate out of the hollow fiber membrane 3b. . In addition, when the hollow fiber membrane module 3a is used as the filter 3 as in this embodiment, it is preferable that a part of the hollow fiber membrane 3b is exposed in the space in the container 2 as shown in FIGS. . If it does in this way, the filtrate F which permeate | transmitted the hollow fiber membrane 3b can be stored in the container 2. FIG. In FIGS. 1 and 2, support members that support these members between the outer member 3 h and the cover 3 f and between the outer member 3 h and the outer member 3 i are not shown, but such a support member is interposed between these members. You may provide and support. This is preferable because not only the filter 3 has high strength but also handling properties are improved. The cultured cells that have not been filtered are stored in the hollow fiber membrane 3b, the space 3k, the second flow path 50, and the pressure generator 30, and are returned to the culture tank 20 by the pressure generator 30 as described later.

Furthermore, as shown in FIG. 1, it is preferable that the plurality of filters 3 described above are all housed in one container 2. In this way, it is possible to prevent contamination of bacteria when switching the filter 3 or the like.
Further, the filtrate F separated by the filter 3 accumulates in the lower portion of the container 2. Therefore, in order to prevent the filtrate F separated by the other filter 3 from adhering to the unused filter 3e (see FIG. 1), the lower portion of the filter 3 is preferably covered with a cover 3f. The cover 3f can be formed of, for example, a synthetic resin or a metal, but is preferably made of a synthetic resin for easy disposal. In addition, when the lower part of the filter 3 is covered with the cover 3f, the filtrate F will be stored in the container 2 after exceeding the upper end of the cover 3f after filling the inside of the cover 3f. In addition, since most of the volume in the cover 3f is occupied by the hollow fiber membrane module 3a, the amount of the filtrate F remaining in the cover 3f is not large, and the decrease in the yield of useful substances due to this is particularly problematic. is not.

  Here, FIG. 3 is a plan view of the cell separation device 1 showing the arrangement position of the filter 3. As shown in FIG. 3, a plurality (five in FIG. 3) of filters 3 are arranged in parallel in the container 2 on the same circumference around the rotation axis 12. As shown in FIGS. 1 and 3, the filter 3 can be arranged so that the introduction direction of the culture medium M is vertical, that is, the longitudinal direction is perpendicular to the horizontal plane. Moreover, the filter 3 can be arrange | positioned so that the introduction direction of the culture solution M may become horizontal, ie, a longitudinal direction may become parallel with respect to a horizontal surface, as shown in FIG.6 and FIG.8. This aspect will be described later.

  As described above, the switching mechanism 4 releases the pressing of the filter 3 by an actuator (not shown). Then, as shown in FIG. 1, one end portion 5 and the other end portion 7 are rotated in the A direction around the rotation shaft 12 by an actuator 13 such as an electric motor, and the first communication portion 6 and the second communication portion. The filter 3 communicating with 8 is switched. The direction of rotation may be opposite to the direction A in the figure. The one end 5 described above is a lid member disposed on the upper side in FIGS. 1 and 2, and the other end 7 is a lid disposed on the lower side in FIGS. 1 and 2. It is a member. That is, in this aspect, the upper and lower lid members are integrally rotated by the actuator 13.

  The actuator 13 can be provided at one end 5 and the other end 7 respectively. However, as shown in FIG. 1, the one end 5 and the other end 7 are connected by a rotating shaft 12 and fixed. It can be set as the mode to do. If it does in this way, since the one edge part 5 and the other edge part 7 can be simultaneously rotated with one actuator 13, it is preferable from a viewpoint of energy saving and simplification of control.

  Further, in this aspect, as shown in FIGS. 1 and 2, positioning members 14 and 15 for positioning the filter 3 are placed close to one end 5 and the other end 7 in the container 2, respectively. Is provided. The positioning members 14 and 15 have a plurality of mounting holes 14 a and 15 a for arranging (fitting) the filter 3 at a predetermined position in the container 2. The attachment holes 14a and 15a have shapes of the outer members 3h and 3i, a shape conforming to the outer diameter, and an opening diameter, respectively. The filter 3 is fixed by fitting the outer members 3h and 3i into the mounting holes 14a and 15a. Instead of fitting the outer member 3h into the mounting hole 14a or fitting the outer member 3i into the mounting hole 15a, each may be screwed, fused, bonded, or the like. In this case, since these members are more reliably fixed, there is no possibility that the filter 3 is detached from the mounting holes 14a and 15a during filtration, which is preferable.

  The main body 21 and the positioning members 14 and 15 are detachably and airtightly fixed by an arbitrary fixing means such as screwing. The positioning member 14 and one end portion 5 are slidably attached through a sealing material 11 such as an O-ring while maintaining airtightness, and the positioning member 15 and the other end portion 7 are also formed such as an O-ring. It is slidably attached via the sealing material 11 while maintaining airtightness (see FIG. 1).

