JP2015012837A - Method for producing cell concentrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve the problem in production of a cell concentrate using a hollow fiber type separation membrane.SOLUTION: Specifically, provided is a method for producing a cell concentrate capable of efficiently recovering cells with lower damage to cells when concentrating a cell suspension. A method for producing a cell concentrate uses a cell suspension treater filled up with a hollow fiber type separation membrane, and comprises (a) concentrating the cell suspension while passing the cell suspension from an introduction port 8a at a linear speed of 370-630 cm/min. and discharging a filtrate, (b) recovering into a recovery container 10 the cell concentrate obtained by passing the cell concentrate from a cell suspension introduction port 8a at a linear speed of 40-150 cm/min. and deriving from a cell suspension derivation port 8b.

Description

  The present invention relates to a technique for concentrating and recovering a cell suspension using a cell suspension treatment device filled with a hollow fiber type separation membrane.

  In the field of cell medicine, a method is used in which cells collected from a living body are directly or in vitro cultured and then administered to a patient. The cells used for these treatments are suspended in a solution suitable for the treatment, or adjusted to an appropriate concentration and transplanted. However, when cells are collected from a living body, the cell concentration may not be sufficient for treatment, and when cells collected from a living body are further cultured outside the body, they may contain tissue-derived contaminants or culture media. Many. Therefore, in order to use cells collected or cultured from a living body for treatment, impurities and culture media are removed, suspended in a solution suitable for treatment (washing), and concentrated to a cell concentration suitable for treatment. It is necessary to

  In order to solve the above object, a concentration and washing operation using centrifugation is known. For example, a method using centrifugation to separate and concentrate regenerative cells from human tissue has been disclosed (Patent Document 1). However, there is a concern that the method using centrifugation as described above may limit the facilities that can be used due to the size of the apparatus, the load on the cells, and the increase in cost. Further, when the supernatant of cells precipitated by centrifugation is removed for washing the cells, the cells may be released to the atmosphere, and there are problems such as contamination.

On the other hand, a cell suspension separation and filtration method using a hollow fiber separation membrane, which is a compact and simple device, has been proposed (Patent Document 2).
When concentrating a cell suspension for cell medical use using the hollow fiber type separation membrane as described above, it is necessary to perform a concentration treatment in a short time in order to reduce damage to the cells. It is considered better to increase the pore size of the separation membrane. However, when the pore diameter of the hollow fiber type separation membrane is increased, there is a concern that clogging of cells and adhesion to the hollow fiber type separation membrane increase, and the number of cells decreases in the process of preparing the concentrate.

Special Table 2007-524396 Japanese Patent No. 2928913

  An object of the present invention is to solve problems in the production of a cell concentrate using the hollow fiber separation membrane. Specifically, the present invention provides a method for producing a cell concentrate that can reduce cell damage and concentrate cells efficiently when concentrating a cell suspension.

  As a result of intensive studies to solve the above problems, the present inventors have designed the linear velocity of the cell concentration step and the cell recovery step within a specific range, thereby reducing damage to cells and recovering cells. The inventors have found that a cell concentrate with a high rate can be produced, and have completed the present invention.

That is, the gist of the present invention is as follows.
(1) Manufacture of a cell concentrate using a cell suspension treatment device in which a hollow fiber separation membrane is filled in a container having a cell suspension inlet, a cell suspension outlet, and a filtrate outlet A method,
(A) passing the cell suspension from the cell suspension inlet at a linear velocity of 370 to 630 cm / min, and concentrating the cell suspension while discharging the filtrate from the filtrate outlet;
(B) passing the cell concentrate through the cell suspension inlet at a linear velocity of 40 to 150 cm / min, and collecting the cell concentrate derived from the cell suspension outlet into a collection container;
A method for producing a cell concentrate comprising:
(2) The method for producing a cell concentrate according to (1), wherein the total cross-sectional area of the hollow fiber type separation membrane filled in the cell suspension treatment device is 0.5 to 1.5 cm ^2. .
(3) The method for producing a cell concentrate according to (1) or (2), wherein the hollow fiber separation membrane packed in the cell suspension treatment device has an inner diameter of 300 to 1000 μm.
(4) Before the step (b), the method includes the step of repeating dilution and concentration using a diluent and washing the cell suspension, according to any one of (1) to (3) A method for producing a cell concentrate.
(5) The method for producing a cell concentrate according to any one of (1) to (4), wherein the collection container has one or more ports through which liquid can flow in and out.
(6) The method for producing a cell concentrate according to (5), comprising a step of taking out the cell concentrate through a port after the step (b).
(7) The method for producing a cell concentrate according to (5) or (6), comprising a step of adding a drug through a port after the step (b).
(8) The method for producing a cell concentrate according to any one of (1) to (7), comprising a step of sealing and dividing the collection container after the step (b).
(9) The method for producing a cell concentrate according to any one of (1) to (8), wherein the cell suspension contains immune cells.
(10) The method for producing a cell concentrate according to any one of (1) to (9), wherein the cell recovery rate is 86% and the cell viability ratio is 95% or more.

