JP2018070519A - Cell formulation production method and apparatus as well as cell formulation produced by production apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide cell formulation production methods and apparatus which efficiently concentrate a liquid comprising cells, and cell formulations produced by the production apparatus.SOLUTION: A cell formulation production apparatus 1 comprises a concentration unit 5 which filters cells by passing a liquid comprising cells through the interior of a hollow fiber membrane and increases the number of the cells comprised in a unit volume, a supply pump 4 which supplies the liquid comprising cells and a washing liquid into the interior of the hollow fiber membrane, and a cell sap bag 2 which stores the liquid comprising cells, being configured so that the liquid circulates the supply pump 4, the concentration unit 5, and the cell sap bag 2 in this order and the liquid level of the cell sap bag 2 is higher or more than the top end portion of the hollow fiber membrane.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for producing a cell preparation, a production apparatus, and a cell preparation.

  Conventionally, methods and devices for separating and proliferating useful cells from cells collected from body fluids and tissues are known. For example, it is like patent document 1. The cell culture device described in Patent Document 1 is a device in which specific cells separated by a cell separation kit are cultured in a cell culture container, and washed and concentrated by a cell recovery kit.

  Here, in the concentration step, it is effective to remove water while passing cells using a hollow fiber membrane. However, Patent Document 1 describes a method using a cell recovery filter in a concentration method, but does not describe a concentration method using a hollow fiber membrane.

  When a hollow fiber membrane is used in the cell concentration step, bubbles may be generated inside the hollow fiber membrane due to a pressure drop or the like. At this time, there is a problem that bubbles become resistance and the flow of the liquid is deteriorated inside the hollow fiber membrane, cells remain in the hollow fiber membrane, and the recovery rate is lowered.

International Publication No. 2012/002497

  An object of the present invention is to provide a method and apparatus for producing a cell preparation that efficiently concentrates a liquid containing cells, and a cell preparation produced by the production apparatus.

  The problem to be solved by the present invention is as described above. Next, means for solving the problem will be described.

  That is, the present invention provides a cell preparation having a concentration step of increasing the number of cells contained in a unit volume by passing a liquid containing cells through the hollow fiber membrane and filtering the cells with the hollow fiber membrane. In this manufacturing method, the inside of the hollow fiber membrane is allowed to pass through without reducing the pressure of the liquid inside the hollow fiber membrane.

  The present invention is characterized in that the liquid passing through the hollow fiber membrane is passed at a pressure equal to or higher than the pressure of the liquid when entering the hollow fiber membrane.

  The present invention is characterized in that the liquid level of the container through which the liquid that has passed through the hollow fiber membrane is discharged is made higher than the upper end of the hollow fiber membrane.

  The present invention is characterized in that the liquid level of the container through which the liquid that has permeated to the outside of the hollow fiber membrane is discharged is higher than the upper end of the hollow fiber membrane.

  The present invention is characterized in that the inside of the container through which the liquid that has passed through the hollow fiber membrane is discharged is pressurized.

  The present invention is characterized in that the inside of a container from which liquid that has permeated to the outside of the hollow fiber membrane is discharged is pressurized.

  The present invention is characterized in that a liquid containing the concentrated cells is diluted and washed with a phosphate buffer.

  The present invention provides a concentration means for increasing the number of cells contained in a unit volume by filtering the cells by passing the liquid containing the cells through the inside of the hollow fiber membrane, the liquid containing the cells and the washing solution A cell preparation comprising: supply means for supplying the hollow fiber membrane; and cell fluid storage means for storing the liquid containing the cells, wherein the supply means, the concentration means, and the cell fluid storage means are circulated in this order. In the manufacturing apparatus, the liquid level of the cell fluid storage means is set to be higher than the upper end portion of the hollow fiber membrane.

  The present invention comprises waste liquid storage means for discharging liquid that has permeated to the outside of the hollow fiber membrane, and the liquid level of the waste liquid storage means is made higher than the upper end of the hollow fiber membrane. To do.

  The present invention provides a concentration means for increasing the number of cells contained in a unit volume by filtering the cells by passing the liquid containing the cells through the inside of the hollow fiber membrane, the liquid containing the cells and the washing solution A cell preparation comprising: supply means for supplying the hollow fiber membrane; and cell fluid storage means for storing the liquid containing the cells, wherein the supply means, the concentration means, and the cell fluid storage means are circulated in this order. In the manufacturing apparatus, the inside of the cell fluid storage means is pressurized.

  The present invention is characterized by comprising waste liquid storage means for discharging liquid that has permeated to the outside of the hollow fiber membrane, and the inside of the waste liquid storage means is pressurized.