  As described above, one end 5 and the other end 7 are applied with pressure in a direction in which they approach each other by an actuator (not shown). Therefore, at the time of filtration, the one end portion 5 and the other end portion 7 are brought close to each other, and the sealing material 11 is crushed between the one end portion 5 and the positioning member 14, and the other end portion 7 and the positioning member 15 are The sealing material 11 is crushed in between, and these are fixed while improving airtightness.

When the filter 3 is switched, the application of pressure by the actuator is released so that the one end portion 5 and the other end portion 7 are not separated from the sealing material 11 provided in the positioning member 14 and the positioning member 15, respectively. Move it slightly away. Then, the actuator 13 is driven to rotate the one end 5 and the other end 7 fixed to the rotating shaft 12 by a predetermined angle around the rotating shaft 12. In this way, since airtightness is ensured also at the time of filtration and switching, contamination with germs can be prevented. In order to prevent cultured cells remaining in the filter 3 from being mixed into the filtrate when the filter 3 is switched, the pressure generator 30 (see FIG. 4) is driven to drive the pressure in the filter 3 as described later. And the culture medium M remaining in the filter 3 is returned to the culture tank 20, and then the filter 3 is switched.

  It is preferable that a sealing material 11 such as an O-ring is also provided between the filter 3 and the positioning members 14 and 15. In this way, even when one end 5 and the other end 7 are rotated, the culture medium M and the filtrate F can be prevented from leaking out of the container 2, and the container 2 It is possible to prevent contamination from outside.

[Culture equipment]
Next, the culture apparatus according to the present embodiment will be described. FIG. 4 is a schematic configuration diagram illustrating the configuration of the culture apparatus according to the present embodiment.
As shown in FIG. 4, the culture device 100 according to the present embodiment includes a culture tank 20, a pressure generator 30, a first flow path 40, in addition to the cell separation device 1 according to the present embodiment described above. And a second flow path 50.
In addition, in the following description, the same code | symbol is attached | subjected about the same component between the cell separation apparatus 1 and the culture apparatus 100, and detailed description is abbreviate | omitted.

(Culture tank)
The culture tank 20 is a container that stores the culture solution M. The culture tank 20 may be formed of an arbitrary volume using an arbitrary material selected from glass, metal, plastic and the like. The culture tank 20 includes a gas supply facility such as air, oxygen, nitrogen and carbon dioxide, a hot / cold water supply facility, a steam supply facility, and a water supply / drainage facility (all not shown). The culture solution M stuck in the culture tank 20 is stirred by a stirrer 22 driven by a driving motor 21 and mixed uniformly. Oxygen required for the culture is ventilated by a conventional method in which oxygen-containing gas is supplied into the liquid from a diffuser (not shown) such as a sparger disposed at the bottom of the tank, and in the gas phase at the top of the tank. It is supplied by two methods of top surface ventilation.

  The culture tank 20 includes a measuring means 23 for measuring the properties of the culture medium M, and obtains measured values of dissolved oxygen concentration, dissolved carbon dioxide concentration, pH, temperature, ammonia concentration, lactic acid concentration, and glutamine concentration. As for the measurement means 23, one detection means is used for each detection item or control item in the actual apparatus, but only one is shown in FIG. 4 for simplification. Moreover, the culture tank 20 is equipped with the liquid level gauge 24, and the liquid level of the culture solution M in the culture tank 20 is managed by measuring the liquid level height of the culture solution M.

Moreover, the culture tank 20 is equipped with the individual operation means (not shown) which controls each supply gas in the liquid and the ventilation | gas_flowing system to an upper surface. This individual operation means has a flow rate control function and a supply amount measurement function for each of air, oxygen, and carbon dioxide supplied in the liquid and on the upper surface. Further, the composition and aeration amount of the supply gas are controlled by the individual operation means.
As an example of the control of the composition of the supply gas and the amount of aeration, in this embodiment, air is aerated at a constant amount, and carbon dioxide gas is mixed corresponding to the pH of the culture medium M. The control of the carbon dioxide concentration can be performed by a normal proportional control using the pH as the control amount and the carbon dioxide flow rate as the operating factor.

  The aeration into the culture medium M by the aeration means can be controlled in its composition and aeration quantity by the individual operation means for controlling the supply gas. As an example of aeration into the culture solution M by the aeration means, in this embodiment, the dissolved oxygen concentration of the culture solution M is used as a control amount, and the oxygen aeration amount is used as an operation factor.

  The culture tank 20 is held at a constant pressure by a pressure regulating valve (not shown) and a pump (not shown) based on the measurement result of the pressure gauge P2. Usually, in order to prevent invasion of germs and the like from the outside, the inside of the culture tank 20 is pressurized to 0.01 to 0.05 MPa by a gauge pressure by a pressure adjusting valve and a pump.