  According to the production method of the present invention, the survival rate of cells contained in the cell concentrate is excellent, and the cell recovery rate can be improved. Furthermore, the cells concentrated by the production method according to the present invention can be provided for therapeutic use.

Cell concentration system provided with cell suspension treatment device used in examples

  The present invention will be described below.

  The cell suspension used in the present invention is not particularly limited as long as it is a suspension containing cells. For example, fat, skin, blood vessel, cornea, oral cavity, kidney, liver, pancreas, heart, nerve, muscle, prostate, Suspensions, blood, and bone marrow fluids that have been subjected to enzyme treatment, crushing treatment, extraction treatment, degradation treatment, ultrasonic treatment, etc. for biological tissues such as intestine, amniotic membrane, placenta, and umbilical cord, density gradient centrifugation, filtration treatment, and enzyme Examples thereof include cell suspensions prepared by pretreatment such as treatment, decomposition treatment, and ultrasonic treatment. Alternatively, a cell suspension after culturing the cells exemplified above in vitro can be used. Examples of the culture solution for culturing cells in vitro include DMEM, α-MEM, MEM, IMEM, RPMI-1640, and the like. A culture solution to which stimulating factors such as cytokines, antibodies and peptides are added can also be used.

  Examples of the cells herein include artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), mesenchymal stem cells, adipose-derived mesenchymal cells, adipose-derived stromal stem cells, and pluripotent adults. Lymphoid systems such as stem cells, bone marrow stromal cells, hematopoietic stem cells, etc., living stem cells having multipotency, T cells, B cells, killer T cells (cytotoxic T cells), NK cells, NKT cells, regulatory T cells, etc. Cells, macrophages, monocytes, dendritic cells, granulocytes, erythrocytes, platelets, neurons, somatic cells such as myocytes, fibroblasts, hepatocytes, cardiomyocytes, or treatments such as gene transfer or differentiation The performed cells are exemplified. Among these, it can be suitably used particularly for immune cells. Examples of immune cells herein include granulocytes, T cells, B cells, killer T cells (cytotoxic T cells), NK cells, NKT cells, regulatory T cells, macrophages, dendritic cells, and the like.

  Moreover, the cell concentrate used for this invention represents the cell suspension concentrated by passing a cell suspension processing device.

  Although the cell suspension processing apparatus of this invention is illustrated as shown in FIG. 1, it is not limited to this. The cylindrical container 1 is composed of a straight body portion 2, head portions 3 on both sides thereof, and header portions 9, and the head portion 3 is provided with a filtrate outlet 4. The filtrate outlet may be provided at one side, at both ends, or at one central portion. In this example, the header portion 9 is provided with a cell suspension inlet 8a and a cell suspension outlet 8b. Inside the cylindrical container 1, a bundle 5 of hollow fiber type separation membranes loaded and a bundle 5 of hollow fiber type separation membranes provided inside the header portion 9 are fixed inside the container and hollow fiber type separation is performed. The resin layer portion 7 forming the opening end 6 of the film and the structure equivalent to this provided inside the head portion 3 are provided. The resin layer part 7 and the open end 6 are covered with a header part 9 (or head part 3), and the cell suspension outlets 8a and 8b and the filtrate outlet 4 are separated by a hollow fiber type. It is separated by wall materials constituting the film and has a non-continuous structure.

  The cell suspension treatment device of the present invention may be used after sterilization. The sterilization method is not particularly limited, and sterilization methods widely used for sterilization of medical devices such as γ-ray sterilization, electron beam sterilization, EOG sterilization, and high-pressure steam sterilization can be suitably used.

  In the example shown in FIG. 1, each part is distinguished as a barrel 2, a head 3, and a header 9 of a cylindrical container, but this is for convenience. Even if the header portion 9 is integrated with the head portion 3 of the cylindrical container by design, or the body portion 2 and the head portion 3 of the cylindrical container are formed of separate parts, the cell suspension inlet And the cell suspension outlet are continuous without being separated by the wall constituting the hollow fiber type separation membrane, and the cell suspension outlet and the filtrate outlet constitute the wall material constituting the hollow fiber type separation membrane. Various structures can be adopted as long as the structure is separated by the structure.

