JP2020174557A - Cell culturing/refining apparatus and cell culturing/refining method - Google Patents

Abstract

To provide a cell culturing/refining apparatus and a cell culturing/refining method in which a cell or a cell aggregate of a target size in culture solution can be recovered while an unnecessary object such as impurity is removed.SOLUTION: A cell culturing/refining apparatus 100 comprises: a culture tank 1; a first filter 2 which separates a target object (isolated cell, cell aggregate) pulled out from the culture tank 1 and a non-target object larger than target object; a second filter 3 which separates the target object separated by the first filter 2 and an impurity smaller than the target object; and a return flow channel 12 which returns the non-target object separated by the first filter 2 to the culture tank 1. A cell culturing/refining method includes: a process of culturing cells in the culture tank 1; a process of performing membrane separation treatment to liquid pulled out from the culture tank 1 and separating into a target object and a non-target object larger than the target object; and a process of performing membrane separation treatment to liquid including the separated target object and separating into a target object and an impurity smaller than the target object, and the non-target object separated by membrane separation treatment is returned to the culture tank 1.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cell culture purification apparatus and a cell culture purification method for separating and purifying cultured cells from impurities and the like.

In recent years, clinical introduction of cell therapy has been promoted. Cell therapy is a treatment method for treating, alleviating, or treating human diseases or injuries by administering own cells or cells of others. In cell therapy, stem cells capable of differentiating into various cells are used, including mesenchymal stem / stromal cells (MSCs) and iPS cells (induced pluripotent stem cells).

Human mesenchymal stem cells (hMSCs) have the ability to proliferate in an undifferentiated state and the ability to differentiate into bone, cartilage, fat, tendons, muscles, neurons and the like. In addition, iPS cells have pluripotency, are produced from somatic cells, and can be autologously transplanted. Stem cells such as MSCs and iPS cells are collected from the patients themselves or others, processed for differentiation induction, activation, gene transfer, etc., or cultured, and then administered or transplanted to the patients.

In general, stem cells such as MSCs and iPS cells proliferate in a cell-adhered state, so that they are planarly cultured (two-dimensionally cultured) in a culture flask, dish, plate or the like. Since these stem cells undergo differentiation and cell death due to slight environmental changes such as oxygen concentration and temperature, they are manually cultured little by little under strict control of culture conditions.

A stable large-scale supply of therapeutic cells is desired. However, it is known that stem cells such as MSCs and iPS cells are difficult to suspend culture due to various reasons such as physical stress, self-aggregation, and difficulty in passage. Since it is difficult to secure the culture area required for mass culture in the current mainstream planar culture, it is necessary to repeat a large number of culture containers and complicated operations for each culture container.

Conventionally, the use of microcarrier culture has been studied as a means for mass-culturing stem cells. Microcarrier culture is a technique in which bead-shaped microcarriers to which cells are adhered are suspended in a culture medium and cultured. Microcarrier culture is expected to be effective for large-scale culture because it is relatively easy to secure a culture area and the culture solution can be shaken and stirred.

In addition, a technique for suspending and culturing aggregated stem cells is also being studied. Originally, it is difficult to suspend culture of stem cells, but in a culture system without an adhesive culture surface, cells adhere and aggregate three-dimensionally to form cell aggregates (spheroids). When cell aggregates are formed, the cells are brought into a state close to in vivo, and proliferative ability and enhancement of cell activity appear. Therefore, culture conditions have been studied.

In general, therapeutic cells need to be product-managed in a state where the target cells have high purity and few impurities from the viewpoint of quality and safety as a cell processed product. Contaminants mixed in cell processed products used for cell therapy include unintended substances derived from cells, substances mixed in during the manufacturing process, and the like. However, it is desired that these unnecessary substances are reduced as much as possible by a physicochemical separation / purification method or a biological separation / purification method.

In addition, when the size of the cell aggregate is small, it is easy to lead to cell death due to apoptosis or the like, and when the size is large, it is easy to differentiate or to lead to cell death due to lack of oxygen or nutrients in the central part. Or become. The size of cell aggregates changes due to self-aggregation during suspension culture, but it is hoped that the same lot of cell aggregates will be uniform in size and shape from the viewpoint of quality as a cell processed product and therapeutic effect. It is rare.

Conventionally, a technique has been proposed in which a plurality of stages of membrane separation treatment are combined with bioprocesses such as cell culture and enzymatic reaction, and separation and purification of a target product produced by the bioprocess.

For example, Patent Document 1 describes a multi-stage filtration system for filtering a biotreated feedstock that has been subjected to treatments such as saccharification and fermentation (see FIG. 3). This multi-stage filtration system collects passages that have passed through both the first and second membrane filter units, while concentrating without passing through the first or second membrane filter units. It is configured to return the goods to the supply side.

Further, Patent Document 2 describes a cell culture device used for producing an antibody (see FIGS. 1, 4, 5, etc.). This cell culture device sends the component that has passed through the first filter section to the second filter section, and returns the component blocked by the first filter section and the component that has passed through the second filter section to the culture vessel. , The structure is such that the antibody or the like blocked by the second filter unit is collected.

Special Table 2017-527267 International Publication No. 2018/159847

When a large amount of target cells are cultured to produce a cell processed product, etc., the culture solution cultured in the culture tank contains, in addition to the target cells, metabolites produced by the cells, medium components, added drugs, etc. , Unnecessary impurities are mixed. In general, these impurities are smaller than MSCs, iPS cells, and the like, so size fractionation using a filter is effective for separation and purification. However, in the case of microcarrier culture or suspension culture of aggregated spheroids, there is a problem that not only impurities smaller than the target cells but also unnecessary substances larger than the target cells are generated.

For example, in the case of microcarrier culture, it is common to add a cell exfoliating agent such as trypsin to the culture solution after culturing to exfoliate the cells adhering to the microcarrier and then recover the cells. In such a case, a large amount of a cell exfoliating agent smaller than the target cell is mixed in the culture solution after culturing, while microcarriers larger than the target cell remain, so that the target cell is recovered. A complicated separation and purification operation is required.

Further, in the case of suspension culture of aggregated spheroids, the culture solution after the culture is in a state in which cell aggregates having different sizes are mixed. After culturing, independent cells that do not adhere to cells may be required, or cell aggregates of a predetermined size may be required. However, in any case, impurities smaller than these target substances and cell aggregates larger than these target products may coexist, which makes it complicated to recover the target products. Separation and purification operation is required.

Therefore, the present invention provides a cell culture purification apparatus and a cell culture purification method capable of removing and recovering unnecessary substances such as impurities from cells in a culture medium and cell aggregates of a desired size. The purpose.

As a result of diligent studies, the present inventors have drawn out the liquid in the culture tank, and then used the first-stage filter to obtain the target substance contained in the cultured liquid, microcarriers larger than the target substance, cell aggregates, etc. By separating the non-target product from the above and separating the target product from impurities smaller than the target product with a second-stage filter, the cells are recultured using the non-target product such as microcarriers and cell aggregates. We have found that it is possible to separate and purify target cells and cell aggregates of a target size with high purity while continuing the above, and have completed the present invention.

That is, in order to solve the above-mentioned problems, the cell culture purification apparatus according to the present invention has a culture tank for culturing cells, a target substance in a liquid drawn from the culture tank, and a non-purpose object larger than the target product. A first filter that separates substances from each other, a second filter that separates the target substance in the liquid separated by the first filter and impurities smaller than the target substance from each other, and the first filter. It is provided with a return flow path for returning the non-target substance separated by the above filter to the culture tank, and the target substance is an isolated cell or a cell aggregate.

Further, the cell culture purification method according to the present invention includes a step of culturing cells in a culture tank and a membrane separation treatment of the liquid drawn from the culture tank to separate the target substance in the liquid and a non-target product larger than the target product. The step of separating the target product from each other and the membrane separation treatment of the liquid containing the target product separated by the membrane separation treatment are performed to separate the target product in the liquid and impurities smaller than the target product from each other. The object is an isolated cell or a cell aggregate.

The cell culture purification apparatus and cell culture purification method according to the present invention can recover cells and cell aggregates of a desired size in a culture medium by removing unnecessary substances such as impurities.

It is a schematic diagram which shows the schematic structure of the cell culture purification apparatus which concerns on embodiment of this invention. It is a schematic diagram which shows the culture tank of a batch culture type. It is a schematic diagram which shows the culture tank of a fed-batch culture type. It is a schematic diagram which shows the culture tank of a perfusion culture type. It is a figure which shows the general characteristic of the filter provided in the filter unit. It is a figure which shows the peristaltic pump used as a liquid feeding device. It is a figure which shows the magnetic levitation type centrifugal pump used as a liquid feeding device. It is a figure which shows the pressurizer used as a liquid feeding device. It is a flowchart which shows the cell culture purification method which concerns on embodiment of this invention. It is a schematic diagram which shows the schematic structure of the cell culture purification apparatus which concerns on the modification of this invention. It is a flowchart which shows the cell culture purification method which concerns on the modification of this invention. It is a schematic diagram which shows the structural example of the cell culture purification apparatus. It is a schematic diagram which shows the structural example of the cell culture purification apparatus. It is a schematic diagram which shows the structural example of the cell culture purification apparatus. It is a micrograph of a microcarrier to which cells have adhered. It is a micrograph of a floating cell. It is a micrograph of a cell aggregate.