  The culture tank 20 is configured to prevent contamination of germs from outside the culture tank 20. For example, the culture vessel main body 25 and the culture vessel lid member 26 may be joined via a sealing material (not shown) such as an O-ring. Further, in the culture tank 20, a portion where the measurement means 23 and the aeration means (not shown) are penetrated may be sealed with a sealing agent or the like.

(Pressure generator)
The pressure generator 30 is a device that generates a pressure for making the inside of the cell separation device 1 a positive pressure or a negative pressure. Note that the pressure generator 30 also generates pressure in the second flow path 50 disposed between the cell separator 1 and the pressure generator 30 in the same manner, and between the cell separator 1 and the culture tank 20. Similarly, a pressure is also generated in the first flow path 40 disposed in the. For example, a diaphragm pump is preferably used as the pressure generating device 30, but any device can be used without being limited thereto as long as the pressure can be generated. The diaphragm pump is a kind of positive displacement pump and transfers fluid by combining the reciprocating motion of a rubber, thermoplastic resin or fluororesin diaphragm with any check valve. The diaphragm pump raises and lowers the diaphragm (diaphragm) instead of the piston, so that no foreign matter is mixed in and a clean operation is possible.

(First channel and second channel)
As for the 1st flow path 40, one opening edge part 41 is arrange | positioned in the culture tank 20, and the other opening edge part 42 is connected with the 1st communication part 6 (refer FIG. 2). In addition, in FIG. 4, the 1st flow path 40 is a mode that the flow path member 40a connected with the 1st communication part 6 and the flow path member 40b provided in the culture tank 20 were connected with the connector 40c. Show. The flow path member 40a is provided with a valve V1, and the flow path member 40b is provided with a valve V2. In this way, for example, when the flow path member 40a is deteriorated, the flow path member 40a can be removed from the connector 40c and replaced with a new flow path member 40a.

  The first flow path 40 may be provided with a pressure pump 10 that applies pressure to the culture medium M during filtration. For example, as shown in FIG. 4, the pressure pump 10 may be provided in the flow path member 40b. As the pressure pump 10, for example, a syringe pump, a peristaltic pump, an air pump, a pressure cylinder, and the like can be used, but the pressure pump 10 is not limited thereto. The first flow path 40 can be composed of a single flow path material. In that case, the number of valves may be one.

The second flow path 50 has one open end 51 connected to the second communication portion 8 and the other open end 52 connected to the pressure generator 30. The second flow path 50 may or may not be provided with a valve (not shown).
As the above-described valves V1 and V2, general ones used for cell culture can be used, and the solution can be transferred by opening and closing them appropriately. The opening / closing operation of these valves V1 and V2 and the transfer of the solution will be described later.

The first flow path 40 and the second flow path 50 are preferably formed using a flexible material such as silicon resin or fluororesin. However, if the above-described elements can be connected to each other, What was formed with what kind of material can be used. In addition, when the 1st flow path 40 and the 2nd flow path 50 are formed with a flexible material, for example, the above-mentioned switching mechanism 4
As described above, even when one end 5 and the other end 7 are rotated, since they have flexibility, they can easily follow these rotations. In this case, if all the filters 3 are used in order, all the used filters 3 are replaced with new ones, so that the rotation angle of one end 5 and the other end 7 is less than one rotation. If the rotation angle of one end 5 and the other end 7 is less than one rotation, the first flow path 40 and the second flow path 50 formed of a flexible material are twisted and the flow path is closed. It is never done. The flow path member 40b may be made of a non-flexible material such as a metal or a hard resin.

(Control device)
The cell separation device 1 and the culture device 100 are connected to the control device C by wire or wirelessly. The control device C switches the filter 3 by the switching mechanism 4, adjusts various components such as the amount of dissolved oxygen based on the properties of the culture medium M measured by the measuring means 23, and adjusts the liquid medium from the medium tank 20 to the culture medium M. Addition and control of the pressure generator 30 are performed. The control device C can be realized by a computer having storage means such as a hard disk drive in which programs necessary for performing these controls and operations are stored.

(Medium tank)
The culture apparatus 100 has a medium tank 60 in which a liquid medium necessary for cell culture is accommodated. The culture medium tank 60 is connected to the culture tank 20 via a third flow path 61 and a valve V3 provided on the third flow path 61. In the present embodiment, the same amount of liquid medium removed from the culture medium M as the filtrate F by the cell separator 1 is supplied from the medium tank 60 into the culture tank 20. The medium tank 60 can be made of, for example, glass, in addition to a metal such as stainless steel or aluminum, or a plastic such as polypropylene or polystyrene.