  The present invention is a cell preparation manufactured by the above-described cell preparation manufacturing apparatus.

  The present invention has the following effects.

  In the present invention, the generation of bubbles inside the hollow fiber membrane is suppressed by allowing the inside of the hollow fiber membrane to pass through without reducing the pressure of the liquid inside the hollow fiber membrane. Thereby, since the flow rate inside the hollow fiber membrane is not hindered by air bubbles and the cell recovery rate is improved, the liquid containing the cells can be concentrated efficiently.

  In the present invention, the liquid passing through the hollow fiber membrane is allowed to pass at a pressure equal to or higher than the pressure of the liquid when entering the hollow fiber membrane, thereby suppressing the generation of bubbles inside the hollow fiber membrane. Thereby, since the flow rate inside the hollow fiber membrane is not hindered by air bubbles and the cell recovery rate is improved, the liquid containing the cells can be concentrated efficiently.

  In the present invention, the liquid level of the container through which the liquid that has passed through the inside of the hollow fiber membrane is discharged is made higher than the upper end of the hollow fiber membrane so that the liquid does not flow into the container due to gravity. The pressure in the hollow fiber membrane increases and the liquid flows into the container. Therefore, even if the flow rate on the container side is not reduced, the liquid is not drawn and discharged to the container side. Therefore, since the pressure applied to the liquid in the hollow fiber membrane does not decrease, the generation of bubbles due to the pressure decrease in the hollow fiber membrane is suppressed.

  In the present invention, the liquid surface of the container through which the liquid that has permeated to the outside of the hollow fiber membrane is discharged is made higher than the upper end of the hollow fiber membrane, so that the liquid does not flow into the container due to gravity. The pressure inside and outside the thread membrane increases and the liquid flows into the container. Therefore, even if the flow rate on the container side is not reduced, the liquid is not drawn and discharged to the container side. Therefore, since the pressure applied to the liquid in the hollow fiber membrane does not decrease, the generation of bubbles due to the pressure decrease in the hollow fiber membrane is suppressed.

  In the present invention, by pressurizing the inside of the container through which the liquid that has passed through the inside of the hollow fiber membrane is discharged, the liquid does not flow into the container due to gravity, and the pressure in the hollow fiber membrane is increased to the container. Liquid flows. Therefore, even if the flow rate on the container side is not reduced, the liquid is not drawn and discharged to the container side. Therefore, since the pressure applied to the liquid in the hollow fiber membrane does not decrease, the generation of bubbles due to the pressure decrease in the hollow fiber membrane is suppressed.

  In the present invention, by pressurizing the inside of the container through which the liquid that has permeated to the outside of the hollow fiber membrane is discharged, the liquid does not flow into the container due to gravity, and the pressure inside and outside the hollow fiber membrane increases to the container. Liquid flows. Therefore, even if the flow rate on the container side is not reduced, the liquid is not drawn and discharged to the container side. Therefore, since the pressure applied to the liquid in the hollow fiber membrane does not decrease, the generation of bubbles due to the pressure decrease in the hollow fiber membrane is suppressed.

  In the present invention, by diluting and washing the liquid containing the concentrated cells with a phosphate buffer, it is possible to suppress the generation of bubbles when the pressure is reduced as compared with the case where the Ringer solution is used.

Schematic which shows the structure of the cell formulation manufacturing apparatus which concerns on 1st embodiment of this invention. (A) Schematic which shows the structure of the concentration unit in the cell formulation manufacturing apparatus which concerns on 1st embodiment of this invention (b) The schematic diagram which shows the state of filtration in a hollow fiber membrane similarly. The figure which shows the flowchart showing the concentration process in the cell formulation manufacturing apparatus which concerns on 1st embodiment of this invention. The figure explaining process K120 of FIG. The figure explaining process K130 of FIG. The figure explaining process K140 of FIG. Schematic which shows the other example of the aperture means which concerns on 1st embodiment of this invention. Schematic which shows the structure of the cell formulation manufacturing apparatus which concerns on 2nd embodiment of this invention. Schematic which shows the structure of the cell formulation manufacturing apparatus which concerns on 3rd embodiment of this invention.

  First, the cell preparation manufacturing apparatus 1 which is 1st embodiment of the manufacturing apparatus of the cell preparation which concerns on this invention is demonstrated using FIGS. 1-2. Unless otherwise specified, each member constituting the cell preparation manufacturing apparatus 1 is made of a material appropriate for use and strength among non-reactive polymers, biocompatible metals, glass, and the like. Specifically, examples of the non-reactive polymer include acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymer, halogenated polymers such as polyvinyl chloride, polyamide, polyimide, polycarbonate, polyethylene, polypropylene, and polystyrene. Examples of the biocompatible metal include stainless steel, titanium, platinum, alloys thereof, and cobalt chromium alloy.