Hereinafter, the cell culture purification apparatus and the cell culture purification method according to the embodiment of the present invention will be described with reference to the drawings. The same reference numerals are given to common configurations in the following figures, and duplicate description will be omitted.

FIG. 1 is a schematic diagram showing a schematic configuration of a cell culture purification apparatus according to an embodiment of the present invention.
As shown in FIG. 1, the cell culture purification apparatus 100 according to the present embodiment includes a culture tank 1, a first filter unit (first filter) 2, a second filter unit (second filter) 3, and a second filter unit (second filter) 3. Extraction pump (first liquid feeding device) P1, recovery pump (second liquid feeding device) P2, discharge pump (third liquid feeding device) P3, recovery tank 5, drawing tube 11 and return A pipe (return flow path) 12, a transfer pipe 13, a recovery pipe 14, and a discharge pipe 15 are provided.

The cell culture purification apparatus 100 cultivates cells in the culture tank 1, draws a liquid containing a culture such as cells from the culture tank 1, and performs a separation and purification treatment. In the cell culture purification apparatus 100, separation and purification are performed by a plurality of stages of membrane separation treatment. The target product, which is the target of separation and purification, is recovered with high purity by removing mixed coarse non-target products and minute impurities. The cell culture purification apparatus 100 is a one-pass type apparatus that sequentially performs the first-stage membrane separation treatment and the second-stage membrane separation treatment.

The cells to be cultured in the cell culture purification device 100 may be floating cells or adherent cells. However, for the purpose of performing a plurality of stages of membrane separation treatment, it is preferable to carry out microcarrier culture or suspension culture of spheroids. That is, as the cells to be cultured, cells adhered to microcarriers or cell aggregates are preferable.

The cells (objects) collected after the separation and purification treatment by the cell culture purification apparatus 100 may be isolated cells such as independent cells in which the cells do not adhere to each other and free cells existing in a suspended state. , It may be a cell aggregate that exists in an aggregated state. When collected in the state of cell aggregates, the cell detachment treatment for detaching the cell adhesion becomes unnecessary, so that it becomes difficult for contaminants to be mixed with the target substance to be separated and purified.

The origin of the cells to be cultured includes animals such as mammals and birds, but is not particularly limited. Specific examples of mammals include primates such as humans, monkeys, orangutans, red-tailed monkeys, cynomolgus monkeys, baboons, marmosets and squirrel monkeys, ungulates such as cows, pigs, horses, sheep and goats, mice, rats and guinea pigs. Examples include rodents such as hamsters, rabbits, dogs, and cats.

Specific examples of cells to be cultured include iPS cells, ES cells (embryonic stem cells), adult stem cells, bone marrow-derived pluripotent stem cells, germline stem cells, mesenchymal stem cells, hepatic stem cells, heart stem cells, and pancreatic stem cells. , Renal stem cells, nerve stem cells, muscle stem cells, epidermal stem cells, hair follicle stem cells, hematopoietic stem cells and other stem cells, hepatocytes, myocardial cells, pancreatic cells, renal cells, fibroblasts, epithelial cells, endothelial cells, epidermal cells, cartilage Examples thereof include somatic cells such as cells, osteoblasts, and synovial cells. The type of cell is preferably a cell whose proliferation depends on cell adhesion, but is not particularly limited.

The culture tank 1 is a tank for culturing cells, and can be formed of an appropriate material such as stainless steel, aluminum alloy, plastic, or glass. The culture tank 1 can be provided with a stirring device for stirring the culture solution, a ventilation device for agitating the culture solution with air, oxygen, nitrogen, carbon dioxide, etc., and a temperature control device for adjusting the temperature of the culture solution.

As the stirring device, various devices such as a mechanical stirring type device, a shaking stirring type device, and an air flow stirring type device that rotate the stirring blade to perform stirring can be provided. Further, as the ventilation device, an air diffuser for liquid ventilation, a sparger, a ventilation pipe for air ventilation, and the like can be provided. As the temperature control device, for example, a water jacket, an electric heater, or the like can be provided.

In addition, the culture tank 1 can be provided with various sensors for monitoring the culture environment in the tank and the culture state of cells. As the sensor, for example, a temperature sensor such as a thermocouple, a dissolved oxygen sensor, a carbon dioxide sensor, a pH sensor, a cell counter using a capacitance meter, an absorptiometer, or the like can be provided.

As shown in FIG. 1, the first filter unit 2 is connected to the culture tank 1 of the cell culture purification apparatus 100 via a pipe or the like. A second filter unit 3 is connected to the first filter unit 2 via a pipe or the like. A recovery tank 5 is connected to the second filter unit 3 via a pipe or the like. The first filter unit 2 and the second filter unit 3 perform membrane separation treatment on the liquid drawn from the culture tank 1 to fractionate the substances in the liquid by size.

The first filter unit 2 and the second filter unit 3 are a filter for performing membrane separation processing, a pressure vessel having a built-in filter, and a primary side provided in the pressure vessel into which the supplied liquid flows. It has an inlet, a primary outlet through which the liquid that has not passed through the filter flows out, and an outlet on the secondary side through which the liquid that has passed through the filter flows out.

The inside of the culture tank 1 of the cell culture purification apparatus 100 is connected to the inlet on the primary side of the first filter unit 2 via a drawing tube 11. The drawing tube 11 is provided with a drawing pump P1 that draws the liquid from the culture tank 1 side and sends it to the first filter unit 2. The drawing tube 11 is a pipe for collecting the target product after culturing, and draws out the liquid in the culture tank 1 and sends it to the primary side of the first filter unit 2.

The outlet on the primary side of the first filter unit 2 is connected to the culture tank 1 via the return pipe 12. The return pipe 12 is a pipe for returning the non-objective material pulled out from the culture tank 1 to the culture tank 1, and the liquid remaining on the primary side of the first filter unit 2 in the membrane separation process is sent to the culture tank 1. send. The return pipe 12 forms a return flow path for returning the non-objective substance separated by the first filter unit 2 to the culture tank 1.

In the cell culture purification apparatus 100, the outlet on the secondary side of the first filter unit 2 is connected to the inlet on the primary side of the second filter unit 3 via a transfer tube 13. The transfer pipe 13 is a pipe for collecting the target product after culturing, and sends the liquid that has permeated to the secondary side of the first filter unit 2 in the membrane separation process to the primary side of the second filter unit 3.

The outlet on the primary side of the second filter unit 3 is connected to the recovery tank 5 via the recovery pipe 14. The recovery tube 14 is provided with a recovery pump P2 that draws liquid from the culture tank 1 side through the secondary side of the first filter unit 2 and the primary side of the second filter unit 3 and sends the liquid to the recovery tank 5. The recovery pipe 14 is a pipe for recovering the target product after culturing, and sends the liquid remaining on the primary side of the second filter unit 3 in the membrane separation process to the recovery tank 5.

The outlet on the secondary side of the second filter unit 3 is connected to an external waste liquid tank or the like of the cell culture purification apparatus 100 via a discharge pipe 15. The discharge pipe 15 is provided with a discharge pump P3 that draws a liquid from the culture tank 1 side through the secondary side of the second filter unit 3 and discharges the liquid to the outside of the cell culture purification device 100. The discharge pipe 15 is a pipe for discarding impurities drawn from the inside of the culture tank 1, and sends the liquid permeated to the secondary side of the second filter unit 3 to the outside of the apparatus in the membrane separation process.

For the first filter unit 2 and the second filter unit 3, a filter having a predetermined pore size is used according to the size of the target object to be separated and purified.

When the target (target product) for separation and purification is an isolated cell, the first filter unit 2 permeates the isolated cell and is larger than the isolated cell (non-target product) or the isolated cell. A filter with a high inhibition rate for large cell aggregates (non-target substances) is used. When the target (target object) for separation and purification is a cell aggregate of a target size or smaller, a microcarrier (non-object) larger than the cell aggregate that permeates the cell aggregate of that size or , A filter having a high inhibition rate for cell aggregates (non-target substances) larger than the cell aggregates is used.

The first filter unit 2 performs a membrane separation treatment on a liquid containing a target product drawn from the culture tank 1 to separate the target product in the liquid and a non-target product larger than the target product from each other. The cultured isolated cells, cell aggregates, and other objects pass through the filter of the first filter unit 2 and are sent to the second filter unit 3. On the other hand, non-target substances such as microcarriers larger than the target product and cell aggregates larger than the target product are returned to the culture tank 1 without passing through the filter of the first filter unit 2.

When the target (object) for separation and purification is an isolated cell, the second filter unit 3 uses a filter that has a high inhibition rate on the isolated cell and permeates impurities smaller than the isolated cell. In addition, when the target (target object) for separation and purification is a cell aggregate of a target size or smaller, the inhibition rate for cell aggregates larger than the target size is high, and impurities smaller than the cell aggregate are permeated. A filter is used.

The second filter unit 3 performs a membrane separation treatment on the liquid containing the target product separated by the first filter unit 2 to separate the target product in the liquid and the contaminants smaller than the target product from each other. The cultured isolated cells, cell aggregates, and other objects are sent to the collection tank 5 without passing through the filter of the second filter unit 3. On the other hand, impurities smaller than the target object pass through the filter of the second filter unit 3 and are discharged to the outside of the apparatus.