  As materials for cylindrical containers of cell suspension treatment equipment, acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymer; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, polyvinyl chloride, etc. Halogenated polymer: Polyamide, polyimide, polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, acrylonitrile, butadiene styrene, polystyrene, polymethylpentene and the like can be used. In particular, a material having sterilization resistance, specifically, polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, polymethylpentene, polystyrene and the like are preferable.

As the material for the resin layer portion for fixing the hollow fiber type separation membrane, general adhesive materials such as polyurethane resin, epoxy resin, and silicon resin can be preferably used.
In the cell suspension treatment apparatus of the present invention, it is preferable to fill a cylindrical container with a bundle of dozens to thousands of hollow fiber type separation membranes. In the present invention, the arrangement of the hollow fiber type separation membrane may be linear, bent or spiral, and the cell suspension inlet and the cell suspension outlet The shape is not particularly limited as long as both ends of the hollow fiber type separation membrane are held between the two.

  As the resin material for the hollow fiber type separation membrane used in the present invention, a synthetic polymer material can be preferably used from the viewpoint of the safety and stability of the material. Among these, polysulfone-based, polyolefin-based, or cellulose-based polymer materials can be preferably used. Furthermore, polyethersulfone, polyethylene, and cellulose ester can be most suitably used because of the safety, stability, and availability of the material.

  The pore diameter of the hollow fiber type separation membrane is preferably 0.05 μm or more, more preferably 0.1 μm or more, and still more preferably 0.2 μm or more. If it is less than 0.05 μm, it is not preferable because a large filtration flow rate cannot be obtained, or unnecessary impurities such as proteins cannot be efficiently removed. On the other hand, if it exceeds 1 μm, the pore diameter close to the size of the cell is present on the hollow fiber type separation membrane, so that the clogging into the pore becomes severe and the cell recovery rate is greatly reduced. Here, the pore diameter of the hollow fiber type separation membrane is a value presented by the manufacturer of the hollow fiber type separation membrane, and is calculated from a pressure value at which bubbles are generated from the entire membrane measured by a general bubble point test.

  The present invention is a cell concentration step in which a cell suspension is passed through a cell suspension introduction port at a linear velocity of 370 to 630 cm / min and the cell suspension is concentrated while discharging the filtrate from the filtrate outlet. Thereafter, the cell containing the cell recovery step of passing the cell concentrate through the cell suspension inlet at a linear velocity of 40 to 150 cm / min and recovering the cell concentrate derived from the cell suspension outlet into a recovery container. This is a method for producing a concentrated solution. In addition to concentrating cells, keeping the linear velocity during cell recovery within a certain range, especially by lowering the linear velocity during the cell recovery step than during the cell concentration step, the cell recovery rate In addition, a cell concentrate having a high survival rate can be produced.

The linear velocity (cm / min) here means the amount of liquid flowing into the hollow fiber separation membrane per unit time, that is, the flow velocity (cm ³ / min = mL / min) is the total cross-sectional area of the hollow fiber separation membrane. It is the value divided by (cm ² ). In the cell concentration step of the present invention, the linear velocity flowing inside the hollow fiber separation membrane is preferably 370 cm / min or more and 630 cm / min or less, more preferably 380 cm / min or more and 610 cm / min or less, More preferably, it is 390 cm / min or more and 590 cm / min or less. When the linear velocity is less than 370 cm / min, there is a concern that the cells are likely to adhere to the surface of the hollow fiber membrane and the recovery rate is lowered, and the concentration process takes too much time and damage to the cells increases. There is concern. If it exceeds 630 cm / min, there is a concern that the pressure applied to the cell suspension treatment device becomes too large and the treatment device is destroyed.

In the cell recovery step of the present invention, the linear velocity flowing inside the hollow fiber separation membrane is preferably 40 cm / min to 150 cm / min, more preferably 45 cm / min to 125 cm / min. Preferably, it is 50 cm / min or more and 100 cm / min or less. If the linear velocity is less than 40 cm / min, there is a concern that the recovery process takes too much time and damage to the cells increases. On the other hand, if it exceeds 150 cm / min, a strong shearing stress is applied to the cells during cell recovery, so that the cells tend to be damaged.
Specifically, the cell concentration process is performed by filtering the cell suspension that has flowed into the hollow fiber type separation membrane provided in the cell suspension treatment device to the outside of the hollow fiber type separation membrane by internal pressure filtration. The cell suspension in which the cell components are concentrated is discharged from the cell suspension outlet. Further, the cell suspension in which the cell components flowing out from the cell suspension outlet are concentrated flows again into the cell suspension treatment device from the cell suspension inlet, and the cell components are further concentrated. By repeating this, the concentration of the cell suspension proceeds.

