JP2016116465A - Production method of cell concentrated solution - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing cell concentrated solution having extremely low content of impurity by improving removal efficiency of contaminant and culture medium component contained in cell suspension and concentration efficiency of cells in a washing process.SOLUTION: By using a hollow fiber membrane module which is equipped with an inlet, an outlet and a filtrate discharge port, and filled with a hollow fiber type separation membrane, cell suspension is sent into the hollow fiber membrane module, and cell suspension is concentrated while filtrate is discharged from the filtrate discharge port arranged on an upper side in a vertical direction of the hollow fiber membrane module. Then, a position of the filtrate discharge port is switched to a lower side in the vertical direction of the hollow fiber membrane module, washing fluid is subsequently sent into the hollow fiber membrane module, cells are washed while filtrate is discharged from the filtrate discharge port, and then cell concentrated solution is recovered from the outlet, by which high-purity cell concentrated solution can be produced efficiently.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a method for producing a cell concentrate using a hollow fiber membrane module filled with a hollow fiber type separation membrane.

  In the field of cell medicine, a method of inoculating a patient with cells collected from a living body directly or after in vitro culture is used. 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 contain tissue-derived contaminants or medium components. There are many. Therefore, in order to use cells collected or cultured from a living body for treatment, remove impurities and medium components, make them suspended in a solution suitable for treatment (washing), and concentrate to a cell concentration suitable for treatment. Need to be done.

  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. Furthermore, when the supernatant of the cell suspension after the cells are precipitated by centrifugation is removed for washing the cells, the cells may be released to the atmosphere, and problems such as contamination may be raised.

  On the other hand, a cell suspension separation and filtration method using a hollow fiber type separation membrane, which is a compact and simple apparatus, has been proposed (Patent Document 2). When concentrating a cell suspension for cell medical use using a hollow fiber type separation membrane, it is necessary to perform a concentration treatment in a short time to reduce damage to the cells, and further, impurities and culture media Since the molecular weight of the component is large, it is considered better to increase the pore diameter of the hollow fiber type separation membrane.

  The present applicant also has a method for producing a cell concentrate using a hollow fiber separation membrane, for example, a cell suspension inlet for receiving a cell suspension, and a method for discharging a liquid substantially free of the cell components. And a hollow fiber type separation having a molecular weight cut-off of 1000 kD or less disposed between the cell suspension inlet and the cell suspension outlet. A cell suspension concentration method (Patent Document 3), comprising a membrane and using a cell suspension treatment device of an internal pressure filtration method, a total cross-sectional area of a hollow fiber type separation membrane, a hollow fiber type separation membrane Has proposed a method for producing a cell concentrate that adjusts the inner membrane area, the linear velocity flowing inside the hollow fiber separation membrane, and the initial filtration flow rate discharged from the filtrate outlet to a predetermined range (Patent Document 4). .

Special Table 2007-524396 Japanese Patent No. 2928913 JP 2012-210187A International Publication No. 2014/034456

  However, in the case of actually producing a cell concentrate using a hollow fiber membrane module, in the washing step, impurities and medium components removed together with the washing solution from the cell suspension are the amount of filtrate in the cell concentration and the washing solution to be introduced. As a result, a large amount of washing solution and a long treatment time were required to sufficiently remove impurities and medium components. There was room. Further, if medium components remain in the produced cell concentrate, it cannot be used for therapeutic purposes as it is, and if it is purified again, the cell viability may be impaired.

  In view of the above improvements, an object of the present invention is to solve the problems in the method for producing a cell concentrate using the hollow fiber membrane module. Specifically, in the washing step, there is a need to provide a method for producing a cell concentrate that improves the removal efficiency of impurities and medium components contained in the cell suspension and has a very low impurity content. Furthermore, the present invention provides a method for producing a cell concentrate in which the cell recovery rate and operability are improved by setting the linear velocity to two stages in the concentration step.

As a result of intensive studies to solve the above problems, the present inventors have found that when the filtrate containing impurities and medium components filtered to the outside of the hollow fiber membrane in the washing step is discharged from the hollow fiber membrane module, While the filtrate stayed in the hollow fiber membrane module for a while, it was found for the first time that a phenomenon in which contaminants and medium components returned to the inside of the hollow fiber membrane again by diffusion occurred. Therefore, as a result of further research, the position of the filtrate outlet used in the concentration step is switched to a predetermined position before the washing step, so that the filtrate containing impurities and medium components can be quickly removed in the subsequent washing step. It was successfully discharged to the outside to prevent foreign substances and medium components from returning to the inside of the hollow fiber membrane.
Furthermore, by setting the linear velocity in the concentration process to two stages, it is possible to produce a cell concentrate without performing a priming operation, and cells with a high cell recovery rate while reducing the processing time required for the concentration process. The inventors have found that a concentrated solution can be obtained, and have completed the present invention.