The pipe connecting the culture tank 1, the first filter unit 2, the second filter unit 3, and the like can be formed of an appropriate material such as a metal such as stainless steel or a synthetic resin such as silicon rubber. Metal pipes can be sterilized by steam sterilization or the like and can be used repeatedly, but equipment for sterilization treatment and cleaning treatment is required. On the other hand, the pipe made of synthetic resin can be sterilized by gamma ray sterilization, ultraviolet sterilization, or the like, and if it has flexibility, the pipe can be routed relatively easily.

The culture tank 1 can also be provided as a flexible culture bag. Flexible culture bags can also be provided by single-use products. The flexible culture bag can be formed using, for example, a multilayer film made of a resin such as ethylene vinyl acetate, ethylene vinyl alcohol, low density polyethylene, or polyamide.

Further, one of a pipe connected to the culture tank 1, a pipe connected to the first filter unit 2, a pipe connected to the second filter unit 3, a first filter unit 2, and a second filter unit 3. The above can also be provided by a single-use product. The use of single-use products reduces the risk of contamination and eliminates the need to install equipment for sterilization and cleaning.

The apparatus configuration and operation method of the culture tank 1 may be any of a batch culture type, a fed-batch culture type, and a perfusion culture type. Batch culture is a culture method in which a medium is prepared for each culture and no medium is supplied during the culture. Fed-Batch Culture is a culture method in which the medium itself or a specific medium component is sequentially or continuously supplied from outside the system during the culture, but the culture solution is not discharged until the culture is completed. Perfusion culture is a culture method in which a medium is continuously supplied during culturing and the same amount of culture broth is continuously discharged.

FIG. 2 is a schematic view showing a batch culture type culture tank.
As shown in FIG. 2, the batch culture type culture tank 21A includes a stirring device 22 for stirring the culture solution, an aeration device 23 for aerating air, oxygen, nitrogen, carbon dioxide, etc. through the culture solution, and the temperature of the culture solution. A temperature control device 24 for adjusting the temperature, a solution tank 25 for preparing an alkaline solution, a supply pump 26 for sending the alkaline solution to the culture tank 21A, a stirrer 22, a ventilation device 23, a temperature control device 24, and a supply pump 26. A control device 27 that controls and adjusts the culture environment can be provided.

In the batch culture type culture tank 21A, a liquid medium is put in at the time of culturing, but the liquid medium is not supplied or the culture solution in the tank is not drawn out during culturing. According to batch culture, the quality of the target product tends to vary from culture to culture, but the risk of contamination can be dispersed and reduced.

The drawing pump P1 is started to operate after batch culture, and the target product batch-cultured in the culture tank 21A is separated and purified by the first filter unit 2 and the second filter unit 3. In the circulation of the liquid between the culture tank 21 and the first filter unit 2, for example, when the liquid in the culture tank 21A is reduced to a predetermined liquid volume, the total liquid feed amount to the first filter unit 2 is predetermined. It can be stopped when the amount of liquid is reached.

FIG. 3 is a schematic view showing a fed-batch culture type culture tank.
As shown in FIG. 3, the fed-batch culture type culture tank 21B has a stirring device 22, a ventilation device 23, a temperature control device 24, a solution tank 25, and an alkali, similarly to the batch culture type culture tank 21A. A supply pump 26 for sending the solution to the culture tank 21B, a control device 27 for adjusting the culture environment by controlling the stirring device 22, the aeration device 23, the temperature control device 24, the supply pump 26, and the like can be provided.

Further, the fed-batch culture type culture tank 21B is provided with a medium tank 28 in which a liquid medium is prepared and a medium pump 29 for sending the liquid medium prepared in the medium tank 28 to the culture tank 21B. The amount of liquid sent by the medium pump 29 is controlled by the control device 27 according to the concentration of the medium component in the culture tank 21B and the like.

A liquid medium is placed in the fed-batch culture tank 21B at the time of culturing, and the liquid medium is sequentially or continuously supplied from the medium tank 28 during culturing. Not discharged. Normally, the temperature, pH, dissolved oxygen concentration, carbon dioxide concentration, etc. in the culture tank 21B are controlled to be constant. According to fed-batch culture, it is possible to produce the desired product while maintaining a low substrate concentration, and it may be possible to reduce the cost of the medium and the like.

The drawing pump P1 is started to operate after fed-batch culture, and the target product fed-batch culture in the culture tank 21B is separated and purified by the first filter unit 2 and the second filter unit 3. In the circulation of the liquid between the culture tank 21B and the first filter unit 2, for example, when the liquid in the culture tank 21B is reduced to a predetermined liquid volume, the total liquid feed amount to the first filter unit 2 is predetermined. It can be stopped when the amount of liquid is reached.

FIG. 4 is a schematic view showing a perfusion culture type culture tank.
As shown in FIG. 4, the perfusion culture type culture tank 21C includes the stirring device 22, the aeration device 23, the temperature control device 24, the solution tank 25, and the alkali, similarly to the batch culture type culture tank 21A. A supply pump 26 for sending the solution to the culture tank 21C, a control device 27 for adjusting the culture environment by controlling the stirring device 22, the aeration device 23, the temperature control device 24, the supply pump 26, and the like can be provided.

Further, the perfusion culture type culture tank 21C is provided with a medium tank 28 in which a liquid medium is prepared, a medium pump 29 for sending the liquid medium prepared in the medium tank 28 to the culture tank 21C, and a cell separation device 8. .. The cell separation device 8 is provided in a circulation flow path for drawing out the culture solution in the culture tank 21C and returning it to the culture tank 21C. The amount of liquid sent by the medium pump 29 is controlled by the control device 27 according to the concentration of the medium component in the culture tank 21C and the like.

The cell separation device 8 is, for example, a tangential flow membrane separation (TFF) method in which a liquid is flowed in a direction orthogonal to the separation membrane, a liquid is flowed in a direction orthogonal to the separation membrane, and suction and release to the separation membrane are performed. Alternate Tangential Flow Filtration (ATF) method, gravity sedimentation separation method, sound wave separation method that separates the difference in size and density by ultrasonic vibration, etc. can be used. it can.

A liquid medium is put into the perfusion culture type culture tank 21C at the time of culturing, the liquid medium is continuously supplied from the medium tank 28 during culturing, and substantially the same amount of the culture broth is continuously discharged from the tank. To. In perfusion culture, the culture solution in the culture tank 21C is sent to the cell separation device 8, and the cells being cultured (object) and unnecessary medium components are separated by the cell separation device 8. The liquid containing cells is returned to the culture tank 21C, and the liquid containing unnecessary medium components is discharged to the outside of the system. Normally, the temperature, pH, dissolved oxygen concentration, carbon dioxide concentration, etc. in the culture tank 21C are controlled to be constant. According to perfusion culture, the culture environment can be easily kept constant, so that the quality and production amount of the target product can be stabilized.

The drawing pump P1 is started to operate during perfusion culture or after perfusion culture, and the target product perfused and cultured in the culture tank 21C is separated and purified by the first filter unit 2 and the second filter unit 3. During the perfusion culture, the total flow rate of the liquid sent to the first filter unit 2 and the liquid discharged from the cell separator 8 to the outside of the system and the liquid medium supplied from the medium tank 28 are substantially the same. Is controlled by. In the circulation of the liquid between the culture tank 21C and the first filter unit 2, for example, when the culture time of the perfusion culture elapses for a predetermined time, the total amount of liquid sent to the first filter unit 2 is a predetermined amount. It can be stopped when it reaches.

FIG. 5 is a diagram showing general characteristics of the filter provided in the filter unit.
In FIG. 5, the horizontal axis represents the relative value of the pore diameter of the filter represented by 1 as the particle diameter of the target object, and the vertical axis represents the blocking rate by the filter. As shown in FIG. 5, the first filter unit 2 needs to be provided with a filter having a pore diameter larger than that of the target object, and the second filter unit 3 needs to be provided with a filter having a pore diameter smaller than that of the target object.

The size of the isolated cells and cell aggregates to be separated and purified depends on the cell processed product to be produced and the like, but is usually about 10 to 30 μm. As shown in FIG. 5, the size fractionation filter used in the first filter unit 2 and the second filter unit 3 generally has a large change in separability with a pore diameter five times that of the object to be separated as a boundary. is there.

Therefore, the pore diameter of the filter of the first filter unit 2 is preferably 5 times or more the particle diameter of the target object. When the pore size of the filter is 5 times or more the particle size of the target object, due to the general characteristics of the size fractionation filter, high transmittance is ensured for the target object and high inhibition is performed for non-target objects larger than the target object. You can get the rate. The pore size of the filter of the first filter unit 2 is preferably 100 μm or less, more preferably 50 μm or more and 100 μm or less, although it depends on the size of the microcarriers and cell aggregates to be removed.

Further, the pore diameter of the filter of the second filter unit 3 is preferably 1/5 or less of the particle diameter of the target object. When the pore size of the filter is 1/5 or less of the particle size of the target object, due to the general characteristics of the size fractionation filter, it is high for impurities smaller than the target object while ensuring a high blocking rate for the target object. The transmittance can be obtained. The pore size of the filter of the second filter unit 3 is preferably 5 μm or less, although it depends on the size of the target object to be separated and purified.