  Moreover, the culture medium tank 60 is maintained at a constant pressure by a pressure regulating valve (not shown) and a pump (not shown) based on the measurement result of the pressure gauge P3. Usually, in order to prevent invasion of germs and the like from the outside, the inside of the medium tank 60 is pressurized to 0.01 to 0.05 MPa as a gauge pressure by a pressure adjusting valve and a pump to be a positive pressure.

(Filtration storage tank)
The culture apparatus 100 according to this embodiment preferably has a filtration storage tank 70. The filtration storage tank 70 is connected to the discharge port 9 in the lower part of the container 2 of the cell separation device 1 via a fourth flow path 71 and a valve V4 provided on the fourth flow path 71. The filtration storage tank 70 temporarily stores the filtrate F separated by the cell separation device 1. The filtration storage tank 70 can be made of, for example, glass in addition to a metal such as stainless steel or aluminum, or a plastic such as polypropylene or polystyrene.

  Further, the filtration storage tank 70 is held at a constant pressure by a pressure regulating valve (not shown) and a pump (not shown) based on the measurement result of the pressure gauge P4. Usually, the inside of the filtration storage tank 70 is pressurized to 0.01 to 0.05 MPa as a gauge pressure by a pressure regulating valve and a pump in order to prevent invasion of germs and the like from the outside.

(Purification equipment)
The culture apparatus 100 according to this embodiment preferably has a purification apparatus 80. The purification device 80 is connected to the filtration storage tank 70 via a fifth flow path 81 and a valve V5 provided on the fifth flow path 81. The purification device 80 purifies useful substances produced by cultured cells contained in the filtrate F transferred from the filtration storage tank 70.

  As the purification apparatus 80, for example, an affinity chromatograph, a high performance liquid chromatograph, an ion exchange chromatograph, a gel filtration chromatograph or the like can be used. In addition, although the refiner | purifier 80 can use only any 1 type selected from these, 2 or more types can also be used together as needed.

  The useful substance purified by the purification apparatus 80 is eluted with an arbitrary eluate and collected in a collection container (not shown). In addition, the washing buffer and the equilibration buffer used in the purification apparatus 80 are stored in a waste container (not shown) and discarded after a predetermined disposal process. In addition, the refiner | purifier 80 may be provided with the pressure gauge P5 which detects an internal pressure.

  In addition, all the above-mentioned 3rd-5th flow paths 61, 71, 81 and valves V3-V5 can use what is generally used for cell culture. For example, any of the above-described valves can be an on-off valve, a ball valve, a clamp valve, an electromagnetic valve, an electric valve, or the like. Further, a buffer tank (not shown) for reducing pressure fluctuation can be provided on the third to fifth flow paths.

[Example of operation of cell separation apparatus and culture apparatus]
An example of the operation of the cell separation device 1 according to the above-described embodiment and the culture device 100 having the same will be described.
As an example of the operation of these apparatuses, specifically, (1) culture step and culture solution transfer step, (2) filtration step and culture solution return step, (3) filtrate transfer step and filter switching step, (4 ) Purification step, (5) Medium transfer step. The culture device 100 according to the present embodiment performs all the steps (1) to (5), and the cell separation device 1 performs the steps (2) and (3).

(1) Culture process and culture solution transfer process First, cells are cultured in the culture tank 20 while the valves V1 to V5 are closed (culture process).
When a predetermined number of days elapses from the start of the culture, or when the concentration of the cultured cells in the culture medium M exceeds a predetermined value, the valve V3 is closed and the valves V2 and V1 are opened. As described above, since the inside of the culture tank 20 is at a positive pressure, the culture solution M is transferred to the cell separation device 1 through the first flow path 40 by the pressure (culture solution transfer step). At this time, if necessary, a pump and a pressure regulating valve provided in the culture tank 20 are driven to make the inside of the culture tank 20 further positive, or a pressure generator 30 such as a diaphragm pump provided in the cell separation device 1. When the pressure in the cell separation apparatus 1 is driven to a negative pressure, the culture medium M can be quickly transferred to the cell separation apparatus 1.

(2) Filtration step and culture solution return step Next, the above-described gas supply equipment that ventilates the gas phase part above the culture tank 20 is driven, the valve V1 is opened, and the culture solution M transferred to the cell separator 1 is transferred. The pressure in the first flow path 40 and the filter 3 is set higher than the pressure in the cell separation device 1. In this way, only the solution of the culture medium M permeates the filter 3 to become the filtrate F, and the cultured cells cannot pass through the filter 3 and remain in the culture medium M in the filter 3 (filtration step). ). Note that the pressure pump 10 may be used instead of driving the gas supply facility that ventilates the gas phase part above the culture tank 20.
In this way, after separating the filtrate F and the cultured cells from the culture solution M for a predetermined amount or for a predetermined time, the valves V3 and V4 are closed, and the valves V1 and V2 are opened. And the pressure generator 30 is driven, the pressure in the filter 3 is raised, and the culture solution M remaining in the filter 3 is returned to the culture tank 20 (culture solution return process). The culture solution M remaining in the filter 3 has a higher concentration of cultured cells than before filtration. By repeating this as many times as necessary, the concentration of cultured cells in the culture tank 20 can be increased.