  The filtrate here means that the number of cells in the filtrate is within a range not exceeding 0.1% of the number of cells in the cell suspension before flowing into the cell suspension treatment device. .

  The cell collection step is a step of switching the flow path around the cell suspension treatment device and collecting the cell concentrate in the cell suspension treatment device in a collection container.

  A value obtained by dividing the linear velocity (cm / min) flowing inside the hollow fiber type separation membrane in the cell concentration step by the linear velocity (cm / min) flowing inside the hollow fiber type separation membrane in the cell recovery step, that is, the linear velocity ratio is It is preferably 3.9 or more and 15 or less, more preferably 4.9 or more and 14 or less, and further preferably 5.9 or more and 13 or less. When the linear velocity ratio is less than 3.9, a strong shear stress is applied to the cells at the time of cell recovery, or the treatment takes time, so that the cells tend to be damaged. Moreover, when the linear velocity ratio exceeds 13, the recovery process takes too much time and damage to the cells increases, or the pressure applied to the cell suspension processing apparatus increases too much and the processor is damaged. Is concerned.

  Hereinafter, although the manufacturing method of this invention is illustrated using FIG. 1, this invention is not limited to this, A various change can be made in the range which is not contrary to the main point of this invention.

  First, a tube or the like is attached to the cell suspension inlet 8a and the cell suspension outlet 8b of the cell suspension processor. At the end of the tube attached to the cell suspension outlet 8b, a bifurcated branch capable of switching the flow path is provided, one of the branch destinations is connected to the collection container 10, and the other is a cell suspension such as a cell bag. It connects with the container 13 containing the liquid. At this time, the flow path closes the two-way cock 15a on the collection container side and opens the two-way cock 15b on the container side containing the cell suspension and the two-way cock 15c on the waste liquid container side. The end of the tube attached to the cell suspension inlet 8a is connected to a container 13 containing the cell suspension, and the cell suspension is placed between the container containing the cell suspension and the cell suspension processor. To be able to circulate. Further, it is conceivable that a machine 14 such as a pump is interposed in the tube so that the liquid can circulate in the attached tube. Further, it is preferable to connect a tube connected to the waste liquid tank 16 to the filtrate outlet 4. The entire attachment of the cell suspension treatment device and the tube is preferably performed in an aseptic environment such as a clean bench. At this time, pressure can be applied to the separation membrane by narrowing the flow path on the cell suspension outlet side 8b, or while applying pressure by installing a pump or the like on the tube on the filtration side. Filtration may be performed, and various filtration methods generally used in hollow fiber type separation membranes can be used in combination.

  In the subsequent cell recovery step, the two-way stopcock 15a on the recovery container side is opened, and the two-way stopcock 15b on the container side containing the cell suspension and the two-way stopcock 15c on the waste liquid container side are closed, so After switching to, the cell suspension present in the cell suspension treatment device or the container 13 is collected in a collection container. The cell suspension collected in the collection container is preferably collected aseptically so that it can be used for subsequent treatment.

The total cross-sectional area of the hollow fiber type separation membrane in the present invention is the total area of the cross-sectional areas of the lumens of the hollow fiber type separation membrane provided in the cell suspension treatment device, and (total cross-sectional area) = (thread) (Number) × π × (hollow fiber inner diameter) × (hollow fiber inner diameter) / 4. The total cross-sectional area of the hollow fiber type separation membranes of the present invention is preferably 0.5 cm ² or more 1.5 cm ² or less, more preferably 0.60 cm ² or more 1.0 cm ² or less, 0.64 cm ² More preferably, it is 1.77 cm ² or less. If it is less than 0.5 cm ² , a sufficient amount of inner membrane area for processing the cell suspension cannot be secured, and there is a concern that the processing takes time. On the other hand, if it exceeds 1.5 cm ² , there is a concern that the cell suspension that has flowed into the cell suspension treatment device may not be dispersed and flow into the hollow fiber, which may reduce the filtration efficiency. .