In the first filter unit 2 and the second filter unit 3, it is preferable that the filter is a hollow fiber membrane. When each filter unit is provided with a hollow fiber membrane, it can be provided as a hollow fiber membrane module having a large number of hollow fiber membranes built in a pressure vessel capable of passing a liquid. According to the hollow fiber membrane, target substances such as isolated cells and cell aggregates that are easily damaged can be efficiently separated and purified with less damage by a tangential flow filtration method membrane separation treatment using a large membrane area. ..

The first filter unit 2 and the second filter unit 3 may be an internal pressure filtration method or an external pressure filtration method. Further, the filter units may have the same filtration method or different filtration methods.

The external pressure filtration type hollow fiber membrane module is provided with an inlet on the primary side on one end side of the pressure vessel, and an outlet on the primary side and an outlet on the secondary side on the other end side of the pressure vessel. One end of the hollow fiber membrane is attached to the pressure vessel so that the primary outlet is located on the outside of the hollow fiber membrane and the secondary outlet is located on the inside of the hollow fiber membrane.

In the membrane separation treatment of the external pressure filtration method, the liquid supplied from the culture tank 1 side flows into the pressure vessel and flows outside the hollow fiber membrane along the length direction of the pressure vessel. A substance smaller than the pore size of the hollow fiber membrane permeates the inside of the hollow fiber membrane and flows out from the outlet on the secondary side. On the other hand, a substance larger than the pore size of the hollow fiber membrane does not permeate inside the hollow fiber membrane and flows out from the outlet on the primary side located outside the hollow fiber membrane.

On the other hand, the internal pressure filtration type hollow fiber membrane module is provided with an inlet on the primary side on one end side of the pressure vessel and an outlet on the primary side and an outlet on the secondary side on the other end side of the pressure vessel. In the hollow fiber membrane, the primary side inlet is located at one end inside the hollow fiber membrane, the primary side outlet is located at the other end inside the hollow fiber membrane, and the secondary side outlet is located outside the hollow fiber membrane. Attached to the pressure vessel so that it is located.

In the membrane separation treatment of the internal pressure filtration method, the liquid supplied from the culture tank 1 side flows into the inside of the hollow fiber membrane and flows inside the hollow fiber membrane along the length direction of the hollow fiber membrane. A substance smaller than the pore size of the hollow fiber membrane permeates the outside of the hollow fiber membrane and flows out from the outlet on the secondary side. On the other hand, a substance larger than the pore size of the hollow fiber membrane does not permeate to the outside of the hollow fiber membrane and flows out from the outlet on the primary side located inside the hollow fiber membrane.

The first filter unit 2 is preferably an internal pressure filtration method from the viewpoint of increasing the speed efficiency of the membrane separation treatment. Further, the second filter unit 3 is preferably an internal pressure filtration method from the viewpoint of increasing the speed efficiency of the membrane separation treatment and reducing cell damage.

The extraction pump P1, the recovery pump P2, and the discharge pump P3 can be provided as an appropriate type of liquid feeding device as long as the liquid can be fed. The liquid feeding devices may be of the same type as each other, or may be different types of devices from each other. As a liquid feeding device, from the viewpoint of stable liquid feeding and aseptic operation, a peristaltic pump, a centrifugal pump, or a pressurization that pressurizes the inside of the culture tank 1 with gas pressure to drive the liquid feeding It is preferably a pump (pressurizer).

FIG. 6 is a diagram showing a peristaltic pump used as a liquid feeding device.
As shown in FIG. 6, the peristaltic pump 30 includes a housing 31, a rotor 32 whose rotational motion is driven by a motor, and a plurality of rollers 33 arranged along the outer circumference of the rotor 32 outside the housing 31. It includes an arc-shaped pressing wall 34 provided so as to face the outer peripheral surface of the rotor 32, and a flexible tube 35.

The housing 31 contains a rotor 32, a base of a roller 33, a motor (not shown), and the like. Each roller 33 is supported on the outside of the housing 31 so as to be rotatable with respect to the rotor 32. The pressing wall 34 is provided on the outside of the housing 31. The tube 35 is made of a flexible material such as silicone rubber. The tube 35 is removable and is attached so as to be sandwiched and extended between the outer peripheral surface of the roller 33 and the pressing wall 34. The peristaltic pump is sometimes called a tube pump, a rotary pump, or the like.

In the peristaltic pump 30, when the rotational movement of the rotor 32 is driven, a plurality of rollers 33 revolve around the rotor 32. The tube 35 is crushed by the roller 32 revolving while pressing the tube 35 against the pressing wall 34. When the tube 35 is crushed, the liquid inside is pushed forward of the orbit of the roller 33. Further, when the crushed tube 35 returns to its original shape by the restoring force, a negative pressure is generated in the tube 35, and the liquid in the rear is sucked to the front side. The liquid is transferred by the peristaltic motion of such a tube 35.

According to the peristaltic pump 30, the liquid to be transferred contacts only the inner surface of the tube 35, so that the risk of contamination can be reduced. Further, since there is no valve structure, a slurry-like liquid, a highly viscous liquid, or the like can be transferred. In addition, since it is a self-priming pump and does not require priming, it has the advantage of being easy to operate and resistant to idle operation. In addition, it is possible to handle and maintain different types of liquids only by replacing the tube 35. However, when a liquid containing cells is sent, the cells may be damaged by crushing the tube 35. Therefore, it may be necessary to evaluate the cell damage in advance and appropriately select the liquid feeding conditions. is there.

FIG. 7 is a diagram showing a magnetic levitation type centrifugal pump used as a liquid feeding device.
As shown in FIG. 7, the magnetically levitated centrifugal pump 40 includes a housing 41, a rotor core 42 with an impeller that generates centrifugal force in the liquid sucked into the housing 41, and a rotor magnet 43 embedded in the rotor core 42 with an impeller. A stator core 44 that functions as a magnetic bearing and a stator coil 45 wound around the stator core 44 are provided.

The housing 41 has a substantially hollow disk shape, and is provided with a suction port opened in the axial direction and a discharge port opened in the radial direction. The rotor core 42 with an impeller is provided with an impeller such as a turbo type, and a rotor magnet 43 formed of a permanent magnet is embedded therein. The stator core 44 is made of a ferromagnetic material such as iron. The stator core 44 has a disk-shaped base portion, and a plurality of pillar portions are vertically provided along the outer periphery of the base portion. A stator coil 45 is wound around a pillar portion of the stator core 44.

The rotor core 42 with an impeller is housed inside the pillar portion of the stator core 44 that functions as a magnetic bearing in the housing 41. In the centrifugal pump 40, the stator core 44 and the stator coil 45, which function as electromagnets, magnetically attract the rotor magnet 43 facing the inner surface of the tip of the column portion, and magnetically levitate the rotor core 42 with an impeller by electrical magnetic control. Rotate. The rotor core 42 with an impeller rotates in a non-contact state with respect to the stator core 44 to generate centrifugal force. The liquid is transferred from the suction port side to the discharge port side by such a rotary motion.

According to the magnetically levitated centrifugal pump 40, the liquid to be transferred contacts only the housing 41 and the rotor core 42 with an impeller, and there is no valve structure or rolling bearing, so that the risk of contamination can be reduced. When the housing 41 in contact with the liquid and the rotor core 42 with an impeller are formed of a chemical resistant material such as fluororesin, the alkaline solution or the like can be safely transferred. In addition, since there is no sliding portion such as a rolling bearing and deterioration due to wear is unlikely to occur, maintenance costs can be reduced. Further, since the liquid can be fed at a constant flow rate, pulsation is reduced, and since there is no sliding portion, vibration is reduced and stable liquid feeding can be performed.

FIG. 8 is a diagram showing a pressurizer used as a liquid feeding device.
As shown in FIG. 8, the pressurizing pump (pressurizer) 50 that drives the liquid feed by gas pressure is between the gas supply source 51 that supplies gas to the culture tank 1 and the gas supply source 51 and the culture tank 1. A gas supply pipe 52 that communicates with the gas regulator 53, a gas regulator 53 that adjusts the pressure of the gas in the culture tank 1, a pressure sensor 54 that measures the pressure in the culture tank 1, and a controller 55 that controls the gas regulator 53. I have.

The gas supply source is provided as, for example, a gas cylinder, a compressor, or the like, and aseptically supplies a high-pressure gas such as nitrogen gas or concentrated air to the culture tank 1. The gas regulator 53 is installed in the middle portion of the gas supply pipe 52, and controls the pressure by operating an actuator such as a valve structure while detecting the pressure on the gas supply source 51 side and the culture tank 1 side.

In the pressurizing pump 50, when the liquid is sent from the culture tank 1 to the first filter unit 2, the high pressure gas is supplied from the gas supply source 51 to the closed culture tank 1. The pressure of the gas in the culture tank 1 is measured by the pressure sensor 54, and an electric signal of the pressure is sent to the controller 55. The controller 55 outputs a control signal to the gas regulator 53 so that the pressure of the gas in the culture tank 1 becomes a predetermined high pressure, and the gas regulator 53 lowers the pressure of the gas supplied from the gas supply source 51. .. The liquid is transferred by the pressure difference between the inside of the culture tank 1 adjusted to a predetermined high pressure and the downstream side.

According to the pressurizing pump 50, the liquid to be transferred does not come into contact with the liquid feeding device, so that the risk of contamination can be reduced. Further, since it is not necessary to provide a liquid pump, the initial cost of the device is suppressed, and maintenance and cleaning are facilitated, so that the running cost is also suppressed. In addition, the culture tank 1 being cultured can be controlled so that the pressure becomes constant according to the fluctuation of the pressure. Further, since the liquid can be fed at a constant flow rate, pulsation is reduced, and since there is no sliding portion, vibration is reduced and stable liquid feeding can be performed.