  Moreover, the cell preparation manufacturing apparatus 1 in this embodiment manufactures a cell preparation by concentrating and washing a liquid containing cells (hereinafter, simply referred to as “cell liquid”).

  As shown in FIG. 1, the cell preparation manufacturing apparatus 1 includes a cell liquid bag 2 that is a cell liquid storage means, a circulation channel 3, a supply pump 4 that is a supply means, a concentration unit 5 that is a concentration means, a washing liquid bag 6, A waste liquid bag 7 and a recovery bag 8 are provided as waste liquid storage means.

  The cell fluid bag 2 stores cell fluid. A cell fluid is sealed in the cell fluid bag 2. Since the cells are initially in the medium, the buffer used for the cell fluid contains carbonic acid. Since carbonic acid is a source of bubbles, a phosphate buffer solution is used instead of Ringer's solution containing carbonic acid as the cleaning solution. That is, the generation of bubbles is suppressed by gradually replacing carbonic acid with phosphoric acid during the concentration step. Further, the cell fluid bag 2 may be connected to a not-shown separation device or the like so as to be able to supply the cell fluid. The circulation channel 3 is connected to the cell fluid bag 2 so as to be detachable. In the cell fluid bag 2, the cell fluid that has passed through the hollow fiber membrane 52 a according to the amount supplied to the concentration unit 5, the amount of restriction of the concentration discharge two-way valve 12, and the amount of restriction of the waste solution two-way valve 14. Is discharged.

  The circulation channel 3 is a channel for cell fluid and washing fluid. The circulation flow path 3 includes an upstream flow path 31 that connects the cell fluid bag 2 and the concentration unit 5 via the cell fluid supply two-way valve 10, the supply pump 4, and the housing two-way valve 11, and throttling means. And a downstream channel 32 connecting the concentration unit 5 and the cell solution bag 2 via the two-way valve 12 for concentration and discharge. Thereby, the cell fluid in the cell fluid bag 2 is supplied to the concentration unit 5 through the circulation channel 3 and is returned to the cell fluid bag 2 again, so that the cell fluid is circulated on the circulation channel 3.

  The supply pump 4 supplies cell fluid and washing fluid to the concentration unit 5. Although the supply pump 4 is comprised, for example from a roller pump or a continuous syringe pump, it is not limited to this. The supply pump 4 is provided on the upstream channel 31 of the circulation channel 3, that is, on the downstream side of the cell fluid bag 2 of the circulation channel 3 and on the upstream side of the concentration unit 5. The supply pump 4 is configured to supply the cell solution in the cell solution bag 2 to the concentration unit 5. The supply pump 4 is configured to be able to supply the cell solution to the concentration unit 5 at an arbitrary flow rate.

  The concentration unit 5 concentrates the cells contained in the cell fluid. As shown in FIG. 2A, the concentration unit 5 includes a housing 51 and a hollow fiber membrane module 52 that is a concentration member. The casing 51 is formed with a hollow fiber membrane supply port 51a and a hollow fiber membrane discharge port 51b.

  The upstream flow path 31 is connected to the hollow fiber membrane supply port 51a. The cell fluid in the cell fluid bag 2 is supplied to the casing 51 from the hollow fiber membrane supply port 51a through the upstream channel 31. As shown in FIG. 1, the downstream flow path 32 is connected to the hollow fiber membrane discharge port 51 b, and the concentration discharge two-way valve 12 is provided in the middle of the downstream flow path 32. The two-way valve 12 for concentration and discharge is a throttle means, and is a means for reducing the flow rate of the liquid that has passed through the inside of the hollow fiber membrane 52a. The hollow fiber membrane outlet 51b is changed to a state in which the liquid and cells inside the hollow fiber membrane 52a of the hollow fiber membrane module 52 are discharged to the downstream channel 32 by switching the two-way valve 12 for concentration and discharge to the open state. It is configured to be able to. Thereby, the housing | casing 51 is comprised so that a cell may be discharged | emitted to the cell fluid bag 2 through the downstream flow path 32 from the hollow fiber membrane discharge port 51b by switching of the two-way valve 12 for concentration discharge.