That is, the gist of the present invention is as follows.
(1) A method for producing a cell concentrate using a hollow fiber membrane module comprising an inlet, an outlet and a filtrate outlet, and filled with a hollow fiber type separation membrane,
A step of feeding the cell suspension into the hollow fiber membrane module, and concentrating the cell suspension while discharging the filtrate from a filtrate outlet arranged on the upper side in the vertical direction of the hollow fiber membrane module;
The step of switching the position of the filtrate outlet to the lower side in the vertical direction of the hollow fiber membrane module, and the washing liquid is fed into the hollow fiber membrane module, and the cells are washed while discharging the filtrate from the filtrate outlet. A method for producing a cell concentrate, comprising the step of recovering the cell concentrate.
(2) Two filtrate outlets are provided in the hollow fiber membrane module, and the position of the filtrate outlet for the filtrate containing the washing liquid is the distance from the lower end of the hollow fiber membrane module to one of the filtrate outlets. The method for producing a cell concentrate according to (1), wherein x is adjusted to a position where L / 2 ≧ x as compared with a length L in the vertical direction of the hollow fiber membrane module.
(3) The ratio of the length L (cm) in the vertical direction of the hollow fiber membrane module to the distance y (cm) between two filtrate outlets is L: y = 3: 1 to 10: 9 The method for producing a cell concentrate according to (2).
(4) The method for producing a cell concentrate according to any one of (1) to (3), wherein the position of the filtrate outlet is switched by changing the direction of the hollow fiber membrane module.
(5) The method for producing a cell concentrate according to any one of (1) to (4), wherein the cell suspension is concentrated at a two-stage linear velocity in the step of concentrating the cell suspension.
(6) In the two-stage linear velocity, the first-stage linear velocity a (cm / min) and the second-stage linear velocity b (cm / min) satisfy the relationship a <b. The manufacturing method of the cell concentrate of description.
(7) The ratio of the linear velocity a (cm / min) in the first step to the linear velocity b (cm / min) in the second step is a: b = 1: 1.2 to 1:32. The method for producing a cell concentrate according to 6).
(8) The cell concentrate according to (6) or (7), wherein the linear velocity a in the first stage is 35 to 300 cm / min, and the linear velocity b in the second stage is 370 to 1100 cm / min. Production method.
(9) Before the step of concentrating the cell suspension, the priming solution is fed into the hollow fiber membrane module while exhausting from the filtrate outlet arranged on the upper side in the vertical direction of the hollow fiber membrane module. The manufacturing method of the cell concentrate in any one of said (1)-(8) including a process.
(10) The method for producing a cell concentrate according to any one of (1) to (9), wherein the hollow fiber separation membrane filled in the hollow fiber membrane module has a pore diameter of 0.1 μm or more.
(11) The method for producing a cell concentrate according to any one of (1) to (10), wherein an inner diameter of the hollow fiber type separation membrane filled in the hollow fiber membrane module is 300 to 1000 μm.

  According to the method for producing a cell concentrate of the present invention, after the cells are concentrated, the filtrate is discharged after switching the position of the filtrate outlet of the hollow fiber membrane module to the lower side in the vertical direction of the hollow fiber membrane module. Since impurities and medium components in the medium can be quickly discharged from the hollow fiber membrane module without returning to the inside of the hollow fiber membrane, cells can be efficiently washed and concentrated in a short time with a small amount of washing liquid. . The cell concentrate obtained after the washing can be provided for therapeutic use because the content of impurities such as impurities and medium components is significantly reduced. Furthermore, by setting the linear velocity in the concentration step to two stages, the priming operation can be omitted, and a cell concentrate having a high cell recovery rate can be obtained in a short time.

It is the schematic which shows an example of embodiment of the hollow fiber membrane module used by this invention. It is the schematic of the hollow fiber membrane module 1 shown to Fig.1 (a). It is the schematic which shows an example of embodiment of the cell concentration method using the hollow fiber membrane module 1 shown to Fig.1 (a). It is the schematic which shows an example of embodiment of the cell concentration method using the hollow fiber membrane module 1 'shown in FIG.1 (b). In the cell concentration method using the hollow fiber membrane module 1 'shown in FIG.1 (b), it is the schematic which shows an example of embodiment which switched the position of the filtrate discharge port.

  The present invention will be described below.

  The cell concentration method of the present invention is a method for producing a cell concentrate using a hollow fiber membrane module having an introduction port, a discharge port and a filtrate discharge port and filled with a hollow fiber type separation membrane.

  The hollow fiber membrane module used in the present invention is of a type in which a hollow fiber membrane is filled in a container having an inlet, an outlet and a filtrate outlet. In the hollow fiber membrane module, a cell suspension is introduced into the hollow fiber membrane from the inlet or outlet. While the cell suspension fills the space inside the hollow fiber membrane, it passes through the micropores provided in the hollow fiber membrane wall, and the filtrate containing impurities and medium components is discharged outside the hollow fiber membrane. The filtrate is discharged from the hollow fiber membrane module through the filtrate discharge port. On the other hand, the concentrated cell suspension inside the hollow fiber membrane is discharged from the hollow fiber membrane module through the outlet or inlet.

The hollow fiber membrane module is not particularly limited as long as it has the above-described configuration. For example, the hollow fiber membrane module 1 shown in FIG. The container constituting the hollow fiber membrane module 1 is composed of a body 2 and heads 3A and 3B on both sides thereof, near the head 3A on the upper side, near the filtrate outlet 5a, and near the head 3B on the lower side. Is provided with a filtrate outlet 5b.
The head 3A is provided with an inlet 6 and the head 3B is provided with a outlet 7. The inlet port 6 and the outlet port 7 are inlets and outlets through which the cell suspension enters and exits the inside of the hollow fiber membrane. The same applies to the washing liquid and the priming liquid as well as the cell suspension. 1 to 4, the hollow fiber membrane modules 1, 1 ′ are described with the upper port in the vertical direction as the inlet 6 and the lower port in the vertical direction as the outlet port 7.

  Inside the barrel portion 2, the bundle 8 of hollow fiber membranes loaded and the bundle 8 of hollow fiber membranes provided inside the head 3 A are fixed inside the barrel portion 2 and the open end of the hollow fiber membrane is provided. 9 has a structure equivalent to that provided in the resin layer portion 10 forming the head 9 and further inside the head portion 3B. The resin layer portion 10 and the open end 9 have a structure covered with a head portion 3A (or a head portion 3B), and the introduction port 6, the discharge port 7 and the filtrate discharge ports 5a and 5b are made of a hollow fiber membrane. It is separated by the wall material which comprises, and has a non-continuous structure.