  The hollow fiber separation membrane used in the present invention preferably has an inner diameter of 300 μm or more and 1000 μm or less, more preferably 350 μm or more and 900 μm or less, and further preferably 400 μm or more and 800 μm or less. . If it is less than 300 μm, a strong shearing stress is applied to the cells, and the cells may be damaged, which is not preferable. On the other hand, when it exceeds 1000 μm, the surface area that can be contacted by the cell suspension becomes small, and it becomes difficult to perform filtration efficiently.

  In addition, before the cell recovery step, dilution and concentration can be repeated using a diluent to wash the cell suspension. Specifically, the cell suspension is replaced with a physiological saline solution or an infusion solution, and the cell suspension after the concentration step is diluted with a diluent, and the diluted cell suspension is concentrated again. In addition, unnecessary impurities such as proteins in the cell concentrate can be suitably removed. By repeating the above steps, the removal rate of unnecessary impurities can be greatly improved, and cells for cell therapy can be provided. Examples of the diluent used for washing include physiological saline solution, infusion solution, medium, distilled water, inorganic salt, saccharide, serum, protein-containing liquid, buffer solution, medium, plasma, etc., particularly from the viewpoint of safety. Therefore, a physiological saline solution or an infusion solution can be preferably used. The addition of the diluent is preferably performed aseptically.

  In addition, cell therapy here includes leukemia treatment, myocardial regeneration and vascular regeneration, stem cell exhaustion disease, bone disease, cartilage disease, ischemic disease, vascular disease, neurological disease, burn, chronic inflammation, heart disease, immune Regenerative medicine such as failure, coulomb disease, breast augmentation, wrinkle removal, cosmetic molding, tissue enlargement such as tissue depression, immunotherapy such as T cell therapy, NKT cell therapy, dendritic cell transfer therapy, gene transfer It can be used for gene therapy using cells, but is not limited thereto. In addition, the separated cells can be seeded on a structural material such as a scaffold and used for treatment.

  Further, the collection container in the present invention is exemplified as shown in FIG. 1, but is not limited to this as long as the container is airtight so that the cell concentrate can be collected in a closed manner. Examples thereof include a culture container, a cryopreservation bag, and a plastic container. 1 is provided with a mixed injection port 11 and a bottle needle connection port 12. A syringe is connected to the mixed injection port 11 to add a drug, or all of the cell concentrate is used for sampling purposes. Some can be recovered. Although the medicine here is not particularly limited, for example, an anticoagulant, nutrient sources such as vitamins, antibiotics, cytokines, stimulating factors such as antibodies and peptides, cryoprotectants and the like can be preferably used. In addition, if the collection container is sealed by heat-sealing the tube upstream of the collection container and disconnected from the cell suspension treatment device, the bottle needle of the infusion set is inserted into the bottle needle connection port 12, and the cell concentration is directly performed on the patient. A liquid can also be administered.

  The cell concentrate recovered according to the present invention may be further cultured, and the cell suspension, cell differentiation, transformation, gene transfer, etc. are performed by replacing the suspended liquid by washing and further culturing. be able to. Examples of the medium used for cell growth include DMEM, α-MEM, MEM, IMEM, and RPMI-1640, and culture may be performed using stimulating factors such as cytokines, antibodies, and peptides. In this case, the collection container is preferably a cell culture bag.

  Cells recovered according to the present invention may be mixed with pharmaceutically acceptable additives to produce a pharmaceutical composition. Examples of pharmaceutically acceptable additives include anticoagulants, nutrient sources such as vitamins, antibiotics, and the like.

  The cell concentrate prepared and washed according to the present invention may be further cryopreserved, and from the point that damage to cells can be reduced, it is better to add a cryoprotectant to the cell concentrate and cryopreserve it using liquid nitrogen. Good. In addition, cryopreserved cells can be thawed and used for transplantation into humans and animals, research, or re-culture. Since the cells produced and washed according to the present invention are less damaged by the concentration operation, they can be suitably used for cryopreservation and use after thawing.

In this case, the collection container is preferably a cryopreservation bag.
As a material for the collection container, polyvinyl chloride, polypropylene, polyethylene, polyimide, polycarbonate, polysulfone, polymethylpentene, polystyrene and the like can be suitably used. The capacity of the collection container is preferably 40 mL or more, more preferably 45 mL or more, and further preferably 50 mL or more. When the volume is less than 40 mL, there is a possibility that the entire cell concentrate cannot be recovered. The upper limit of the volume varies depending on the use after collection. When culturing with additional medium added to the cell concentrate, it is preferably 1500 mL or less so that a large amount of medium can be added. In this case, it is preferably 200 mL or less so that the cell concentrate in the collection container can be taken out without loss.