(3) Filtrate transfer step and filter switching step The filtrate F filtered by the cell separation device 1 is temporarily stored in the cell separation device 1. This filtrate F contains useful substances produced by the cultured cells, waste products produced by the cultured cells, and the like.
When the filtrate F stored in the cell separator 1 reaches a predetermined amount, the valves V1 and V2 are closed and the valve V4 is opened. Then, the inside of the cell separation apparatus 1 is set to a positive pressure by the pressure generator 30 or the inside of the filtration storage tank 70 is set to a negative pressure by driving a pump and a pressure regulating valve provided in the filtration storage tank 70. The filtrate F is transferred into the filtration storage tank 70 (filtrate transfer process).

Here, when the cell separator 1 separates the cultured cells from the filtrate F, the filter 3 is clogged and the filtration cannot be performed smoothly. This can be grasped, for example, by monitoring the pressure fluctuation with the pressure gauge P1 provided in the cell separation device 1. When the pressure in the cell separation device 1 reaches a predetermined value, the switching mechanism 4 is operated to switch to a new filter 3 that is not used (filter switching step). In the example shown in FIGS. 1 and 4, the switching mechanism 4 switches the filter 3 by rotating one end portion 5 and the other end portion 7 about the rotation shaft 12 around an axis by an actuator 13 such as an electric motor. .
Note that switching to the new filter 3 may be performed when a predetermined period has elapsed. Moreover, the aspect which monitors a pressure fluctuation with the pressure gauge P1 and switches the filter 3 and the aspect which switches the filter 3 after a predetermined period can also be used together.

(4) Purification step When the filtrate F is transferred into the filtration storage tank 70 and reaches a predetermined amount, the valve V4 is closed and the valve V5 is opened. Then, the pump and pressure regulating valve provided in the filtration storage tank 70 are driven to make the inside of the filtration storage tank 70 positive pressure, or the pump and pressure regulating valve provided in the purification apparatus 80 are driven to make the inside of the purification apparatus 80 positive pressure. Or At the same time, an eluate capable of eluting the target useful substance is passed through the purifier 80. By doing in this way, the useful substance contained in the filtrate F is refine | purified (purification process).
When the purification of the useful substance is completed, a washing buffer for washing the inside of the purification device 80 and an equilibration buffer for equilibrating the purification device 80 are passed through the purification device 80 to prepare for the next purification step.

(5) Medium transfer process Moreover, when a solution is removed from the culture solution M as the filtrate F by the cell separation device 1 in this way, the liquid level of the culture solution M in the culture tank 20 decreases. Therefore, in the present embodiment, when this is detected by the liquid level gauge 24, the control device C opens the valve V3 and closes the valve V2 by an actuator (not shown). Then, the pump and pressure adjustment valve provided in the medium tank 60 are driven to make the inside of the medium tank 60 positive pressure, and the pressure adjustment valve provided in the culture tank 20 is driven so that the medium tank 60 is transferred to the culture tank 20. A liquid culture medium is transferred (medium transfer process).
When the liquid level gauge 24 detects that the liquid level of the culture medium M in the culture tank 20 has reached a predetermined height, the control device C closes the valve V3 by an actuator (not shown). At the same time, the pump provided in the medium tank 60 is stopped, and the supply of the liquid medium from the medium tank 60 to the culture tank 20 is stopped.
Next, returning to (1) again, the same operation is repeated.

After performing this multiple times, the concentration of the waste product in the culture medium M becomes high, so that cell culture cannot be performed, the growth rate of cultured cells decreases, and the production efficiency of useful substances decreases. If so, cell culture is stopped, and the culture medium M is completely filtered to purify useful substances.
Then, each tank, flow path, valve and the like are washed or replaced by a conventional method to prepare for the next cell culture.

  As described above, when the above-described series of operations are repeatedly performed using the cell separation device 1 and the culture device 100 according to the present embodiment, the filter 3 is switched to the new filter 3 before clogging. The useful substance contained in the filtrate F can be continuously and long-term purified while increasing the concentration of the cultured cells. That is, the cell separation device 1 and the culture device 100 according to the present embodiment can perform a separation operation for separating the cultured cells and the solution in the culture solution M for a long period of time.