  As another embodiment of the hollow fiber membrane module, as shown in FIG. 1 (b), there is a hollow fiber membrane module 1 'having only a filtrate outlet 5a (no filtrate outlet 5b).

  The hollow fiber membrane modules 1 and 1 ′ shown in FIGS. 1 (a) and 1 (b) are distinguished from each other as the body 2 of the container, the head 3A, and the head 3B. It is convenient. Even if the body 2 of the container, the head 3A, and the head 3B are formed of separate parts by design, the inlet 6 and outlet 7 are continuous without being separated by the wall material that forms the hollow fiber membrane. In addition, various structures can be adopted as long as the introduction port 6 and the discharge port 7 and the filtrate discharge ports 5a and 5b are separated by a wall material constituting the hollow fiber membrane. .

In the hollow fiber membrane module, the number of filtrate outlets may be single or two, and there is no particular limitation. However, when two filtrate outlets are provided, the position of the filtrate outlet for the filtrate containing the cleaning liquid is determined. The distance x from the lower end of the hollow fiber membrane module to any one of the filtrate outlets is adjusted to a position where L / 2 ≧ x compared to the vertical length L of the hollow fiber membrane module. In addition, the volume that can be retained until the impurities and medium components in the filtrate are discharged can be reduced.
For example, in the hollow fiber membrane module 1 shown in FIG. 2, filtrate outlets 5a and 5b are provided, and the filtrate outlet 5b on the lower side in the vertical direction is used as the filtrate outlet for the filtrate containing the cleaning liquid. In this case, the “distance x from the lower end of the hollow fiber membrane module to the filtrate outlet” refers to the distance from the lower end of the outlet 7 in the hollow fiber membrane module 1 to the center of the filtrate outlet 5b. .
x is not particularly limited as long as it is adjusted to a position where L / 2 ≧ x compared to the length L in the vertical direction of the hollow fiber membrane module, but a specific value of x is preferably 4 to 25 cm. -20 cm is more preferable, and 4-10 cm is more preferable.

“Vertical length L of the hollow fiber membrane module” refers to the distance from the lower end of the outlet 7 in the hollow fiber membrane module 1 to the upper end of the inlet 6.
The value of L is not particularly limited, but specifically is preferably 15 to 50 cm, and more preferably 20 to 40 cm.

Further, the ratio of the length L (cm) in the vertical direction of the hollow fiber membrane module to the distance y (cm) between the two filtrate outlets is adjusted to a range of L: y = 3: 1 to 10: 9. By doing so, the cleaning effect by switching the filtrate outlet can be easily exhibited. The ratio is more preferably adjusted to a range of L: y = 3: 1 to 3: 2.
The distance y between the two filtrate outlets is, for example, the distance from the center of the upper filtrate outlet 5a to the center of the lower filtrate outlet 5b in the hollow fiber membrane module 1 shown in FIG. Say.
The value of y is preferably 5 to 45 cm, and more preferably 15 to 45 cm.

  The hollow fiber membrane module may be sterilized from the viewpoint of preventing bacteria from entering the cells. 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.

  As materials for hollow fiber membrane module containers, acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymer; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride; 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 membrane, general adhesive materials such as polyurethane resin, epoxy resin, and silicon resin can be preferably used.

The hollow fiber membrane module is preferably filled with a bundle of about 10 to 1000 hollow fiber membranes. In the present invention, the arrangement of the hollow fiber membrane may be linear, bent, or spiral, and both ends of the hollow fiber membrane are held between the inlet and the outlet. If it is done, the shape is not particularly limited.
In addition, the hollow fiber membrane at the time of cell concentration may be arranged in the vertical direction, arranged in the horizontal direction, or arranged in the oblique direction, but the washing effect by switching the filtrate outlet It is more preferable that the hollow fiber membranes are arranged in the vertical direction from the viewpoint of easily exhibiting.

  As the resin material for the hollow fiber 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 or cellulose-based hydrophilic polymer materials can be more preferably used. Furthermore, polyethersulfone and cellulose ester can be most suitably used because of the safety, stability, and availability of the material.

Moreover, the hole diameter of the hole provided in the wall of the hollow fiber membrane is preferably 0.1 μm or more and 1.5 μm or less. When the pore size is less than 0.1 μm, it is not preferable because impurities such as unnecessary proteins cannot be efficiently removed when the cell suspension is concentrated. On the other hand, when the pore diameter exceeds 1.5 μm, pores having a pore size close to the size of the cells are present in the hollow fiber membrane, so that clogging occurs in the step of concentrating the cell suspension, and the cell recovery rate is large. Since it may be lowered, it is not preferable.
The pore diameter is preferably 1.5 μm or less, more preferably 1.2 μm or less, and further preferably 1.0 μm or less.
The said hole diameter points out the average hole diameter of a hollow fiber membrane, and is generally measured and calculated with a palm porometer.

  Examples of cells in the present invention include artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), mesenchymal stem cells, adipose-derived mesenchymal stem cells, adipose-derived stromal stem cells, and pluripotent adult stem cells. Lymphocytes such as bone marrow stromal cells, hematopoietic stem cells, and other multipotent biological stem cells, 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, nerve cells, muscle cells, fibroblasts, hepatocytes, cardiomyocytes, and other cells that have undergone treatment such as gene transfer or differentiation And rare cells such as tumor cells, endothelial cells and fetal cells.