FIG. 9 is a flowchart showing a cell culture purification method according to an embodiment of the present invention.
FIG. 9 describes a flow of separating and purifying the target product such as isolated cells and cell aggregates after batch culturing in the one-pass type cell culture purification apparatus 100 shown in FIG.

As shown in FIG. 9, in the cell separation and purification apparatus 100, first, cells are cultured in the culture tank 1 (step S10). At the start of culturing, culturing conditions such as temperature, pH, dissolved oxygen concentration, carbon dioxide concentration, and stirring speed of the culturing solution are set.

During culturing, the culturing state is monitored by a temperature sensor, a dissolved oxygen sensor, a carbon dioxide sensor, a pH sensor and the like. The culture temperature can be controlled within a predetermined range by a temperature controller. Further, the dissolved oxygen concentration and the carbon dioxide concentration of the culture solution can be controlled within a predetermined range by aerating air, oxygen, carbon dioxide, nitrogen or the like by an air diffuser or adding an alkaline solution. In addition, the pH of the culture solution can be controlled within a predetermined range by adding an alkaline solution or aerating carbon dioxide.

Subsequently, the number of cultured cells is measured during the culture in the culture tank 1 (step S20). The cell number may be measured online using a sensor, or the culture medium may be sampled and performed offline. As for the number of cells, when the target substance to be separated and purified is an isolated cell, the cell density of the isolated cell, the dielectric constant (capacitance) of the culture solution, and the like may be measured. When the target substance to be separated and purified is cell aggregates, the number of cell aggregates, the density of cell aggregates, the dielectric constant (capacitance) of the culture solution, and the like may be measured.

Subsequently, it is determined whether or not the number of cells in the measured culture solution has reached the target value (step S30). As the target value, for example, an arbitrary value corresponding to the culture time at which dead cells do not accumulate excessively, the culture time and cell density at which cell differentiation is unlikely to occur, the culture time and cell density at which spheroids reach the desired size, etc. are set in advance. It can be obtained and set in a culture test or the like.

As a result of the determination, if the number of cells in the culture solution has not reached the target value (step S30; No), the culture in the culture tank 1 is continued (step S10).

On the other hand, as a result of the determination, when the number of cells in the culture solution has reached the target value (step S30; Yes), the operation of the extraction pump P1 is started (step S40). When the number of cells reaches the target value and the culture is terminated, the culture solution in the culture tank 1 can be replaced with a liquid, if necessary. For example, extracting only the liquid in the culture tank 1, supplying a buffer or the like for washing the cultured cells, supplying a cell release agent solution for peeling the cell adhesion of the cultured cells, and the like. Can be done.

In the case of microcarrier culture or suspension culture of spheroids, the culture medium cultivated in the culture tank 1 contains target substances such as isolated cells and cell aggregates, and non-target substances such as microcarriers and cell aggregates larger than the target substances. The target object and impurities smaller than the target object are mixed. Examples of impurities include, but are not limited to, cell-generated biotransforms, trypsin, heparin, antibiotics, serum components, endotoxins, bacteria, mycoplasma, viruses and the like.

Subsequently, the operation of the recovery pump P2 is started when the flow rate of the liquid passing through the first filter unit 2 is stable (step S50).

Subsequently, the operation of the discharge pump P3 is started when the flow rate of the liquid passing through the second filter unit 3 is stable (step S60).

Membrane separation processing is performed in the first filter unit 2 and the second filter unit 3 by the operation of the extraction pump P1, the recovery pump P2, and the discharge pump P3 (step S70). In the first filter unit 2, a membrane separation treatment is performed to separate the target product in the liquid drawn from the culture tank 1 and the non-target product larger than the target product from each other. In the second filter unit 3, a membrane separation process is performed to separate the target object in the liquid separated by the first filter unit 2 and the contaminants smaller than the target object from each other. The non-target product separated by the membrane separation treatment is returned to the culture tank 1, and the target product separated by the membrane separation treatment is recovered in the recovery tank 5.

Subsequently, during the membrane separation treatment, the amount of the liquid that has been membrane-separated by the first filter unit 2 and the second filter unit 3 is measured (step S80). The amount of the liquid that has been membrane-separated can be measured by measuring the liquid level in the culture tank 1, the total flow rate flowing through the drawing pipe 11 and the return pipe 12, the operating time of the drawing pump P1 which is a predetermined transfer flow rate, and the like. Good.

Subsequently, it is determined whether or not the amount of the liquid membrane-separated by the first filter unit 2 and the second filter unit 3 has reached the target value (step S90). As the target value, for example, an arbitrary value corresponding to the amount of the culture solution cultivated in the culture tank 1, the circulation time of the liquid between the culture tank 1 and the first filter unit 2, and the like can be set.

As a result of the determination, if the amount of the liquid membrane-separated by the first filter unit 2 and the second filter unit 3 does not reach the target value (step S90; No), the first filter unit 2 and the second filter unit 3 The membrane separation process is continued at (step S70).

On the other hand, as a result of the determination, when the amount of the liquid membrane-separated by the first filter unit 2 and the second filter unit 3 reaches the target value (step S90; Yes), the extraction pump P1, the recovery pump P2 and the discharge The operation of the pump P3 is stopped (step S100). The separation and purification treatment of the target product by the membrane separation treatment is completed, and the target product recovered in the recovery tank 5 can be subjected to other purification treatment, cell processing treatment and the like.

According to the above-mentioned one-pass type cell culture purification apparatus 100 and cell culture purification method, in the first-stage membrane separation treatment, from target substances such as isolated cells and cell aggregates to coarse microcarriers and cells larger than the target product. Non-objectives such as aggregates can be separated and removed. Then, in the second-stage membrane separation treatment, contaminants can be separated and removed from the target substances such as isolated cells and cell aggregates from which the non-target substances have been removed. After the first-stage membrane separation treatment, non-target substances such as coarse microcarriers and cell aggregates larger than the target product are returned to the culture tank 1, and are therefore subjected to the cell detachment treatment for peeling the cell adhesion again. Can be done and can be easily collected. In the case of the perfusion culture type, non-target substances such as microcarriers and cell aggregates returned to the culture tank 1 can be subjected to reculture.

Further, according to the one-pass type cell culture purification device 100 and the cell culture purification method, since the membrane separation treatment is performed in a plurality of stages in one pass, the number of liquid feeding devices and pipes to be installed can be reduced. It is possible to suppress the initial cost of the device, and in particular, when the drawing pipe 11, the return pipe 12, the first filter unit 2, the second filter unit 3, etc. are formed by a single-use product, running costs and maintenance can be reduced. It is effective in that it can be reduced.

Next, the cell culture purification apparatus and the cell culture purification method according to the modified example of the present invention will be described with reference to the drawings.

FIG. 10 is a schematic view showing a schematic configuration of a cell culture purification apparatus according to a modified example of the present invention.
As shown in FIG. 10, the cell culture purification apparatus 200 according to the modified example includes a culture tank 1, a first filter unit (first filter) 2, a second filter unit (second filter) 3, and extraction. Pump (first liquid feeding device) P1, discharge pump (third liquid feeding device) P3, transfer pump (fourth liquid feeding device) P4, return pump (fifth liquid feeding device) P5 The storage tank 6, the drawing pipe 11, the return pipe 12, the upstream transfer pipe 16, the downstream transfer pipe 17, the downstream return pipe 18, and the discharge pipe 15 are provided.

Similar to the cell culture purification device 100, the cell culture purification device 200 is configured to culture cells in the culture tank 1 and draw out a liquid containing a culture such as cells from the culture tank 1 for separation and purification treatment. ing. The difference between the cell culture purification device 200 and the cell culture purification device 100 is that after the first-stage membrane separation treatment, the separated liquid is temporarily stored and circulated with respect to the stored liquid. The point is that it is a circulation type device that performs the second stage membrane separation process.

As shown in FIG. 10, the first filter unit 2 is connected to the culture tank 1 of the cell culture purification apparatus 200 via a pipe or the like. A storage tank 6 is connected to the first filter unit 2 via a pipe or the like. The second filter unit 3 is connected to the storage tank 6 via a pipe or the like.

Similar to the cell culture purification device 100, the inside of the culture tank 1 of the cell culture purification device 200 is connected to the inlet on the primary side of the first filter unit 2 via a drawing tube 11. The drawing tube 11 is provided with a drawing pump P1 that draws the liquid from the culture tank 1 and sends it to the first filter unit 2. Further, the outlet on the primary side of the first filter unit 2 is connected to the culture tank 1 via the return pipe 12.

In the cell culture purification apparatus 200, the outlet on the secondary side of the first filter unit 2 is connected to the storage tank 6 via the upstream transfer pipe 16. The upstream transfer pipe 16 is provided with a transfer pump P4 that draws a liquid from the secondary side of the first filter unit 2 and sends it to the storage tank 6. The upstream transfer pipe 16 is a pipe for collecting the target product after culturing, and sends the liquid that has permeated to the secondary side of the first filter unit 2 to the storage tank 6 in the membrane separation process.