  Further, as shown in FIG. 1 and FIG. 2A, the housing 51 is formed with a housing discharge port 51 c. A waste liquid flow path 13 is connected to the housing discharge port 51 c, and a waste liquid two-way valve 14 is provided in the middle of the waste liquid flow path 13. The waste liquid two-way valve 14 is a throttle means, and is a means for reducing the flow rate of the liquid permeated to the outside of the hollow fiber membrane 52a. The casing 51 is configured such that the liquid that has permeated to the outside of the hollow fiber membrane 52a is discharged from the casing discharge port 51c through the waste liquid passage 13 (see thin ink arrow).

  In the present embodiment, the housing 51 is formed in a substantially cylindrical shape. The casing 51 is arranged such that its axis extends in the vertical direction. A hollow fiber membrane supply port 51a is formed on the lower surface of the casing 51, and a hollow fiber membrane discharge port 51b is formed on the upper surface. Further, a housing discharge port 51c is formed on the side surface of the housing 51 on the hollow fiber membrane discharge port 51b side.

  The hollow fiber membrane module 52 filters cells from the cell fluid. The hollow fiber membrane module 52 includes a hollow fiber membrane 52a, a partition member 52b, and the like. The hollow fiber membrane 52a is formed of, for example, a polysulfone hollow fiber membrane, and is configured in consideration of necessary membrane exchange performance and permeation resistance.

  As shown in FIG. 2 (b), the hollow fiber membrane 52a is disposed inside the housing 51 in a bundle of an amount necessary to ensure membrane exchange performance. The hollow fiber membrane 52a is formed with pores smaller than the size of the cells so that the cells remain inside the hollow fiber membrane 52a. Both end portions of the hollow fiber membrane 52a are fixed to the partition wall member 52b with both end surfaces opened. In the present embodiment, the hollow fiber membrane 52a is arranged in a cylindrical module composed of the partition wall member 52b, and is configured to be able to supply the cell fluid into the hollow fiber membrane 52a from both ends thereof.

  As shown in FIG. 2A, the hollow fiber membrane module 52 is detachably provided inside the housing 51. In the hollow fiber membrane module 52, the partition members 52 b at both ends are arranged at the end portion on the hollow fiber membrane supply port 51 a side and the end portion on the hollow fiber membrane discharge port 51 b side of the casing 51. The partition member 52b is fixed so as to partition the inside of the housing 51 in a watertight state.

  Thus, in the concentration unit 5, the supply chamber 51 d is configured by the partition member 52 b at the end of the housing 51 on the hollow fiber membrane supply port 51 a side, and the partition member 52 b is formed at the end of the housing 51 on the hollow fiber membrane discharge port 51 b side. Thus, a discharge chamber 51e is formed. That is, the concentration unit 5 includes a supply chamber 51d in which a hollow fiber membrane supply port 51a is formed and a discharge chamber 51e in which a hollow fiber membrane discharge port 51b is formed so as to sandwich the hollow fiber membrane module 52 inside the housing 51. And are configured.

  The supply chamber 51d and the discharge chamber 51e are communicated with each other by a hollow fiber membrane 52a. Therefore, the concentration unit 5 is configured such that the cell liquid in the supply chamber 51d flows into the discharge chamber 51e through the hollow fiber membrane 52a. Further, the concentration unit 5 is formed with a housing outlet 51c at a portion where the hollow fiber membrane module 52 is disposed. In this specification, the pressure loss due to the permeation of the liquid through the hollow fiber membrane 52a is not considered as a pressure drop.

  As shown in FIG. 1, in this embodiment, the cell fluid bag 2 is provided at a position higher than the concentration unit 5. This is because the liquid surface of the cell fluid bag 2 from which the liquid that has passed through the inside of the hollow fiber membrane 52a is discharged is made higher than the upper end of the hollow fiber membrane 52a. By making the liquid level of the cell fluid bag 2 higher than the upper end of the hollow fiber membrane 52a, the liquid does not flow from the hollow fiber membrane outlet 51b to the cell fluid bag 2 due to gravity, and the inside of the housing 51 The liquid flows into the cell fluid bag 2 by increasing the pressure and pushing out from the hollow fiber membrane outlet 51b. That is, the liquid passing through the hollow fiber membrane 52a is allowed to pass at a pressure equal to or higher than the pressure of the liquid when entering the hollow fiber membrane 52a.

  Accordingly, the liquid in the casing 51 is not drawn and discharged from the hollow fiber membrane outlet 51b. Therefore, since the pressure applied to the liquid in the casing 51 does not decrease, the generation of bubbles due to the pressure decrease in the casing 51 is suppressed.