The cell suspension is not particularly limited as long as it is a suspension containing the cells. For example, fat, skin, blood vessel, cornea, oral cavity, kidney, liver, pancreas, heart, nerve, muscle, prostate, intestine Suspension, blood, and bone marrow fluid after enzymatic treatment, crushing treatment, extraction treatment, decomposition treatment, ultrasonic treatment, etc. of biological tissue such as amniotic membrane, placenta, and umbilical cord, density gradient centrifugation, filtration treatment, and enzyme treatment And cell suspensions prepared by pretreatment such as decomposition treatment and ultrasonic treatment.
Moreover, the cell suspension after culturing the cell illustrated 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. In addition, a culture solution to which a stimulating factor such as cytokine, antibody or peptide is added can also be used.

  Below, each process of this invention is demonstrated in detail.

(Concentration process)
In the method for producing a cell concentrate of the present invention, first, a cell suspension is fed from the inlet or outlet of the hollow fiber membrane module, and the filtrate is disposed on the upper side in the vertical direction of the hollow fiber membrane module. Concentrate the cell suspension as it drains from the outlet.

In this step, the hollow fiber membrane module is arranged so that the filtrate outlet for the filtrate filtered from the cell suspension is on the upper side in the vertical direction. By disposing the filtrate outlet on the upper side in the vertical direction in this way, the pressure of the filtrate applied to the filtrate outlet is reduced as compared with the case where it is arranged on the lower side in the vertical direction. Efficient cell concentration can be performed by suppressing clogging of the hollow fiber membrane by cells flowing through the lumen.
For example, in the hollow fiber membrane modules 1 and 1 ′ shown in FIGS. 1 (a) and 1 (b), the filtrate outlet 5a on the upper side in the vertical direction is the filtrate outlet used in this step.

  Further, as an embodiment of this step, for example, as shown in FIG. 3, the introduction port 6 of the hollow fiber membrane module 1 and the outlet port 7 are connected via a circuit 11 including a storage container 12, and the hollow fiber The cell suspension discharged from the outlet 7 of the membrane module 1 is made to flow again inside the hollow fiber membrane in the hollow fiber membrane module 1, and the liquid in the cell suspension is discharged from the hole of the hollow fiber membrane. Thus, the cell components in the cell suspension can be further concentrated. By circulating the cell suspension in this way and repeating the concentration, a cell suspension concentrated to a desired cell concentration is generated.

  The cell suspension is stored in a storage container 12 connected to the circuit 11. At the start of concentration, the storage container 12 filled with the cell suspension is connected to the circuit 11, and then the pump 14a in the circuit 11 between the storage container 12 and the hollow fiber membrane module 1 is driven to A predetermined amount of cell suspension is introduced from the inlet 6 or outlet 7 of the thread membrane module 1. As the position of the pump 14a, as shown in FIG. 3, the circuit 11 between the inlet 6 in the head 3 of the hollow fiber membrane module 1 and the storage container 12 or the outlet of the header 4 is not shown. What is necessary is just to be installed in either of the circuits 11 between 7 and the storage container 12.

  Tubes are connected to the filtrate discharge port 5a on the upper side in the vertical direction and the filtrate discharge port 5b on the lower side in the vertical direction of the hollow fiber membrane module 1, and the tubes join together to be connected to the waste liquid container 15. A valve 13a is installed on the outlet side of the filtrate outlet 5a, and a valve 13b is installed on the outlet side of the filtrate outlet 5b so that the flow of the filtrate can be adjusted. Moreover, the pump 14b may be provided in the tube after the said joining.

The filtrate discharged from the hollow fiber membrane 8 of the hollow fiber membrane module 1 is discharged to the waste liquid container 15 by driving the pump 14b from the filtrate discharge port 5a on the upper side in the vertical direction.
In this case, the valve 13b near the outlet of the filtrate outlet 5b is closed so that the filtrate is not discharged from the lower filtrate outlet 5b.

  The concentration rate of the cell suspension in this step is determined by measuring the volume of the cell suspension returned from the outlet 7 or the inlet 6 of the hollow fiber membrane module 1 to the storage container 12 via the circuit 11, The cell concentration may be measured by sampling the cell suspension in the container 12.

  A branch part 16 is provided in the circuit 11 between the outlet 7 of the hollow fiber membrane module 1 and the storage container 12, and a recovery container 17 is connected to the branch part 16 via a tube. Further, a valve 13c is provided in the circuit 11 between the branch part 16 and the storage container 12, and a valve 13d is provided in the tube between the branch part 16 and the collection container 17, so that the cell suspension in the circuit 11 is provided. It is possible to adjust the flow of.

  Moreover, as another embodiment of this process, as shown in FIG. 4, the form using the hollow fiber membrane module 1 'is mentioned. In the embodiment shown in FIG. 4, since the filtrate outlet 5b on the lower side in the vertical direction is not provided, it is not necessary to close the valve 13b as in the hollow fiber membrane module 1 shown in FIG. The same operation as that of the hollow fiber membrane module 1 shown in FIG.

  Further, in this step, the linear velocity of the cell suspension when introduced into the hollow fiber membrane module may be constant, but the priming operation is performed by concentrating by changing the linear velocity in two stages. The cell recovery rate can be improved while shortening the operation time.