  Some useful substances are secreted (encapsulated) into cells without being secreted outside the cells. In such a case, after obtaining the culture solution M containing the cultured cells at high density using the cell separation device 1 and the culture device 100 according to the present embodiment, the culture is performed using a centrifuge (not shown) or the like. What is necessary is just to concentrate a cell and to extract a useful substance by destroying a cell membrane etc. by arbitrary methods. Even in this case, the cell separation device 1 and the culture device 100 according to the present embodiment perform the separation operation for separating the cultured cells and the solution in the culture medium M for a long time when culturing the cultured cells. be able to.

Next, with reference to FIG. 5 to FIG. 9, another aspect of the switching mechanism for the cell separation device and the culture device according to the present embodiment will be described. Here, FIG. 5 to FIG. 9 are schematic configuration diagrams for explaining another aspect of the switching mechanism of the cell separation device and the culture device according to the present embodiment.
In addition, the same code | symbol is attached | subjected about the same component in the one aspect | mode of an above described switching mechanism, and the other aspect of the switching mechanism demonstrated below, and detailed description is abbreviate | omitted.

(Other aspects of switching mechanism)
Other aspects of the switching mechanism 4 of the cell separation device 1 and the culture device 100 include the following.
For example, as shown in FIGS. 5 and 3, the switching mechanism 4 </ b> A arranges a plurality of filters 3 in the container 2 in parallel on the same circumference around the rotation shaft 12.
Specifically, disk-shaped positioning members 14 and 15 are provided in the container 2 so as to be close to the one end portion 5 and the other end portion 7 respectively. A plurality of mounting holes 14 a and 15 a for positioning and fixing the filter 3 are provided on one surface of the positioning members 14 and 15. Further, the mounting holes 14 a and 15 a are provided on the same circumference around the rotation shaft 12. The mounting holes 14a and 15a are fixed to the rotary shaft 12 disposed at the center of rotation by making the surfaces on which they are provided face each other and matching the positions of the opposing mounting holes 14a and 15a. Yes.

  In the case of this aspect, one end 5 and the other end 7 are fixed with airtightness between the main body 21 by any fixing means such as screwing. Further, the positioning members 14 and 15 are installed between the inner wall of the main body 21 with a slight gap that allows rotation around the axis. And the main body 21 is being fixed to the wall part or support | pillar of a plant | facility with the support member which is not shown in figure. Furthermore, the rotating shaft 12 is connected to an actuator 13 that rotates the rotating shaft 12. The filter 3 is fixed by being fitted into the mounting holes 14a and 15a of the opposing positioning members 14 and 15.

Since the switching mechanism 4A drives the actuator 13 to rotate the positioning members 14 and 15 in the container 2 together with the rotating shaft 12, the filter 3 fixed to the switching mechanism 4A also rotates the rotating shaft 12. It rotates in the B direction on the same circumference with the center.
That is, by setting it as such an aspect, the switching mechanism 4A can rotate the filter 3 centering on the rotating shaft 12, and can switch the filter 3 connected to the 1st communication part 6 and the 2nd communication part 8. FIG. .

  Moreover, as a further modification of this aspect, the switching mechanism 4B can install the opposing rotating plate perpendicularly to the horizontal plane, for example, as shown in FIG. In this case, the rotating shaft 12 to which the positioning members 14 and 15 are fixed is installed in parallel to the horizontal plane (for convenience, this aspect may be referred to as “laterally”). When the hollow fiber membrane module 3a is used as the filter 3, in the hollow fiber membrane module 3a fixed to the positioning members 14 and 15, the flow path of the culture solution M is parallel to the horizontal plane.

In this modification, the switching mechanism 4B is provided sideways above the container 2, so that when the switching mechanism 4B rotates the positioning members 14 and 15 around the rotating shaft 12, the filter 3 fixed to this is fixed. Rotate in the D direction on the same circumference around the rotation axis 12 as well.
That is, by setting it as such an aspect, the switching mechanism 4B can rotate the filter 3 centering on the rotating shaft 12, and can switch the filter 3 connected to the 1st communication part 6 and the 2nd communication part 8. FIG. .

  In this modification, preferably, the first communication part 6 is provided at one end 5 at the position where the rotating filter 3 is lowest, and the second communication part 8 is provided at the other end 7. And if the 1st communication part 6, the filter 3, and the 2nd communication part 8 are connected by this position and it filters, the filtrate F filtered by the filter 3 will be used for the new filter 3e (not shown in FIG. 6). It can be made to flow down the container 2 without making contact. Therefore, in the case of this aspect, it is not necessary to provide the cover 3f.

  The container 2 in this modification can be made into an arbitrary shape. For example, the shape of one surface 27 on which one end 5 is disposed and the shape of the other surface 28 on which the other end 7 is disposed are both oval, oval, elliptical, and other oval shapes. To do. In this case, the area of one surface 27 may be reduced and the area of the other surface 28 may be increased. And when these are sealed with the surrounding wall 29, as shown in FIG. 6, the bottom part of the container 2 becomes low as the container 2 goes to the other surface 28 from the one surface 27. As shown in FIG. Therefore, it is preferable to provide the discharge port 9 in the vicinity of the other surface 28 where the bottom of the container 2 is lowest because the filtrate F can be easily discharged out of the cell separation device 1.