The inside of the storage tank 6 is connected to the inlet on the primary side of the second filter unit 3 via the downstream transfer pipe 17. The downstream transfer pipe 17 is a pipe for transferring the target object separated by the first filter unit 2 to the second filter unit 3, and draws out the liquid in the storage tank 6 to the primary side of the second filter unit 3. Send to.

The outlet on the primary side of the second filter unit 3 is connected to the storage tank 6 via the return pipe 18 on the downstream side. The downstream return pipe 18 is provided with a return pump P5 that draws liquid from the primary side of the storage tank 6 and the second filter unit 3 and returns it to the storage tank 6. The downstream return pipe 18 is a pipe for returning the target object pulled out from the storage tank 6 to the storage tank 6, and the liquid remaining on the primary side of the second filter unit 3 in the membrane separation process is stored in the storage tank 6. Send to.

The outlet on the secondary side of the second filter unit 3 is connected to an external waste liquid tank or the like of the cell culture purification device 200 via a discharge pipe 15 as in the cell culture purification device 100. The discharge pipe 15 is provided with a discharge pump P3 that draws a liquid from the secondary side of the second filter unit 3 and discharges the liquid to the outside of the cell culture purification device 200.

The transfer pump P4 and the return pump P5 can be provided as an appropriate type of liquid feeding device as long as the liquid can be fed. The liquid feeding devices may be of the same type as each other, or may be different types of devices from each other. From the viewpoint of stable liquid feeding and aseptic operation, each liquid feeding device pressurizes the inside of the culture tank 1 with a peristaltic pump, a centrifugal pump, or gas pressure to drive the liquid feeding. It is preferably a pump (pressurizer).

FIG. 11 is a flowchart showing a cell culture purification method according to a modified example of the present invention.
FIG. 11 describes a flow of separating and purifying the target product such as isolated cells and cell aggregates after batch culturing in the circulation type cell culture purification apparatus 200 shown in FIG.

As shown in FIG. 11, in the cell separation / purification device 200, first, the cells are cultured in the culture tank 1 in the same manner as in the cell separation / purification device 100 (step S10). Subsequently, the number of cultured cells is measured during the culture in the culture tank 1 (step S20). Subsequently, it is determined whether or not the number of cells in the measured culture solution has reached the target value (step S30). As a result of the determination, if the number of cells in the culture solution has not reached the target value (step S30; No), the culture in the culture tank 1 is continued (step S10). On the other hand, as a result of the determination, when the number of cells in the culture solution has reached the target value (step S30; Yes), the operation of the extraction pump P1 is started (step S110).

In the cell separation / purification apparatus 200, the operation of the transfer pump P4 is started when the flow rate of the liquid passing through the first filter unit 2 is stable (step S120).

Due to the operation of the drawing pump P1 and the transfer pump P4, the membrane separation process is performed in the first filter unit 2 (step S130). In the first filter unit 2, a membrane separation treatment is performed to separate the target product in the liquid drawn from the culture tank 1 and the non-target product larger than the target product from each other.

Subsequently, during the membrane separation treatment, the amount of the liquid that has been membrane-separated by the first filter unit 2 and stored in the storage tank 6 is measured (step S140). The amount of liquid stored in the storage tank 6 is the liquid level in the culture tank 1, the total flow rate flowing through the drawing pipe 11 and the return pipe 12, the total flow rate flowing through the upstream transfer pipe 16, and a predetermined transfer flow rate. The operating time of a certain drawing pump P1, the operating time of the transfer pump P4 having a predetermined transfer flow rate, the liquid level in the storage tank 6, and the like may be measured.

Subsequently, it is determined whether or not the amount of the liquid membrane-separated by the first filter unit 2 has reached the target value (step S150). As the target value, for example, an arbitrary value corresponding to the amount of the culture solution cultivated in the culture tank 1, the circulation time of the liquid between the culture tank 1 and the first filter unit 2, and the like can be set.

As a result of the determination, if the amount of the liquid subjected to the membrane separation treatment by the first filter unit 2 does not reach the target value (step S150; No), the membrane separation treatment is continued by the first filter unit 2 (step S130).

On the other hand, as a result of the determination, when the amount of the liquid membrane-separated by the first filter unit 2 has reached the target value (step S150; Yes), the operation of the return pump P5 is started (step S160).

Subsequently, the operation of the discharge pump P3 is started when the flow rate of the liquid passing through the second filter unit 3 is stable (step S170).

Due to the operation of the return pump P5 and the discharge pump P3, the membrane separation process is performed in the second filter unit 3 (step S180). In the second filter unit 3, a membrane separation treatment is performed to separate the target product in the liquid separated by the membrane separation treatment of the first filter unit 2 and the contaminants smaller than the target product from each other. The non-target product separated by the membrane separation treatment is returned to the culture tank 1, and the target product separated by the membrane separation treatment is collected in the storage tank 6.

Subsequently, during the membrane separation treatment, the amount of the liquid that has been membrane-separated by the second filter unit 3 is measured (step S190). The amount of the liquid separated by the membrane is the liquid level in the storage tank 6, the total flow rate flowing through the downstream transfer pipe 17 and the downstream return pipe 18, the total flow rate flowing through the discharge pipe 15, and a predetermined transfer flow rate. The operating time of a certain return pump P5, the operating time of the discharge pump P3 which is a predetermined transfer flow rate, and the like may be measured.

Subsequently, it is determined whether or not the amount of the liquid membrane-separated by the second filter unit 3 has reached the target value (step S200). As the target value, for example, an arbitrary value corresponding to the amount of liquid stored in the storage tank 6, the circulation time of the liquid between the storage tank 6 and the second filter unit 3, and the like can be set.

As a result of the determination, if the amount of the liquid that has been membrane-separated by the second filter unit 3 has not reached the target value (step S200; No), the membrane separation treatment is continued by the second filter unit 3 (step S180).

On the other hand, as a result of the determination, when the amount of the liquid membrane-separated by the second filter unit 3 has reached the target value (step S200; Yes), the extraction pump P1, the transfer pump P4, the return pump P5 and the discharge pump P3 (Step S210). The separation and purification treatment of the target product by the membrane separation treatment is completed, and the target product recovered in the storage tank 6 can be subjected to other purification treatment, cell processing treatment and the like.

According to the above-mentioned circulation type cell culture purification apparatus 200 and cell culture purification method, as in the case of the one-pass type, in the first-stage membrane separation treatment, from the target substance such as isolated cells and cell aggregates, coarse microcarriers can be obtained. Non-target substances such as cell aggregates larger than the target product can be separated and removed. Then, in the second-stage membrane separation treatment, contaminants can be separated and removed from the target substances such as isolated cells and cell aggregates from which the non-target substances have been removed.

Further, according to the circulation type cell culture purification apparatus 200 and the cell culture purification method, the target product separated by the first-stage membrane separation treatment is temporarily stored and periodically undergoes the second-stage membrane separation treatment. Therefore, the concentration of unnecessary contaminants can be further reduced. Since the concentration of contaminants can be diluted to a level according to the performance of the filter, target products such as isolated cells and cell aggregates can be obtained with high purity to produce high-quality processed cell products. be able to.

Next, a specific configuration example of the cell culture purification apparatus according to the present invention will be described with reference to the drawings.

FIG. 12 is a schematic view showing a configuration example of a cell culture purification device.
As shown in FIG. 12, a preferred form of a one-pass cell culture purification apparatus is configured with a hollow fiber membrane module, a centrifugal pump, and a peristaltic pump. The one-pass type cell culture purification apparatus 100A shown in FIG. 12 includes a culture tank 101, a first hollow fiber membrane module 102 having a built-in filter, a second hollow fiber membrane module 103 having a built-in filter, and a drawing pump P11. A recovery pump P12, a discharge pump P13, and a recovery tank 105 are provided.

In the one-pass type cell culture purification apparatus 100A shown in FIG. 12, the extraction pump P11 for extracting the liquid from the culture tank 101 side is composed of a centrifugal pump. Further, the recovery pump P12 for pulling out the target object from the first hollow fiber membrane module 102 is composed of a centrifugal pump. Further, the discharge pump P13 for pulling out impurities from the second hollow fiber membrane module 103 is composed of a peristaltic pump. Other equipment, piping configurations, etc. are the same as those of the cell culture purification apparatus 100.

FIG. 13 is a schematic view showing a configuration example of a cell culture purification device.
As shown in FIG. 13, a preferred form of a circulating cell culture purification apparatus is configured with a hollow fiber membrane module, a centrifugal pump, and a peristaltic pump. The circulation type cell culture purification apparatus 200A shown in FIG. 13 includes a culture tank 101, a first hollow fiber membrane module 102 having a built-in filter, a second hollow fiber membrane module 103 having a built-in filter, and a drawing pump P11. The upstream transfer pump P14, the downstream transfer pump P15, the discharge pump P13, and the storage tank 106 are provided.

In the circulation type cell culture purification apparatus 200A shown in FIG. 13, the extraction pump P11 for extracting the liquid from the culture tank 101 side is composed of a centrifugal pump. Further, the upstream transfer pump P14 for pulling out the target object from the first hollow fiber membrane module 102 is composed of a centrifugal pump. Further, the downstream transfer pump P15 for pulling out the target object from the storage tank 106 is composed of a centrifugal pump. Further, the discharge pump P13 for pulling out impurities from the second hollow fiber membrane module 103 is composed of a peristaltic pump. Other equipment, piping configurations, etc. are the same as those of the cell culture purification apparatus 200.