As the two-stage linear velocity, the first-stage linear velocity a (cm / min) and the second-stage linear velocity b (cm / min) are adjusted so as to satisfy the relationship of a <b. The stage is performed for the purpose of priming, and since the hollow fiber membrane can be filled with the cell suspension, all the hollow fiber membranes can be used effectively. Thereafter, increasing the linear velocity in the second stage is preferable because the cell suspension can be efficiently concentrated while shortening the concentration time.
Among them, from the viewpoint of sufficiently extruding the air in the hollow fiber membrane outside the membrane in the first stage and efficiently concentrating in the second stage, the linear velocity a (cm / min) in the first stage and the second stage It is preferable to adjust the ratio of the linear velocity b (cm / min) to a range of a: b = 1: 1.2 to 1:32, and a ratio of a: b = 1: 10 to 1:20. Is more preferable.
In this way, when the concentration is performed at two stages of linear velocity, the air in the hollow fiber membrane can be pushed out of the membrane efficiently for the first stage, and the damage to the cells can be reduced as much as possible for the second stage. From the viewpoint of producing a cell concentrate with a high cell recovery rate in a short time, the first stage linear velocity a is in the range of 35 to 300 cm / min, and the second stage linear velocity b is in the range of 370 to 1100 cm / min. The linear velocity b is more preferably adjusted in the range of 390 to 590 cm / min.

  The linear velocity is the amount of cell suspension that passes through the hollow fiber lumen cross section in the hollow fiber membrane module per unit time. The flow rate (flow rate) of the cell suspension into the hollow fiber membrane module is the cell suspension. It can be calculated by dividing by the total cross-sectional area of the hollow fiber lumen in the liquid processor.

  Moreover, there is no limitation in particular about other concentration conditions, such as the filtration rate in this process, and a hollow fiber membrane area.

  The end of this step is, for example, confirming that the amount of the filtrate discharged from the cell suspension has reached a predetermined amount, sampling the cell suspension in the storage container 12, and measuring the cell concentration. It may be judged by things. Specifically, this step may be completed by stopping the flow of the concentrated cell suspension into the hollow fiber membrane module.

(Switching process)
In the present invention, after the concentration step, the position of the filtrate outlet is switched to the lower side in the vertical direction of the hollow fiber membrane module. By switching the position of the filtrate outlet in this way, the filtrate containing impurities and medium components remaining in the hollow fiber membrane module is quickly transferred to the outside of the hollow fiber membrane module when the washing process described later is performed. Can be discharged.

  As embodiment which switches a filtrate discharge port as mentioned above, the form shown in FIG. 3 is mentioned, for example. In the form shown in FIG. 3, by opening and closing the valves 13a and 13b on the outlet side of the filtrate outlet 5a provided on the upper side in the vertical direction of the hollow fiber membrane module 1 and the filtrate outlet 5b provided on the lower side in the vertical direction, respectively. Switch. That is, during the concentration step, the valve 13b is closed and the filtrate is discharged from the filtrate outlet 5a on the upper side in the vertical direction. During the subsequent switching step, the valve 13a is closed, and the valve 13b is opened instead. The filtrate outlet is switched by discharging the filtrate from the filtrate outlet 5b. The filtrate outlets 5a and 5b may be opened and closed by a known method other than a valve.

  Moreover, as another embodiment which switches a filtrate discharge port, the form shown in FIG. 5 is mentioned, for example. The form shown in FIG. 5 is the form after concentration in the form shown in FIG. 4. The hollow fiber membrane module 1 ′ shown in FIG. Connected to. For this reason, the position of the filtrate outlet 5a is switched from the vertical upper side in FIG. 4 to the vertical lower side in FIG. At the same time, the positions of the inlet 6 and outlet 7 are also reversed up and down.

  In another embodiment, the position may be switched by sliding one filtrate outlet. As such a hollow fiber membrane module, although not shown, for example, a hollow fiber membrane module configured such that the position of the filtrate discharge port can be slid and moved in the long axis direction of the hollow fiber membrane module. In the hollow fiber membrane module of this aspect, as shown in FIGS. 4 and 5, switching can be performed without removing the hollow fiber membrane module 1 ′ from the circuit 11. The slide mechanism is not particularly limited as long as the filtrate can be discharged from the hollow fiber membrane module.

(Washing / recovery process)
In the present invention, after the switching step, the washing liquid is fed from the inlet or outlet, and the cells are washed while discharging the filtrate from the filtrate outlet, and then the cell concentrate is recovered from the outlet.

  In this step, impurities and medium components contained in the concentrated cell suspension in the hollow fiber membrane module and the circuit connecting the inlet and outlet are introduced by introducing the washing liquid into the hollow fiber membrane module. To the outside of the hollow fiber membrane as a filtrate. Then, the filtrate and the medium component are again removed from the filtrate by quickly discharging the filtrate from the filtrate outlet switched to the lower side in the vertical direction of the hollow fiber membrane module by the switching step. Therefore, it is possible to efficiently perform washing and concentration, and to obtain a cell concentrate having a very low impurity content.

For example, in the embodiment shown in FIG. 3, a solution bag filled with a cleaning liquid is connected to the storage container 12 (not shown), and the cleaning liquid is introduced into the storage container 12. Next, the pump 14 a is driven to introduce the cleaning liquid into the hollow fiber membrane module 1 from the inlet 6 or the outlet 7. The filtrate discharged to the outside of the hollow fiber membrane is discharged to the waste liquid container 15 from the filtrate discharge port 5b on the lower side in the vertical direction by driving the pump 14b. The generated cell concentrate is returned to the storage container 12 via the circuit 11.
While continuing the washing and concentration, the cell concentrate in the storage container 12 is sampled to confirm that the concentrations of impurities and culture components are below a predetermined amount, and the washing is finished.
Next, the valve 13c between the branch part 16 and the storage container 12 is closed, the valve 13d between the branch part 16 and the recovery container 17 is opened, and the pump 14a is driven again to recover the cell concentrate. It is introduced into the container 17 and collected.