  The cleaning liquid bag 6 stores a phosphate buffer that is a cleaning liquid. The cleaning liquid channel 15 is connected to the cleaning liquid bag 6 so as to be detachable. The washing liquid flow path 15 is connected to the upstream side of the supply pump 4 and between the supply pump 4 and the two-way valve 10 for supplying cell liquid. A cleaning liquid supply two-way valve 16 is provided in the middle of the cleaning liquid flow path 15. By switching the two-way valve 10 for supplying cell fluid to a fully closed state and the two-way valve 16 for supplying cleaning fluid to a fully opened state, the cleaning solution bag 6 is communicated with the upstream flow path 31. As described above, either the cell fluid from the cell fluid bag 2 or the cleaning fluid from the cleaning fluid bag 6 is selectively supplied to the upstream channel 31.

  The waste liquid bag 7 is a container that collects the liquid that has permeated to the outside of the hollow fiber membrane 52 a in the concentration unit 5. A waste liquid flow path 13 is connected to the waste liquid bag 7 so as to be detachable. Liquid that has permeated to the outside of the hollow fiber membrane 52a is discharged into the waste liquid bag 7 in accordance with the supply amount to the concentration unit 5, the throttle amount of the waste liquid two-way valve 14, and the throttle amount of the concentration discharge two-way valve 12. Is done. Note that another container may be connected to the waste liquid bag 7 so that the waste liquid is discharged into the container when the waste liquid bag 7 becomes full.

  In the present embodiment, the waste liquid bag 7 is provided at a position higher than the concentration unit 5. This is because the liquid level of the waste liquid bag 7 from which the liquid that has permeated to the outside of the hollow fiber membrane 52a is discharged is made higher than the upper end of the hollow fiber membrane 52a. By making the liquid level of the waste liquid bag 7 higher than the upper end of the hollow fiber membrane 52a, the liquid does not flow from the housing discharge port 51c to the waste liquid bag 7 due to gravity, and the pressure in the housing 51 increases. Then, the liquid flows into the waste liquid bag 7 by being pushed out from the housing discharge port 51c.

  Therefore, the liquid in the casing 51 is not drawn and discharged from the casing discharge port 51c. Therefore, since the pressure applied to the liquid in the casing 51 does not decrease, the generation of bubbles due to the pressure decrease in the casing 51 is suppressed.

  The collection bag 8 collects the concentrated cell solution. A collection channel 17 is connected to the collection bag 8 so that it can be attached and detached. The collection channel 17 is connected to the cell fluid bag 2. A collecting two-way valve 18 is provided in the middle of the collecting channel 17. By switching the collection two-way valve 18 to the fully open state, the collection bag 8 is communicated with the cell solution bag 2, and the cell solution in the cell solution bag 2 is discharged to the collection bag 8.

  Next, a method for producing a cell preparation by the cell preparation production apparatus 1 according to the present invention will be described with reference to FIGS. 4 to 6, the black valve indicates a fully opened state or a state where the flow rate is reduced, and the white valve indicates a fully closed state. The method for producing a cell preparation according to the present invention relates to a concentration step for concentrating a cell solution.

  As shown in FIG. 3, in step K <b> 110 of the concentration step, priming of the cell preparation manufacturing apparatus 1 is performed, and the circulation channel 3 and the concentration unit 5 are filled with a cleaning liquid. At this time, the inside and the outside of the hollow fiber membrane 52a are filled with the cleaning liquid.

  In step K120, as shown in FIG. 4, the cell preparation manufacturing apparatus 1 fully opens the two-way valve 10 for supplying the cell fluid, the two-way valve 11 for the casing, and the two-way valve 12 for concentration and discharge. The inside of the hollow fiber constituting the hollow fiber membrane 52a of the hollow fiber membrane module 52 is switched from the cell fluid bag 2 by the supply pump 4 by switching the valve 14, the two-way valve 16 for supplying the cleaning liquid, and the two-way valve 18 for recovery to the fully closed state. Supply cell fluid to. The amount of cell fluid enclosed in the cell fluid bag 2 can be 1000 ml, for example, and the flow rate of the supply pump 4 can be 50 ml / min, for example. In step K120, the cell fluid circulates in the circulation channel 3.