  Further, in the present invention, by reducing the linear velocity at the time of cell recovery than at the time of cell concentration, a value obtained by dividing the cell viability in the cell concentrate by the cell viability in the cell suspension, that is, the cell viability ratio Is maintained at 95% or more, but the cell viability ratio here refers to the cell viability in the cell suspension collected in the collection container, and the cell viability in the cell suspension before treatment. The value divided by the cell viability is expressed as a percentage, and the higher the value, the less damage to the cells. The cell viability is calculated by {cell viability (%) = 100−dead cell rate (%)}. The dead cell rate (%) is determined by adding 10 μL of via-probe (BD) to 100 μL of each cell suspension prepared so that the cell concentration becomes 10 6 per 1 mL, and let stand for 10 minutes in the dark at room temperature. Then, it can be taken into a flow cytometer (BD, FACSCanto) and the positive rate (%) of via-probe can be measured.

  In the present invention, the cell viability ratio is preferably 95% or more, more preferably 96% or more, and still more preferably 97% or more. If it is less than 95%, the cells are heavily damaged in the cell concentration step or cell recovery step. For example, when administered to a patient as a cell therapy application, the cells may not function normally and may not exhibit therapeutic effects. There is sex.

  Further, in the present invention, by reducing the linear velocity at the time of cell recovery than at the time of cell concentration, a value obtained by dividing the number of cells contained in the cell concentrate by the number of cells contained in the cell suspension, that is, the cells The recovery rate can be maintained at 85% or more. The cell recovery rate (%) here is the number of cells in the cell suspension recovered in the recovery container, and the cell suspension before processing. The value divided by the number of cells in the solution is expressed as a percentage, and the higher the value, the better the recovery efficiency. The number of cells was determined by measuring the cell suspension with a blood cell counter (Sysmex, K-4500), calculating the cell concentration (cells / mL) of the leukocyte fraction as the cell concentration in this Example, It can be calculated by the product of (mL) and cell concentration (cells / mL).

  The cell recovery rate (%) is preferably 86% or more, more preferably 87% or more, and further preferably 89% or more. If it is less than 85%, it may not be possible to secure the number of cells required for administration to a patient for cell therapy.

  Hereinafter, the present invention will be described using experimental results. The processing time described in the examples is the time (seconds) from the time when the cell suspension is passed through the cell suspension inlet to the time when the cell concentrate is completely collected in the collection container. It is.

  In the method for cell concentration in each experimental example, a PVC tube was connected to the inlet and outlet of the cell suspension treatment device described in each example. The tube on the outlet side was provided with a bifurcated branch capable of switching the flow path, one connected to the collection container and the other suspended from a plastic container for storing the cell suspension. The inlet tube of the cell suspension processor was also hung on the plastic container so that the cell suspension in the plastic container could circulate through the module and the tube. A pump was installed in the PVC tube so that the flow of the cell suspension could be set to an appropriate flow rate. A two-way stopcock was installed on the PVC tube so that the flow path on the cell suspension treatment device outlet side could be switched. A tube was attached to the outlet for the filtrate of the cell suspension treatment device, and this was set up to be poured into a waste container. While adjusting the flow rate with a pump, the cell suspension was passed through a cell suspension processor, and filtration was performed while circulating the cell suspension. At this time, the flow rate on the cell suspension inlet side and the filtration flow rate discharged from the filtrate outlet were recorded. After concentration to a certain amount (60 to 80 mL), 250 mL of physiological saline was added to the plastic container to dilute the cell concentrate. Subsequently, as in the past, the cell suspension was passed through the cell suspension processor while adjusting the flow rate with a pump, and filtered until it was concentrated to a certain volume (60 mL to 80 mL) while circulating. It was. Thus, the dilution and concentration were repeated using 250 mL of physiological saline, and the operation of washing the cell suspension was performed 4 times. Finally, the flow path was switched to the collection container side, and the cell concentrate in the plastic container, tube, and hollow fiber type separation membrane was pushed out at a constant flow rate by a pump and collected in the collection container. Subsequently, the number of cells in the cell concentrate in the collection container was measured, and the cell recovery rate and the survival rate ratio were calculated. Moreover, the cell suspension used for an Example was made into 1000 mL of cell suspension (RPMI1640 culture medium containing 10% FBS) containing the cultured Jurkat cell. Examples are shown below.