In the embodiment shown in FIG. 5 as well, the cleaning / recovery operation is performed as in the embodiment shown in FIG. That is, a solution bag filled with a cleaning liquid is connected to the storage container 12 (not shown), and after introducing the cleaning liquid into the storage container 12, the pump 14a is driven and the cleaning liquid is hollowed out from the inlet 6 or the outlet 7 It introduces into the yarn membrane module 1. The filtrate discharged to the outside of the hollow fiber membrane is discharged to the waste liquid container 15 by driving the pump 14b from the filtrate discharge port 5a on the lower side in the vertical direction. The generated cell concentrate is returned to the storage container 12 via the circuit 11.
While continuing the washing and concentration, the cell concentrate in the storage container 12 is sampled to confirm that the concentrations of impurities and culture components are below a predetermined amount, and the washing is finished.
Next, the valve 13c between the branch part 16 and the storage container 12 is closed, the valve 13d between the branch part 16 and the recovery container 17 is opened, and the pump 14a is driven again to recover the cell concentrate. It introduce | transduces into the container 17 and collect | recovers.

In this step, the linear velocity of the washing liquid when introduced into the hollow fiber membrane module is not particularly limited as long as it is the same as the linear velocity of the cell suspension in the concentration step.
Moreover, as washing conditions of this process, what is necessary is just to be able to adjust to the said linear velocity, For example, there is no limitation in particular about other conditions, such as a filtration rate and a hollow fiber membrane area.

  The washing solution is not particularly limited as long as the cells to be collected are not damaged, but a general buffer solution such as physiological saline, Ringer's solution, a medium used for cell culture, and phosphate buffer is preferable.

(Priming process)
Further, in the present invention, before the concentration step, the priming liquid may be sent from the inlet or outlet while exhausting from the filtrate outlet arranged vertically above the hollow fiber membrane module. Good.

  In this step, the air pushed out by introducing the priming liquid into the inside of the hollow fiber membrane is quickly discharged from the filtrate outlet arranged on the upper side in the vertical direction of the hollow fiber membrane module. Can be done well.

  Examples of the priming solution include infusion solutions typified by physiological saline, distilled water, Ringer's solution, liquids containing inorganic salts, saccharides, serum, proteins, buffers, plasma, media, and the like. In particular, a physiological saline solution or an infusion solution can be preferably used from the viewpoint of safety. In addition, the liquid containing serum, plasma, and protein can be used as a priming solution because the surface of the hollow fiber membrane is coated with protein, thereby preventing cell adhesion to the hollow fiber membrane and improving the cell recovery rate. It can be used suitably. The priming liquid does not have to be one kind of liquid, and two or more liquids from these groups can be mixed and used. As an example of mixing and using two liquids, a combination of a physiological saline solution added with albumin or a culture medium added with serum can be mentioned.

  For example, in the embodiment shown in FIG. 3, the priming liquid is introduced into the storage container 12, the pump 14 a is driven, and the priming liquid is introduced into the hollow fiber membrane module 1 from the inlet 6 or the outlet 7 via the circuit 11. To do. In the hollow fiber membrane module 1, the valve 13a on the outlet side of the filtrate outlet 5a on the upper side in the vertical direction may be opened, and exhausted from here.

  In the embodiment shown in FIG. 4, the priming liquid is introduced into the storage container 12, the pump 14 a is driven, and the priming liquid is introduced into the hollow fiber membrane module 1 from the inlet 6 or the outlet 7 via the circuit 11. To do. In the hollow fiber membrane module 1, the valve 13a at the outlet of the filtrate outlet 5a on the upper side in the vertical direction may be opened and exhausted from here.

The cells in the cell concentrate obtained as described above have an extremely high purity of 99% or more, and can be directly provided for therapeutic use for various diseases.
Therefore, the present invention relates to a method for treating various diseases in which the cell concentrate obtained by the production method is administered to humans or non-human animals.
As the treatment method, immune cell therapy for cancer using immune cells such as cytotoxic T cells, NK cells, NKT cells, dendritic cells, and pluripotent biological stem cells such as mesenchymal stem cells are used. And cell transplantation treatment methods for regeneration of tissues such as bones, joints, blood vessels, and organs, and graft-versus-host disease (GVHD) treatment methods using mesenchymal stem cells. Cells used for these treatments can be cells derived from iPS cells, ES cells, and cells into which various genes have been introduced. Furthermore, the cell concentrate obtained by the above production method can also be suitably used as a medium exchange application in which a fresh medium is added to the cell concentrate and the culture is performed again.

  The present invention is not limited to the hollow fiber membrane module shown in FIGS. 1 (a), 1 (b) and 2 and the embodiments shown in FIGS. 3, 4, and 5, but contrary to the gist of the present invention. Various changes can be made without departing from the scope.

  Hereinafter, the present invention will be described using experimental results.

  In this example, the value obtained by dividing the total amount of protein or albumin or IgG contained in the cell concentrate by the total amount of protein or albumin or IgG contained in the cell suspension, expressed as a percentage, ie, total protein The removal rate, albumin removal rate, and IgG removal rate can be maintained at 99% or more, and the higher these values, the better the cleaning efficiency. The total protein amount in the cell concentrate can be calculated by measuring the protein concentration (mg / mL) and multiplying the value with the cell suspension amount (mL). The same applies to albumin and IgG.

  The “cell recovery rate” as used herein is the percentage obtained by dividing the number of cells in the cell concentrate collected in the collection container by the number of cells in the cell suspension before processing. 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).

In addition, the “cell survival ratio” here refers to a value obtained by dividing the cell survival rate in the cell concentrate collected in the collection container by the cell survival rate in the cell suspension before treatment. 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 based on via-probe (manufactured by BD Japan, trade name: BD Via-Probe Cell Viability) with respect to 100 μL of each cell suspension prepared so that the cell concentration becomes 10 6 cells per mL. 10 μL of (Solution) is added and allowed to stand at room temperature in the dark for 10 minutes, and then taken into a flow cytometer (trade name: FACSCanto, manufactured by Japan BD) and measured as a positive rate (%) of via-probe.