In the cell culture purification device 100A shown in FIG. 12 and the cell culture purification device 200A shown in FIG. 13, a centrifugal pump is installed at a place where an object such as an isolated cell or a cell aggregate passes, and the isolated cell , A peristaltic pump is installed at a place where an object such as a cell aggregate does not pass and unnecessary impurities pass through. Centrifugal pumps are liquid delivery devices that are less likely to damage cells than peristaltic pumps.

Therefore, according to such a form, by using a centrifugal pump, it is possible to perform a stable separation and purification process by continuously flowing a liquid to reduce pulsation while minimizing cell damage. .. Further, by using the peristaltic pump, it is possible to surely discharge a slurry-like liquid having a high concentration of contaminants, a liquid having a high viscosity, etc. to the outside of the device while reducing the risk of contamination. The location where the peristaltic pump is installed is advantageous when it is configured with a single-use product because the piping (tube) can be easily replaced.

FIG. 14 is a schematic view showing a configuration example of a cell culture purification device.
As shown in FIG. 14, the circulation type cell culture purification apparatus can also be configured to recover the target product in the circulating liquid based on sensing. The circulation type cell culture purification device 200B shown in FIG. 14 includes a culture tank 101, a first hollow fiber membrane module 102, a second hollow fiber membrane module 103, and a drawing pump P11, similarly to the cell culture purification device 200A. The upstream transfer pump P14, the downstream transfer pump P15, the discharge pump P13, and the storage tank 106 are provided.

The cell culture purification device 200B differs from the cell culture purification device 200A in that the recovery tank 105, the particle size measuring device 121, the return valve (flow path switch) 123, and the recovery side valve (flow path switch). It is a point that includes 124 and a control device 130. The configuration of other equipment and piping is the same as that of the cell culture purification apparatus 200A.

As shown in FIG. 14, the first hollow fiber membrane module 102 is connected to the culture tank 101 of the cell culture purification apparatus 200B via a pipe or the like. A storage tank 106 is connected to the first hollow fiber membrane module 102 via a pipe or the like. The second hollow fiber membrane module 103 is connected to the storage tank 106 via a pipe or the like.

A recovery pipe 119 is connected to a downstream return pipe 118 that connects the outlet on the primary side of the second hollow fiber membrane module 103 and the storage tank 106. A recovery tank 105 for recovering the target object separated by the second hollow fiber membrane module 103 is connected to the other end of the recovery pipe 119. The recovery pipe 119 sends the liquid separated from the downstream return pipe 118 to the recovery pipe 119 to the recovery tank 105.

In the cell culture purification apparatus 200B, the branch flow path formed by the recovery pipe 119 branches from the circulation flow path that returns from the storage tank 106 to the storage tank 106 via the second hollow fiber membrane module 103, and is downstream of the branch flow path. Is connected to the recovery tank 105.

A particle size measuring device 121 is installed in the downstream return pipe 118. The particle size measuring device 121 measures the particle size of the particles contained in the liquid containing the target substance separated by the second hollow fiber membrane module 103. Examples of the particle size measuring device 121 include a laser diffraction / scattering type particle size measuring device, a dynamic light scattering measuring device, an electric particle size measuring device that measures the particle size by changing the capacitance and electric resistance, and a field. A flow fractionation (FFF) type particle size measuring device or the like can be used.

Further, at the branch point between the downstream return pipe 118 and the recovery pipe 119, the return valve 123 that can open and close the circulation flow path toward the storage tank 106 side and the branch flow path toward the recovery tank 105 side can be opened and closed. A recovery side valve 124 is installed. The return-side valve 123 and the recovery-side valve 124 are relative to the circulation flow path from the storage tank 106 to the storage tank 106 via the second hollow fiber membrane module 103 and the branch flow path formed by the recovery pipe 119. It is possible to switch the main flow path with high flow rate.

Further, the cell culture purification device 200B is provided with a control device 130 that controls the return side valve 123 and the recovery side valve 124 based on the measurement by the particle size measuring device 121. The control device 130 obtains a volume-based or number-based particle size distribution of the liquid circulating in the circulation flow path from the particle size measured by the particle size measuring device 121. Further, the opening and closing of the return valve 123 and the recovery valve 124 is controlled based on the particle size distribution of the liquid circulating in the circulation flow path.

In the cell culture purification apparatus 200B shown in FIG. 14, after the membrane separation treatment is performed by the first hollow fiber membrane module 102, the liquid containing the target substance separated by the first hollow fiber membrane module 102 is stored in the storage tank 106. Be done. The liquid stored in the storage tank 106 circulates between the storage tank 106 and the second hollow fiber membrane module 103, and in the meantime, the concentration of unnecessary contaminants is increased by the membrane separation treatment by the second hollow fiber membrane module 103. It will be reduced.

In the cell culture purification apparatus 200B, while the membrane separation treatment is being continued by the second hollow fiber membrane module 103, the particle size of the particles contained in the liquid containing the target substance separated by the second hollow fiber membrane module 103 is changed. , Measured by the particle size measuring device 121. The particle size measured by the particle size measuring device 121 is stored in a database as, for example, a volume-based or number-based particle size distribution for each predetermined liquid amount unit.

The spectrum of the particle size distribution of the liquid containing the target substance shows the signal of the target substance such as isolated cells and cell aggregates in addition to the signal of unnecessary contaminants at the initial stage when the membrane separation treatment is started. However, when the membrane separation treatment is continued, the signal of the target product remains, while the signal of unnecessary contaminants weakens.

The return valve 123 and the recovery valve 124 are automatically controlled to switch the main flow path from the circulation flow path to the branch flow path when the particles smaller than the target substance decrease to a predetermined concentration. Can be done.

Particles smaller than the target product can be distinguished from the target product such as isolated cells and cell aggregates by the region where the signal is located on the particle size distribution. For example, in the control device 130, a predetermined particle size range can be set in advance as information for detecting particles corresponding to impurities. As the range of the particle size, for example, a detection limit or more and a size or less of the target object can be set.

Further, when the flow path is switched between the return valve 123 and the recovery valve 124, the half width of the signal of the particles corresponding to the contaminants, the peak height of the signal, or the peak area of the signal is determined on the particle size distribution. It can be controlled by determining whether or not the value is equal to or less than a preset target value.

As a result of the determination, when the signal value of the particles corresponding to the contaminants decreases below the target value, the opening degree of the return valve 123 is reduced and the opening degree of the recovery side valve 124 is increased. On the other hand, as a result of the determination, when the signal value of the particles corresponding to the contaminants exceeds the target value, the return valve 123 is opened and the recovery valve 124 is closed.

According to such a cell culture purification device 200B or a cell culture purification method, the liquid containing the target substance is collected in the collection tank 105 when the signal of the contaminant becomes weak and the concentration of the contaminant becomes lower than a predetermined concentration. can do. Unlike the storage tank 106, which may allow high-concentration contaminants to flow through, the recovery tank 105 can be prepared without contaminants, and therefore has higher purity and higher quality than when collected in the storage tank 106. Isolated cells and cell aggregates can be obtained.

Although the present invention has been described above, the present invention is not limited to the above-described embodiments and modifications, and various modifications can be made without departing from the spirit of the present invention. For example, the present invention is not necessarily limited to those having all the configurations included in the above-described embodiments and modifications. Part of the configuration of a certain embodiment or modification is replaced with another configuration, a part of the configuration of a certain embodiment or modification is added to another configuration, or a configuration of a certain embodiment or modification. You can omit a part of.

For example, the cell culture purification devices 100 and 200 are particularly limited in the shape and model of the culture tank, recovery tank, storage tank, etc., the model and arrangement of equipment such as the liquid feeding device and valve, and the connection of pipes. It is not something that is done. The batch culture type culture tank 21, the feeding culture type culture tank 31, and the perfusion culture type culture tank 41 may be applied to the one-pass type cell culture purification device 100, or the circulation type cell culture purification device 200. May be applied to. The number and connections of the medium tanks 38, 48 and the solution tanks 25, 35, 45 are not particularly limited.

The peristaltic pump 30 (see FIG. 6), the centrifugal pump 40 (see FIG. 7), and the pressurizing pump 50 (see FIG. 8) have other shapes and models as long as they have the same main functions. May be good. These liquid feeding devices may be applied to any part of the one-pass type cell culture purification device 100, or may be applied to any part of the circulation type cell culture purification device 200.

Further, the control device 130 both acquires the particle size distribution and controls the return side valve 123 and the recovery side valve 124, but the device for acquiring the particle size distribution and the return side valve 123 and the recovery side valve A device for controlling 124 may be provided separately. The return side valve 123 and the recovery side valve 124 may be composed of a three-way valve or the like instead of individual valves.

Further, in the above-mentioned cell culture purification method (see FIGS. 9 and 11), the operation of each pump is started in order and stopped at the same time, but the start time and stop time of the operation are particularly limited. It's not a thing. The operation of the liquid feeding device may be started at a place where priming is not required. The operation stop time may be changed to different times such as stopping the culture tank side first.

Hereinafter, the present invention will be specifically described with reference to examples, but the technical scope of the present invention is not limited thereto.

As a cell culture purification device, a circulation type cell culture purification device shown in FIG. 10 was used to test cell culture and separation and purification by membrane separation treatment. As the cell culture purification test, microcarrier culture, suspension culture of suspension cells, and suspension culture of spheroids were performed.