  In step K130, as shown in FIG. 5, the cell preparation manufacturing apparatus 1 switches the waste two-way valve 14 to a fully open state, and the concentration discharge two-way valve 12 has a predetermined flow rate (for example, 5 ml / min). Squeeze like so. Thereby, the cells contained in the cell fluid pass through the hollow fiber membrane 52a. On the other hand, impurities such as protein are discharged to the outside of the hollow fiber membrane 52 a and discharged to the waste liquid bag 7 together with the cleaning liquid in the housing 51. At this time, since the cell liquid bag 2 and the waste liquid bag 7 are arranged higher than the casing 51, liquid does not flow from the casing 51 to the cell liquid bag 2 and the waste liquid bag 7 due to gravity, and the inside of the casing 51 When the pressure is increased and pushed out from the casing 51, the liquid flows into the cell fluid bag 2 and the waste fluid bag 7. That is, the pressure of the liquid inside and outside the hollow fiber membrane 52a in the housing 51 is not reduced. In addition, reducing the flow rate with the two-way valve 12 for concentration and discharge also has the effect of increasing the pressure of the cell fluid inside the hollow fiber membrane 52a. In this way, the cell fluid is concentrated in step K130. Then, when the amount of liquid in the cell fluid bag 2 is reduced to a predetermined amount (for example, 100 ml), all the valves are switched to the fully closed state.

  As described above, the generation of bubbles inside the hollow fiber membrane 52a is suppressed by allowing the hollow fiber membrane 52a to pass through without reducing the pressure of the cell fluid inside the hollow fiber membrane 52a. As a result, the flow inside the hollow fiber membrane 52a is not hindered by bubbles, and the cell recovery rate is improved. On the other hand, when the pressure of the cell fluid inside the hollow fiber membrane 52a is reduced, bubbles are likely to be generated inside the hollow fiber membrane 52a. As a result, air bubbles become resistance and the flow of cell fluid becomes poor inside the hollow fiber membrane 52a, and the cells remaining inside the hollow fiber membrane 52a cannot be recovered, resulting in a low recovery rate.

  In step K140, the cell preparation manufacturing apparatus 1 switches the cleaning liquid supply two-way valve 16, the housing two-way valve 11, and the concentration discharge two-way valve 12 to the fully open state as shown in FIG. Thus, the cleaning liquid is supplied from the cleaning liquid bag 6 to the inside of the hollow fiber constituting the hollow fiber membrane 52a of the hollow fiber membrane module 52. Thereby, the cell liquid inside the hollow fiber membrane 52a is replaced with the washing liquid, and the cell liquid is collected in the cell liquid bag 2. In step K140, the cell solution is diluted and washed with a washing solution. And when the liquid amount of the cell fluid bag 2 increases to a predetermined amount (for example, 1000 ml), all the valves are switched to the fully closed state. In addition, when there are air bubbles inside the hollow fiber membrane 52a, it is difficult to collect the cells remaining inside the hollow fiber membrane 52a even if the cleaning liquid is supplied.

  Step K120 to step K140 are repeated a predetermined number of times, and in the last step K140, the amount of cells contained in the unit volume is increased by reducing the amount of liquid in the cell solution bag 2 from the original amount of liquid, thereby concentrating the cell solution. Complete. For example, the predetermined number of times can be 5 times, and in the fifth step K140, the amount of the cell fluid bag 2 can be 250 ml.

  In step K150, the cell preparation manufacturing apparatus 1 switches the collection two-way valve 18 to a fully open state, and discharges (collects) the cell solution concentrated from the cell solution bag 2 to the collection bag 8. The recovery rate of the cells recovered by such a concentration step is, for example, about 70%. That is, a cell preparation can be obtained with high efficiency. On the other hand, when the pressure is reduced inside the hollow fiber membrane 52a, bubbles are generated inside the hollow fiber membrane 52a, and the cell recovery rate is lowered.

  Next, referring to FIG. 7, a throttling means replacing the two-way valve 12 for concentration and discharge will be described.

  As shown in FIG. 7, the cell preparation manufacturing apparatus 1 may use a pipe 21 having a channel that is narrower than the channel on the upstream side of the concentration unit 5, instead of the two-way valve 12 for concentration and discharge. That is, the pipe 21 constituting the downstream flow path 32 connected to the hollow fiber membrane discharge port 51b is made thinner than the pipe 22 constituting the upstream flow path 31 connected to the hollow fiber membrane supply port 51a. In this case, in step K130, the flow rate is throttled by the pipe 21, whereby the pressure of the cell fluid increases inside the hollow fiber membrane 52a. Therefore, the same effect as the case where the concentration exhaust two-way valve 12 is used can be obtained.

  Next, the cell preparation manufacturing apparatus 30 which is 2nd embodiment of the manufacturing apparatus of the cell preparation which concerns on this invention is demonstrated using FIG. In the following embodiments, the same points as those of the above-described embodiments will not be specifically described, and different portions will be mainly described.