As the cell suspension used in the examples, 1500 mL of a cell suspension (RPMI 1640 medium containing 10% FBS) containing cultured Jurkat cells was used, and 1000 mL of physiological saline was used as a washing solution.
Examples are shown below.

Example 1
The manufacturing apparatus of the aspect shown in FIG. 3 was used. That is, a vinyl chloride tube was connected to the inlet 6 and outlet 7 of the hollow fiber membrane module 1, and the tube on the outlet 7 side was provided with a bifurcated branching portion 16 capable of switching the flow path. One end of the branch portion 16 was connected to the collection container 17, and the other end was connected to the storage container 12. The tube on the inlet 6 side was also connected to the storage container 12, and the circuit 11 was formed so that the solution could circulate through the hollow fiber membrane module 1 and the tube. A pump 14a is installed in the tube of the circuit 11 so that the flow of the cell suspension can be set to an appropriate flow rate.
A two-way stopcock 13c is installed between the branch portion 16 and the storage container 12, and a two-way stopcock 13d is installed between the branch portion 16 and the recovery container 17, so that cells are stored in the storage container 12 or the recovery container 17. The flow path of the suspension can be switched.
Furthermore, tubes having two-way stopcocks 13a and 13b are attached to the filtrate outlet 5a on the upper side in the vertical direction and the filtrate outlet 5b on the lower side in the vertical direction of the hollow fiber membrane module 1, respectively. 14b was attached and this was installed so that it might pour into the waste liquid container 15. In addition, the two-way cock 13a was opened in advance and the two-way cock 13b was closed so that the filtrate was discharged from the filtrate outlet 5a on the upper side in the vertical direction.

While adjusting the flow rate to 450 mL / min (linear velocity of 580 cm / min) by the pump 14 a, the cell suspension in the storage container 12 was passed through the hollow fiber membrane module 1 from the inlet 6. While circulating the cell suspension, filtration was performed until all the cell suspension was removed from the storage container 12 storing the cell suspension (concentration). The filtrate was introduced into the waste liquid container 15 by driving the pump 14b from the filtrate outlet 5a.
After the concentration step, the two-way stopcock 13a was closed and the two-way stopcock 13b was opened, so that the filtrate could be discharged from the filtrate outlet 5b on the lower side in the vertical direction of the hollow fiber membrane module.
Next, 1000 mL of physiological saline is placed in the storage container 12, and is similarly passed through the hollow fiber membrane module 1 from the inlet 6 at a flow rate of 450 mL / min (linear velocity of 580 cm / min). The cell suspension was washed by filtration while circulating the suspension.
After filtration until the physiological saline solution in the storage container 12 runs out, the flow path is switched to a tube connected to the collection container 17, and the cell concentrate in the hollow fiber membrane module 1 and the circuit 11 is supplied at a rate of 75 mL / min. The product was extruded and collected in a collection container 17.

The hollow fiber membrane module 1 was prepared using 300 polyethersulfone hollow fiber separation membranes (product name: MF020 TYPE-1, hollow fiber inner diameter 570 μm, hole diameter 0.2 μm, manufactured by Toyobo Co., Ltd.).
The total length L was 27 cm, the position x of the filtrate outlet 5b from the end of the hollow fiber membrane module 1 was 4 cm, and the distance y between the filtrate outlets 5a and 5b was 18 cm.
The produced hollow fiber membrane module 1 had a filtration area of 0.12 m ² , and the total cross-sectional area of the hollow fiber type separation membrane was 0.77 cm ² .
The total protein removal rate of the obtained cell concentrate was 99.89%, the albumin removal rate was 99.83%, and the IgG removal rate was 99.96%. The cell recovery rate was 88%, and the survival rate was 97%.

(Example 2)
The experimental system, liquid feeding speed, and hollow fiber membrane module were the same as in Example 1. After completion of the concentration step, a step of switching the filtrate outlet 5a to the filtrate outlet 5b was carried out for washing. At this time, the total length L of the hollow fiber membrane module 1 was 27 cm, the position x of the filtrate outlet 5b was 13 cm, and the distance y between the filtrate outlets 5a and 5b was 9 cm.
As a result, the total protein removal rate in the obtained cell concentrate was 99.88%, the albumin removal rate was 99.57%, and the IgG removal rate was 99.43%. The cell recovery rate was 84%, and the survival rate was 93%.

(Example 3)
The experimental system, liquid feeding speed, and hollow fiber membrane module were the same as in Example 1. After completion of the concentration step, a step of switching the filtrate outlet 5a to the filtrate outlet 5b was carried out for washing. At this time, the total length L of the hollow fiber membrane module 1 was 27 cm, the position x of the filtrate outlet 5b was 4 cm, and L: y = 3: 2. As a result, the total protein removal rate in the obtained cell concentrate was 99.88%, the albumin removal rate was 99.82%, and the IgG removal rate was 99.96%. The cell recovery rate was 86%, and the survival rate was 93%.

Example 4
The experimental system and the hollow fiber membrane module were the same as in Example 3. In the concentration step, the linear velocity until the cell suspension was filled up to the position of the filtrate outlet 5a on the upper side in the vertical direction of the hollow fiber membrane module 1 was set. a, The linear velocity until all the cell suspensions disappear from the storage container 12 storing the cell suspension is b, and the linear velocity at the time of concentration is switched to two stages. At this time, a: b = 1: 26. As a result, the total protein removal rate in the obtained cell concentrate was 99.89%, the albumin removal rate was 99.92%, and the IgG removal rate was 99.97%. The cell recovery rate was 84%, the survival rate was 98%, and the treatment time was 10 minutes.