<Microcarrier culture>
In the microcarrier culture, human mesenchymal stem cells (hMSC) were used as the cells corresponding to the target substances. The cells corresponding to the target product were cultured by adhering them to microcarrier beads according to the following procedure, and after culturing, they were collected as isolated cells.

First, a liquid medium was prepared by adding 10% fetal bovine serum (FBS) to Dulbecco's Modified Eagle's Medium (DMEM medium). Microcarrier beads "Cytodex 1" (manufactured by GE Healthcare) were added to this liquid medium, hMSC was inoculated, and a batch culture method was performed by stirring in a culture tank.

FIG. 15 is a photomicrograph of microcarriers to which cells have adhered.
As shown in FIG. 15, on the 9th day after the start of the culture, a state in which human mesenchymal stem cells (hMSC) 502 was attached to the spherical microcarrier beads 501 was observed.

Subsequently, stirring in the culture tank was stopped, and the microcarrier beads in the culture solution were precipitated. Next, only the culture solution was withdrawn from the culture tank, a washing buffer was added to the culture tank in which the microcarrier beads remained, and the cells were washed with stirring. Then, the stirring in the culture tank was stopped again, the trypsin solution was added into the culture tank, and the mixture was reacted for 5 minutes. Then, the liquid was drawn out from the culture tank, the membrane separation treatment was started in the first filter unit, and when the storage tank reached a predetermined liquid volume, the membrane separation treatment was started in the second filter unit. As a result, the target hMSC was recovered in a separated and purified state in the storage tank.

<Floating culture of floating cells>
In the suspension culture of suspended cells, Chinese Hamster Ovary cells (CHO cells) were used as cells corresponding to the target substances. The cells corresponding to the target product were cultured in a suspended state according to the following procedure, and after culturing, they were collected as isolated cells.

First, a liquid medium of DMEM medium (10% FBS) was prepared. CHO cells were seeded in this liquid medium, and a fed-batch culture method was performed by stirring in a culture tank.

FIG. 16 is a photomicrograph of floating cells.
As shown in FIG. 16, the number of cells increased as the culture time elapsed, and on the 5th day after the start of the culture, independent floating cells that did not adhere to each other were observed.

Subsequently, when the inside of the culture tank reaches a predetermined number of cells, the liquid is withdrawn from the culture tank, the membrane separation treatment is started by the first filter unit, and when the storage tank reaches the predetermined amount of liquid, The membrane separation process was started in the second filter unit. As a result, the target CHO cells were collected in a storage tank in a separated and purified state.

<Floating culture of spheroids>
In the suspension culture of spheroids, iPS cells were used as the cells corresponding to the target substances. The cells corresponding to the target product were aggregated and cultured according to the following procedure, and recovered as spheroids after culturing.

First, a liquid medium for iPS cells was prepared. IPS cells were seeded in this liquid medium, and a perfusion culture method was performed by stirring in a culture tank.

FIG. 17 is a photomicrograph of cell aggregates.
As shown in FIG. 17, the spheroids became larger as the culture time elapsed, and on the 5th day after the start of the culture, spheroids having a diameter of several hundred μm were observed.

Subsequently, the liquid was withdrawn from the culture tank, the membrane separation treatment was started in the first filter unit, and when the storage tank reached a predetermined liquid volume, the membrane separation treatment was started in the second filter unit. As a result, the target iPS cell spheroid was recovered in a separated and purified state in the storage tank.

1 Culture tank 2 1st filter unit (1st filter)
3 Second filter unit (second filter)
5 Recovery tank 6 Storage tank 8 Cell separation device 11 Extraction pipe 12 Return pipe 13 Transfer pipe 14 Recovery pipe 15 Discharge pipe 16 Upstream side transfer pipe 17 Downstream side transfer pipe 18 Downstream side return pipe 21 Culture tank 22 Stirrer 23 Ventilation device 24 Temperature control device 25 Solution tank 26 Supply pump 27 Control device 28 Medium tank 29 Medium pump 30 Peristaltic pump 31 Housing 32 Rotor 33 Roller 34 Pressing wall 35 Tube 40 Centrifugal pump 41 Housing 42 Rotor core with impeller 43 Rotor magnet 44 Stator core 45 Stator coil 50 Pressurization pump (pressurizer)
51 Gas supply source 52 Gas supply pipe 53 Gas regulator 54 Pressure sensor 55 Controller 100 Cell culture purification device 105 Recovery tank 106 Storage tank 118 Downstream return pipe 119 Recovery pipe 121 Particle size measuring device 123 Return side valve (flow path switch)
124 Recovery side valve (flow path switch)
130 Control device P1 Extraction pump (first liquid delivery device)
P2 recovery pump (second liquid feeder)
P3 discharge pump (third liquid feeder)
P4 transfer pump (fourth liquid feeder)
P5 return pump (fifth liquid delivery device)
P11 Extraction pump P12 Recovery pump P13 Discharge pump P14 Upstream transfer pump P15 Downstream transfer pump

Claims (15)

A culture tank for culturing cells and
A first filter that separates the target product in the liquid drawn from the culture tank and the non-target product larger than the target product from each other.
A second filter that separates the target substance in the liquid separated by the first filter and impurities smaller than the target substance from each other.
It is provided with a return flow path for returning the non-target substance separated by the first filter to the culture tank.
The target product is a cell culture purification device that is an isolated cell or a cell aggregate. The cell culture purification apparatus according to claim 1.
The cells are cells adhered to microcarriers or cell aggregates, and are
The target substance is an isolated cell or a cell aggregate having a size smaller than the desired size.
The non-target substance separated by the first filter is a cell culture purification device which is a microcarrier or a cell aggregate larger than the target size. The cell culture purification apparatus according to claim 1.
A cell culture purification apparatus in which the pore size of the first filter is 5 times or more the particle size of the target product. The cell culture purification apparatus according to claim 1.
A cell culture purification apparatus in which the pore size of the second filter is one-fifth or less of the particle size of the target product. The cell culture purification apparatus according to claim 1.
The first filter is a hollow fiber membrane, and is a cell culture purification device of an internal pressure filtration method in which a liquid drawn from the culture tank flows inside the hollow fiber membrane along the length direction. The cell culture purification apparatus according to claim 1.
The culture tank is a cell culture purification device of a batch culture type, a fed-batch culture type, or a perfusion culture type. The cell culture purification apparatus according to claim 1.
A liquid feeding device for drawing a liquid from the culture tank and sending it to the first filter is provided.
The liquid feeding device is a peristaltic pump, a centrifugal pump, or a cell culture purification device which is a pressurizer that pressurizes the pressure in the culture tank to drive the liquid feeding. The cell culture purification apparatus according to claim 1.
One or more of the pipe connected to the culture tank, the pipe connected to the first filter, the pipe connected to the second filter, the first filter, and the second filter. A cell culture purification device that is a single-use product. The cell culture purification apparatus according to claim 1.
A cell culture purification apparatus including a particle size measuring device for measuring the particle size distribution of particles contained in a liquid containing the target substance separated by the second filter. The cell culture purification apparatus according to claim 1.
A recovery tank for recovering the target object separated by the second filter is provided.
Each of the first filter and the second filter has an inlet on the primary side where the supplied liquid flows in, an outlet on the primary side where the liquid that has not passed through the filter flows out, and a liquid that has passed through the filter. It has an outlet on the secondary side that flows out,
The outlet on the secondary side of the first filter is connected to the inlet on the primary side of the second filter, and the outlet on the primary side of the second filter is a cell connected to the collection tank. Culture purification device. The cell culture purification apparatus according to claim 1.
A storage tank for storing the target object separated by the first filter is provided.
Each of the first filter and the second filter has an inlet on the primary side where the supplied liquid flows in, an outlet on the primary side where the liquid that has not passed through the filter flows out, and a liquid that has passed through the filter. It has an outlet on the secondary side that flows out,
The outlet on the secondary side of the first filter is connected to the storage tank, the storage tank is connected to the inlet on the primary side of the second filter, and the primary side of the second filter is connected. The outlet on the side is a cell culture purification device connected to the water tank. The cell culture purification apparatus according to claim 11.
A recovery tank for recovering the target object separated by the second filter is provided.
A cell culture purification apparatus in which a branch flow path branched from a circulation flow path that returns from the storage tank to the storage tank via the second filter is connected to the collection tank. The cell culture purification apparatus according to claim 12.
A cell culture purification device including a flow path switcher for switching a flow path between the circulation flow path and the branch flow path. The cell culture purification apparatus according to claim 13.
A particle size measuring device for measuring the particle size of particles contained in the liquid containing the target substance separated by the second filter is provided.
The flow path switcher is a cell culture purification device that switches a flow path from the circulation flow path to the branch flow path when particles smaller than the target substance have a preset target concentration or less. The process of culturing cells in a culture tank and
A step of membrane-separating the liquid drawn from the culture tank to separate the target product in the liquid and the non-target product larger than the target product from each other.
The process includes a step of membrane-separating a liquid containing the target substance separated by the membrane separation treatment to separate the target substance in the liquid and impurities smaller than the target substance from each other.
The non-objective substance separated by the membrane separation treatment is returned to the culture tank and returned to the culture tank.
The object is a cell culture purification method in which isolated cells or cell aggregates are used.