(Other aspects of switching mechanism)
Other aspects of the switching mechanism 4 of the cell separation device 1 and the culture device 100 include the following.
For example, as illustrated in FIG. 7, the switching mechanism 4 </ b> C arranges a plurality of filters 3 in parallel on the straight line in the container 2.
Specifically, rectangular fixing plates 14 c and 15 c are provided in the container 2 so as to be close to the one end 5 and the other end 6, respectively. As with the positioning members 14 and 15, the fixing plates 14c and 15c are provided with a plurality of mounting holes 14a and 15a for positioning and fixing the filter 3 on one surface. The fixing plates 14c and 15c are fixed by the fixing members 16 and 16 with the positions of the opposing mounting holes 14a and 15a being matched. This is preferable because the strength of the switching mechanism 4C is improved. The fixing of the positioning members 14 and 15 by the fixing members 16 and 16 is not limited to this aspect and can be performed in other aspects.

The fixing plates 14c and 15c are connected to moving means (not shown) for moving the fixing plates 14c and 15c. Examples of the moving means include a rack and pinion structure connected to an electric motor and an air cylinder. Accordingly, when the moving means is driven to move the fixing plates 14c and 15c on a straight line, the filter 3 fixed to the moving plate also moves on the straight line.
That is, by setting it as such an aspect, the switching mechanism 4C moves the filter 3 arrange | positioned in parallel on the straight line by the moving means to the E direction on a straight line, and is the 1st communication part 6 and the 2nd communication part. The filter 3 communicating with 8 can be switched.

Further, as a further modification of this aspect, the switching mechanism 4D can install the fixed plates 14D and 15D facing each other perpendicularly to the horizontal plane, for example, as shown in FIG. When the hollow fiber membrane module 3a is used as the filter 3, the hollow fiber membrane module 3a fixed to the fixing plates 14D and 15D is configured so that the flow path of the culture solution M (not shown in FIG. 8) is parallel to the horizontal plane. Become. That is, the switching mechanism 4D is turned sideways.
That is, by setting it as such an aspect, switching mechanism 4D moves the filter 3 arrange | positioned in parallel on the straight line by the moving means to the G direction on a straight line, and is the 1st communication part 6 and the 2nd communication part. The filter 3 communicating with 8 can be switched.
In this aspect, as described above, since the plurality of filters 3 are arranged in parallel on a straight line, when the first communication portion 6, the filter 3 and the second communication portion 8 are connected to perform filtration, the filter The filtrate F filtered at 3 can be made to flow down below the container 2 without being brought into contact with a new filter 3e (not shown in FIG. 8). Therefore, it is not necessary to provide the cover 3f in this case. Further, the fixing plate 14D and the fixing plate 15D may be connected and fixed by a fixing member (not shown). This is preferable because the strength of the switching mechanism 4D is improved.

  The container 2 in this modification can be made into an arbitrary shape. For example, as shown in FIG. 8, the shape of one surface 27 where one end 5 is disposed and the shape of the other surface 28 where the other end 7 is disposed are both hexagonal. It is done. As described above, for example, the area of one surface 27 may be reduced and the area of the other surface 28 may be increased. And when these are sealed with the surrounding wall 29, as shown in FIG. 8, the bottom part 29a of the container 2 becomes low as the container 2 goes to the other surface 28 from the one surface 27. As shown in FIG. Therefore, it is preferable to provide the discharge port 9 in the vicinity of the other surface 28 where the bottom 29a of the container 2 is lowest because the filtrate F can be easily discharged out of the cell separation device 1.

(Other aspects of switching mechanism)
Other aspects of the switching mechanism of the cell separation device 1 and the culture device 100 include the following.
For example, as shown in FIG. 9, the switching mechanism 4 </ b> E has a plurality of filters 3 in the container 2, and the same number of first communication parts 6 and second communication parts 8. All of the filters 3 in the container 2 are connected to a set of the first communication part 6 and the second communication part 8.
Further, the first switching valve 4E1 is provided in the first flow path 40 connected to the first communication part 6 or the first communication part 6. In addition, the second switching valve 4E2 is provided in the second flow path 50 connected to the second communication portion 8 or the second communication portion 8. In FIG. 9, the first switching valve 4 </ b> E <b> 1 is provided in the first flow path 40, and the second switching valve 4 </ b> E <b> 2 is provided in the second flow path 50. As described above, the first switching valve 4E1 may be provided in the first communication part 6, and the second switching valve 4E2 may be provided in the second communication part 8 (both not shown). )