(Example 5)
The experimental system and the hollow fiber membrane module were the same as in Example 4, with a = 40 cm / min and b = 580 cm / min. As a result, the total protein removal rate in the obtained cell concentrate was 99.92%, the albumin removal rate was 99.91%, and the IgG removal rate was 99.98%. The cell recovery rate was 87%, the survival rate was 93%, and the treatment time was 13 minutes.

(Example 6)
The experimental system and the hollow fiber membrane module were the same as in Example 4, with a: b = 1: 3.5, a = 40 cm / min, and b = 140 cm / min. As a result, the total protein removal rate in the obtained cell concentrate was 99.94%, the albumin removal rate was 99.92%, and the IgG removal rate was 99.96%. The cell recovery rate was 73%, the survival rate was 95%, and the treatment time was 31 minutes.

(Comparative Example 1)
The experimental system, liquid feeding speed, and hollow fiber membrane module were the same as in Example 1. After completion of the concentration step, the filtrate was discharged from the filtrate outlet 5a on the upper side in the vertical direction in the washing step without switching the position of the filtrate outlet. At this time, the total length L of the hollow fiber membrane module 1 was 27 cm, and the position x of the filtrate outlet was 22 cm. L: y = 27: 0. As a result, the total protein removal rate in the obtained cell concentrate was 99.84%, the albumin removal rate was 99.14%, and the IgG removal rate was 98.81%. The cell recovery rate was 87%, and the survival rate was 94%. The processing time was 13 minutes.

  In Examples 1-6, since the filtrate outlet was switched from the upper side to the lower side in the vertical direction after concentration, the total protein removal rate and albumin removal of the obtained cell concentrate were compared with Comparative Example 1 in which switching was not performed. Rate and IgG removal rate are more excellent, especially in Examples 1 to 6, albumin removal rate is 99.57% or more, and IgG removal rate is 99.43% or more, and the content of impurities It can be seen that a highly pure cell concentrate is produced in which is significantly reduced.

  In Example 5, the linear velocity of the first stage in the concentration step was 40 cm / min, and the linear velocity of the second stage was 580 cm / min, so that the linear velocity of the second stage was 140 cm / min as in Example 6. It can be seen that the cell recovery rate can be significantly increased in a short time compared to the minute.

1. Hollow fiber membrane module Torso 3A, 3B. Head 5a, 5b. Filtrate outlet 6. Inlet 7 Outlet 8. 8. bundle of hollow fiber membranes Open end 10. Resin layer part 11. Circuit 12. Storage container 13a, 13b, 13c, 13d. Valves 14a, 14b. Pump 15. Waste liquid container 16. Bifurcation 17. Collection container

Claims (11)

A method for producing a cell concentrate using a hollow fiber membrane module comprising an inlet, an outlet and a filtrate outlet and filled with a hollow fiber separation membrane,
A step of feeding the cell suspension into the hollow fiber membrane module, and concentrating the cell suspension while discharging the filtrate from a filtrate outlet arranged on the upper side in the vertical direction of the hollow fiber membrane module;
The step of switching the position of the filtrate outlet to the lower side in the vertical direction of the hollow fiber membrane module, and the washing liquid is fed into the hollow fiber membrane module, and the cells are washed while discharging the filtrate from the filtrate outlet. A method for producing a cell concentrate, comprising the step of recovering the cell concentrate.   In the hollow fiber membrane module, two filtrate outlets are provided, and the position of the filtrate outlet for the filtrate containing the washing liquid is a distance x from the lower end of the hollow fiber membrane module to one of the filtrate outlets. 2. The method for producing a cell concentrate according to claim 1, wherein the cell concentrate is adjusted to a position where L / 2 ≧ x as compared with the length L in the vertical direction of the yarn membrane module.   The ratio between the vertical length L (cm) of the hollow fiber membrane module and the distance y (cm) between two filtrate outlets is L: y = 3: 1 to 10: 9. The manufacturing method of the cell concentrate of description.   The method for producing a cell concentrate according to any one of claims 1 to 3, wherein the position of the filtrate outlet is switched by changing the direction of the hollow fiber membrane module.   The method for producing a cell concentrate according to any one of claims 1 to 4, wherein in the step of concentrating the cell suspension, the cell suspension is concentrated at two stages of linear velocity.   6. The cell concentration according to claim 5, wherein the linear velocity a (cm / min) in the first step and the linear velocity b (cm / min) in the second step satisfy a relationship of a <b. Liquid manufacturing method.   The ratio of the linear velocity a (cm / min) in the first stage and the linear velocity b (cm / min) in the second stage is a: b = 1: 1.2 to 1:32. A method for producing a cell concentrate.   The method for producing a cell concentrate according to claim 6 or 7, wherein the first stage linear velocity a is 35 to 300 cm / min, and the second stage linear velocity b is 370 to 1100 cm / min.   Before the step of concentrating the cell suspension, a step of feeding a priming solution into the hollow fiber membrane module while exhausting from the filtrate outlet arranged on the upper side in the vertical direction of the hollow fiber membrane module. The manufacturing method of the cell concentrate of any one of Claims 1-8.   The method for producing a cell concentrate according to any one of claims 1 to 9, wherein the hollow fiber separation membrane filled in the hollow fiber membrane module has a pore diameter of 0.1 µm or more. The method for producing a cell concentrate according to any one of claims 1 to 10, wherein an inner diameter of the hollow fiber type separation membrane filled in the hollow fiber membrane module is 300 to 1000 µm.

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