Example 1
A cell suspension treatment device was prepared using 300 polyethersulfone hollow fiber separation membranes [product name: MF020 TYPE-1 hollow fiber inner diameter 570 μm, pore diameter 0.2 μm, manufactured by Toyobo Co., Ltd.]. The filtration area of the prepared processor was 0.12 m ² and the total cross-sectional area of the hollow fiber type separation membrane was 0.77 cm ² . The concentration conditions were such that the flow rate to the cell suspension inlet was 452 mL / min (linear velocity: 590 cm / min) and the filtration flow rate was 200 mL / min discharged from the filtrate outlet. The flow rate during recovery was 31 mL / min (linear velocity 40 cm / min). As a result, the cell recovery rate was 94%, the survival rate ratio was 100%, and the treatment time was 440 seconds.

(Example 2)
The same cell suspension treatment apparatus as in Example 1 was used. The concentration conditions were such that the flow rate to the cell suspension inlet was 452 mL / min (linear velocity: 590 cm / min) and the filtration flow rate was 200 mL / min discharged from the filtrate outlet. The flow rate during recovery was 38 mL / min (linear velocity 50 cm / min). As a result, the cell recovery rate was 90%, the survival rate ratio was 98%, and the treatment time was 404 seconds.

Example 3
The same cell suspension treatment apparatus as in Example 1 was used. The concentration conditions were such that the flow rate to the cell suspension inlet was 452 mL / min (linear velocity: 590 cm / min) and the filtration flow rate was 200 mL / min discharged from the filtrate outlet. The flow rate at the time of recovery was 77 mL / min (linear velocity 100 cm / min). As a result, the cell recovery rate was 89%, the survival rate ratio was 98%, and the treatment time was 334 seconds.

Example 4
The same cell suspension treatment apparatus as in Example 1 was used. The concentration conditions were such that the flow rate to the cell suspension inlet was 452 mL / min (linear velocity: 590 cm / min) and the filtration flow rate was 200 mL / min discharged from the filtrate outlet. The flow rate during recovery was 115 mL / min (linear velocity: 150 cm / min). As a result, the cell recovery rate was 91%, the survival rate ratio was 97%, and the treatment time was 310 seconds.

(Example 5)
The same cell suspension treatment apparatus as in Example 1 was used. The concentration conditions were such that the flow rate to the cell suspension inlet was 482 mL / min (linear velocity: 630 cm / min), and the filtration flow rate was 200 mL / min discharged from the filtrate outlet. The flow rate during recovery was 38 mL / min (linear velocity 50 cm / min). As a result, the cell recovery rate was 90%, the survival rate ratio was 96%, and the treatment time was 396 seconds.

(Example 6)
The same cell suspension treatment apparatus as in Example 1 was used. The concentration conditions were such that the flow rate to the cell suspension inlet was 299 mL / min (linear velocity: 390 cm / min) and the filtration flow rate was 200 mL / min discharged from the filtrate outlet. The flow rate during recovery was 38 mL / min (linear velocity 50 cm / min). As a result, the cell recovery rate was 87%, the survival rate ratio was 95%, and the treatment time was 540 seconds.

(Example 7)
The same cell suspension treatment apparatus as in Example 1 was used. The concentration conditions were such that the flow rate to the cell suspension inlet was 283 mL / min (linear velocity: 370 cm / min), and the filtration flow rate was 200 mL / min discharged from the filtrate outlet. The flow rate during recovery was 38 mL / min (linear velocity 50 cm / min). As a result, the cell recovery rate was 86%, the survival rate ratio was 98%, and the treatment time was 554 seconds.

(Example 8)
A separation membrane similar to that in Example 1 was used. The number of hollow fibers used was 250, the filtration area of the prepared processing device was 0.10 m ² , and the total cross-sectional area of the hollow fiber type separation membrane was 0.64 cm ² . The concentration conditions were such that the flow rate to the cell suspension inlet was 377 mL / min (linear velocity 590 cm / min) and the filtration flow rate was 200 mL / min discharged from the filtrate outlet. The flow rate at the time of recovery was 64 mL / min (linear velocity 100 cm / min). As a result, the cell recovery rate was 89%, the survival rate ratio was 98%, and the treatment time was 404 seconds.

(Comparative Example 1)
The same cell suspension treatment apparatus as in Example 1 was used. The concentration conditions were such that the flow rate to the cell suspension inlet was 452 mL / min (linear velocity: 590 cm / min) and the filtration flow rate was 200 mL / min discharged from the filtrate outlet. The flow rate during recovery was 149 mL / min (linear velocity: 195 cm / min). As a result, the cell recovery rate was 84%, the survival rate ratio was 97%, and the treatment time was 300 seconds.