  As shown in FIG. 8, the cell preparation manufacturing apparatus 30 provides the cell solution bag 2 and the waste solution bag 7 at a position lower than the upper end portion of the concentration unit 5, and sets the waste solution channel 13 to the hollow fiber membrane supply port 51 a of the casing 51. It is connected to a case outlet 51f formed on the side surface. The cell fluid bag 2 passed through the hollow fiber membrane 52a according to the supply amount to the concentration unit 5, the throttle amount of the concentration discharge two-way valve 12, the throttle amount of the waste liquid two-way valve 14, and the gravity. The cell liquid is discharged, and the liquid that has permeated to the outside of the hollow fiber membrane 52a is discharged into the waste liquid bag 7. Therefore, if the flow rate is not reduced by the two-way valve 12 for concentration and discharge and the two-way valve 14 for waste liquid, the liquid in the casing 51 is drawn and discharged from the hollow fiber membrane outlet 51b and the casing outlet 51f by gravity. The pressure applied to the liquid in the housing 51 is reduced. Therefore, in the present embodiment, the process is executed as follows in the process K130 shown in FIG.

  In step K130, the cell preparation production apparatus 30 throttles the concentration / discharge two-way valve 12 to a predetermined flow rate (for example, 5 ml / min) and the waste liquid two-way valve 14 to a predetermined flow rate (for example, 45 ml / min). min). By restricting the flow rate with the two-way valve 12 for concentration discharge and the two-way valve 14 for waste liquid, the pressure of the cell liquid does not decrease inside and outside the hollow fiber membrane 52a. Then, when the amount of liquid in the cell fluid bag 2 is reduced to a predetermined amount (for example, 100 ml), all the valves are switched to the fully closed state.

  Thus, even when the cell fluid bag 2 and the waste solution bag 7 are provided at a position lower than the concentration unit 5, the flow rates of the concentration discharge two-way valve 12 and the waste solution two-way valve 14 on the downstream side of the hollow fiber membrane 52a. The pressure of the cell fluid does not decrease inside the hollow fiber membrane 52a. As a result, the generation of bubbles is suppressed inside the hollow fiber membrane 52a, the flow inside the hollow fiber membrane 52a is not hindered by the bubbles, and the cell recovery rate is improved.

  In addition, the form which provided one of the cell liquid bag 2 or the waste liquid bag 7 in the position higher than the concentration unit 5, and provided the other in the position lower than the concentration unit 5 by combining 1st embodiment and 2nd embodiment suitably. It can also be.

  Next, the cell preparation manufacturing apparatus 40 which is 3rd embodiment of the manufacturing apparatus of the cell preparation which concerns on this invention is demonstrated using FIG.

  As shown in FIG. 9, the cell preparation manufacturing apparatus 40 is provided with the cell solution bag 2 and the waste solution bag 7 at a position lower than the upper end of the concentration unit 5, and the waste solution channel 13 is provided in the hollow fiber membrane supply port 51 a of the casing 51. It is the same as that of 2nd embodiment by the point connected to the case discharge port 51f formed in the side surface of the side. On the other hand, the cell preparation production apparatus 40 is different from the second embodiment in that the downstream flow path 32 is connected to the lower part of the cell liquid bag 2 and the waste liquid flow path 13 is connected to the lower part of the waste liquid bag 7. The cell preparation manufacturing apparatus 40 is different from the second embodiment in that pressurizing means 41 and 42 are provided on the upper portions of the cell fluid bag 2 and the waste fluid bag 7, respectively.

  The pressurizing unit 41 maintains the inside of the cell fluid bag 2 in a state of being pressurized to a predetermined pressure, and includes a pressurizing channel 411 and a pressure adjusting channel 412 connected to the upper part of the cell fluid bag 2. On the pressure channel 411, a sterile filter 413 and an air pump 414 are provided in this order from the cell fluid bag 2 side. A pressure regulating valve 415 and a sterile filter 416 are provided on the pressure regulating channel 412 in order from the cell fluid bag 2 side.

  The pressurizing unit 41 pressurizes the inside of the cell fluid bag 2 by supplying the outside air supplied from the air pump 414 through the pressurizing channel 411 to the cell fluid bag 2 through the aseptic filter 413. Further, the pressurizing means 41 opens the cell when the pressure inside the cell fluid bag 2 exceeds a predetermined pressure, and releases the air in the cell fluid bag 2 to the outside through the pressure regulation channel 412. The pressure inside the liquid bag 2 is maintained at a predetermined pressure.

  The pressurizing means 42 maintains the inside of the waste liquid bag 7 at a predetermined pressure. The pressurizing channel 421, the pressure adjusting channel 422, the aseptic filter 423, the air pump 424, the pressure adjusting valve 425, the aseptic A filter 426 is provided. Since these configurations are the same as the pressurizing means 41 described above, detailed description thereof is omitted.