The first switching valve 4E1 and the second switching valve 4E2 are connected to an actuator (not shown) such as an electric motor or an air pump that operates them. Then, the filter is connected to the first communication part 6 and the second communication part 8 by driving the actuator to operate the pair of first switching valve 4E1 and second switching valve 4E2 synchronously for each filter 3. Switch.
That is, only the filter 3 that performs filtration operates the first switching valve 4E1 and the second switching valve 4E2 synchronously to open them, and the filter 3 that does not perform filtration performs the first switching valve 4E1 and the second switching valve 4E1. The switching valve 4E2 is operated synchronously to be in a closed state. By doing in this way, only the filter 3 which is performing filtration communicates with the first switching valve 4E1 and the second switching valve 4E2, and the culture medium M can be filtered. In addition, the filter 3 which is not filtering means the new filter 3e and the filter 3 (used filter 3) used for filtration and switched by the switching mechanism 4E.

  Then, when the filter 3 that is performing filtration is clogged and it becomes difficult to filter the culture medium M, the first switching valve 4E1 and the second switching valve that are connected to the filter 3 that is difficult to filter. 4E2 is operated synchronously to a closed state, and the first switching valve 4E1 and the second switching valve 4E2 of the new filter 3e are operated synchronously to an open state. In this way, the new filter 3e communicates with the first switching valve 4E1 and the second switching valve 4E2, and the culture medium M can be filtered with the new filter 3e.

  As described above, the cell separation device and the culture device according to the present invention have been described in detail according to the embodiments. However, the present invention is not limited to the above-described embodiments, and includes various modifications. 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. Further, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

DESCRIPTION OF SYMBOLS 1 Cell separator 2 Container 3 Filter 3a Hollow fiber membrane module 3f Cover 4, 4A-4E Switching mechanism 4E1 1st switching valve 4E2 2nd switching valve 5 One end part 6 1st communication part 7 The other end part 8 2nd communication part 12 Rotating shaft 13 Actuator 40 1st flow path 50 2nd flow path 100 Culture apparatus F Filtrate M Culture liquid

Claims (11)

A container having a first communicating portion having an opening communicating with the outside at one end and a second communicating portion having an opening communicating with the outside at the other end;
A plurality of filters arranged in parallel in the container and connected to the first communication part and the second communication part;
A cell separation apparatus comprising: a switching mechanism that arbitrarily switches the filter to communicate the first communication part, the filter, and the second communication part. In claim 1,
The switching mechanism is
A plurality of the filters are arranged in parallel on the same circumference around the rotation axis,
The cell separation device, wherein the first and second communication portions are switched by rotating the one end portion and the other end portion around the rotation axis by an actuator. In claim 1,
The switching mechanism is
A plurality of the filters are arranged in parallel on the same circumference around the rotation axis,
The cell separation device, wherein the filter is rotated about the rotation axis by an actuator, and the filter connected to the first communication portion and the second communication portion is switched. In claim 1,
The switching mechanism is
A plurality of the filters are arranged in parallel on a straight line;
The cell separation device, wherein the filter arranged in parallel on a straight line by an actuator is moved on the straight line to switch the filter connected to the first communication part and the second communication part. In claim 1,
The switching mechanism is
The same number of the first communication portions and the second communication portions as the plurality of filters are provided, and all of the plurality of filters are connected to a set of the first communication portion and the second communication portion,
A first flow path connected to the first communication part, or a first switching valve provided in the first communication part;
A second flow path connected to the second communication part, or a second switching valve provided in the second communication part;
An actuator for operating the first switching valve and the second switching valve,
The cell separation device, wherein the first switching valve and the second switching valve are actuated by the actuator to switch a filter connected to the first communication portion and the second communication portion. In claim 1,
A cell separation device, wherein a lower part of the filter is covered with a cover. In claim 1,
A cell separation apparatus, wherein a plurality of the filters are all contained in one container. In claim 1,
The cell separation device, wherein the filter is a hollow fiber membrane module. In claim 1,
A cell separation apparatus, wherein a discharge port for discharging the filtrate filtered by the filter to the outside of the container is provided at a lower part of the container. A container having a first communication part having an opening communicating with the outside at one end and a second communication part having an opening communicating with the outside at the other end, and a plurality of the containers in parallel in the container A filter disposed and connected to the first communication part and the second communication part; and a switching mechanism for arbitrarily switching the filter to connect the first communication part, the filter and the second communication part; A cell separation device comprising:
A culture vessel containing a culture solution;
A pressure generator for generating pressure in the cell separator;
A first flow path in which one open end is disposed in the culture tank and the other open end is connected to the first communication portion;
A culture apparatus comprising: a second flow path having one open end connected to the second communication portion and the other open end connected to the pressure generating device. In claim 10,
The culture apparatus, wherein both the first flow path and the second flow path are formed of a flexible material.

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