(Comparative Example 2)
The same cell suspension treatment apparatus as in Example 1 was used. The concentration conditions were such that the flow rate to the cell suspension inlet was 452 mL / min (linear velocity: 590 cm / min) and the filtration flow rate was 200 mL / min discharged from the filtrate outlet. The flow rate at the time of recovery was 299 mL / min (linear velocity: 390 cm / min). As a result, the cell recovery rate was 83%, the survival rate ratio was 94%, and the treatment time was 282 seconds.

(Comparative Example 3)
The same cell suspension treatment apparatus as in Example 1 was used. The concentration conditions were such that the flow rate to the cell suspension inlet was 149 mL / min (linear velocity: 195 cm / min) and the filtration flow rate was 200 mL / min discharged from the filtrate outlet. The flow rate during recovery was 38 mL / min (linear velocity 50 cm / min). As a result, the cell recovery rate was 74%, the survival rate ratio was 98%, and the treatment time was 940 seconds.

From Table 1, the step of concentrating the cell suspension while passing the cell suspension at a linear velocity of 370 to 630 cm / min and discharging the filtrate from the outlet for the filtrate, and the cell concentration of 40 to 150 cm Cell concentration at a high cell recovery rate and survival rate by using a method for producing a cell concentrate that includes a step of collecting the cell concentrate extracted from the cell suspension outlet through a recovery container. It is clear that a liquid can be produced.

  As described above, the cell suspension remaining in plastic containers, tubes, and hollow fiber type separation membranes is less damaging and efficient by lowering the linear velocity during cell recovery than during cell concentration. Cells can be recovered. Furthermore, contaminants such as proteins other than cells in the cell suspension can be efficiently removed by washing with physiological saline after cell concentration. Therefore, if the cell concentrate produced by the method of the present invention is used, only target cells can be efficiently concentrated and collected in a short time by an aseptic operation, so that cells for cell therapy can also be provided. .

1. Tubular container Torso part 3. Head 4. 4. Filtrate outlet 5. bundle of hollow fiber type separation membranes Open end (dotted line)
7). Resin layer (shaded area)
8a. Cell suspension inlet 8b. Cell suspension outlet 9. Header part 10. Collection container 11. Mixed injection port 12. Bottle needle connection port 13. Container with cell suspension 14. Pump 15a. Two-way stopcock on the collection container side 15b. Two-way stopcock on the side of the container containing the cell suspension 15c. 15. Two-way stopcock on the waste liquid container side Waste container

Claims (10)

A method for producing a cell concentrate using a cell suspension processor in which a hollow fiber separation membrane is packed in a container having a cell suspension inlet, a cell suspension outlet, and a filtrate outlet. And
(A) passing the cell suspension from the cell suspension inlet at a linear velocity of 370 to 630 cm / min, and concentrating the cell suspension while discharging the filtrate from the filtrate outlet;
(B) passing the cell concentrate through the cell suspension inlet at a linear velocity of 40 to 150 cm / min, and collecting the cell concentrate derived from the cell suspension outlet into a collection container;
A method for producing a cell concentrate comprising: Method for producing a cell concentrate according to claim 1, the total cross-sectional area of the hollow fiber type separation membranes filled in the cell suspension processor is characterized in that it is a 0.5 to 1.5 cm ^2. The method for producing a cell concentrate according to claim 1 or 2, wherein the hollow fiber separation membrane filled in the cell suspension treatment device has an inner diameter of 300 to 1000 µm. The production of the cell concentrate according to any one of claims 1 to 3, further comprising a step of washing the cell suspension by repeating dilution and concentration using a diluent before the step (b). Method. The method for producing a cell concentrate according to any one of claims 1 to 4, wherein the collection container has one or more ports through which liquid can flow in and out. The method for producing a cell concentrate according to claim 5, comprising a step of taking out the cell concentrate through a port after the step (b). The method for producing a cell concentrate according to claim 5 or 6, comprising a step of adding a drug through a port after the step (b). The manufacturing method of the cell concentrate in any one of Claims 1-7 including the process of sealing and dividing a collection container after the process of (b). The method for producing a cell concentrate according to any one of claims 1 to 8, wherein the cell suspension contains immune cells.

The method for producing a cell concentrate according to any one of claims 1 to 9, wherein the cell recovery rate is 86% and the cell viability ratio is 95% or more.

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