  In the present embodiment, since it is necessary to prevent the pressure applied to the liquid in the casing 51 from decreasing as in the second embodiment, the cell fluid is applied by the pressurizing means 41 and 42 in the process K130 shown in FIG. The inside of the bag 2 and the waste liquid bag 7 is pressurized to a predetermined pressure.

  As described above, even when the cell solution bag 2 and the waste solution bag 7 are provided at a position lower than the concentration unit 5, the cell solution inside the hollow fiber membrane 52 a is pressurized by pressurizing the inside of the cell solution bag 2 and the waste solution bag 7. The pressure does not drop. As a result, the generation of bubbles is suppressed inside the hollow fiber membrane 52a, the flow inside the hollow fiber membrane 52a is not hindered by the bubbles, and the cell recovery rate is improved.

  In addition, the form which provided one side of the cell liquid bag 2 or the waste liquid bag 7 in the position higher than the concentration unit 5, and provided the other in the position lower than the concentration unit 5 by combining 1st embodiment and 3rd embodiment suitably. It can also be.

1 Cell preparation manufacturing equipment 2 Cell fluid bag (cell fluid storage means)
4 Supply pump (supply means)
5 Concentration unit (concentration means)
7 Waste liquid bag (waste liquid storage means)
52a Hollow fiber membrane

Claims (12)

A method for producing a cell preparation comprising a concentration step of passing a liquid containing cells through a hollow fiber membrane and increasing the number of cells contained in a unit volume by filtering the cells through the hollow fiber membrane. ,
A method for producing a cell preparation, wherein the inside of the hollow fiber membrane is allowed to pass through without reducing the pressure of the liquid inside the hollow fiber membrane.   The method for producing a cell preparation according to claim 1, wherein the liquid passing through the hollow fiber membrane is passed at a pressure equal to or higher than the pressure of the liquid when entering the hollow fiber membrane.   3. The cell according to claim 1, wherein the liquid surface of the container through which the liquid that has passed through the hollow fiber membrane is discharged has a height equal to or higher than the upper end of the hollow fiber membrane. Preparation method of the preparation.   4. The liquid level of a container from which liquid that has permeated to the outside of the hollow fiber membrane is discharged is set to a height equal to or higher than the upper end portion of the hollow fiber membrane. 5. A method for producing the cell preparation described in 1.   The method for producing a cell preparation according to claim 1 or 2, wherein the inside of the container through which the liquid that has passed through the hollow fiber membrane is discharged is pressurized.   The method for producing a cell preparation according to claim 1 or 2, wherein the inside of the container from which the liquid that has permeated to the outside of the hollow fiber membrane is discharged is pressurized.   The method for producing a cell preparation according to any one of claims 1 to 6, wherein the liquid containing the concentrated cells is diluted and washed with a phosphate buffer. A concentration means for increasing the number of cells contained in a unit volume by filtering the cells by passing the liquid containing the cells through the inside of the hollow fiber membrane;
Supply means for supplying the liquid containing the cells and the washing liquid into the hollow fiber membrane;
Cell fluid storage means for storing a liquid containing the cells,
An apparatus for producing a cell preparation that circulates in the order of the supply means, the concentration means, and the cell fluid storage means,
An apparatus for producing a cell preparation, characterized in that the liquid surface of the cell fluid storage means is made higher than the upper end of the hollow fiber membrane. Comprising waste liquid storage means for discharging the liquid that has permeated to the outside of the hollow fiber membrane,
The apparatus for producing a cell preparation according to claim 8, wherein the liquid level of the waste liquid storage means is set to be higher than the upper end of the hollow fiber membrane. A concentration means for increasing the number of cells contained in a unit volume by filtering the cells by passing the liquid containing the cells through the inside of the hollow fiber membrane;
Supply means for supplying the liquid containing the cells and the washing liquid into the hollow fiber membrane;
Cell fluid storage means for storing a liquid containing the cells,
An apparatus for producing a cell preparation that circulates in the order of the supply means, the concentration means, and the cell fluid storage means,
An apparatus for producing a cell preparation, wherein the inside of the cell fluid storage means is pressurized. Comprising waste liquid storage means for discharging the liquid that has permeated to the outside of the hollow fiber membrane,
The apparatus for producing a cell preparation according to claim 10, wherein the inside of the waste liquid storage means is pressurized.   The cell preparation manufactured with the manufacturing apparatus of the cell preparation as described in any one of Claims 8-11.

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