JP2012035004A - Cell concentration device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell concentration device, capable of concentrating cells at high concentration by removing impurities from a cell suspension while downsizing of the device.SOLUTION: The cell concentration device 1 includes: a first hollow yarn module 10 and a second hollow yarn module 20 each including a hollow yarn membrane 11 or a hollow yarn membrane 21 which transmits materials smaller than cells in a cell suspension A, and an outer cylinder receiving the same; a tube 36 for connecting the inside of the hollow yarn membrane 11 of the first hollow yarn module 10 to the inside of the hollow yarn membrane 21 of the second hollow yarn module 20; a pump P1 for supplying the cell suspension A into the hollow yarn membrane 11 of the first module 10; and a pump P2 for pressurizing the cell suspension A in the first module 10 to move the cell suspension A from the first module 10 to the second module 20 through the tube 36. The second module 20 has a membrane capacity smaller than that of the first module 10.

Description

  The present invention relates to a cell concentration apparatus.

Currently, cell therapy using stem cells contained in fat is expected. In response to this demand, a system for separating and collecting angiogenic therapy cells that concentrates cells (monocytes) used for cell therapy from blood using a dialysis apparatus has been known (for example, see Patent Document 1). .
Moreover, a hollow fiber membrane type leukocyte removal filter device that removes leukocytes in blood by permeating through the hollow fiber membrane is known (see, for example, Patent Document 2).

JP 2005-336080 A JP-A-9-122231

  Here, when cells are locally injected in cell therapy, the cell volume is required to be small, but the separation and recovery system disclosed in Patent Document 1 is insufficient in concentration rate, so that further centrifugation is required. Such a concentration step is required, which causes problems such as generation of cell masses and enlargement of the apparatus.

  On the other hand, in the hollow fiber membrane type leukocyte removal filter device disclosed in Patent Document 2, since the concentration rate by the hollow fiber membrane filter is insufficient, a concentration step such as centrifugal separation is eventually required, resulting in the generation of cell clusters. This causes a problem that the apparatus becomes large.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a cell concentrator capable of concentrating cells at a high concentration while reducing the size of the device.

In order to achieve the above object, the present invention employs the following means.
The present invention provides a first hollow fiber module and a second hollow fiber module each having a hollow fiber membrane that allows a substance smaller than cells in a cell suspension to permeate and an outer cylinder that accommodates the hollow fiber membrane, A pipe connecting the inside of the hollow fiber membrane of the first hollow fiber module and the inside of the hollow fiber membrane of the second hollow fiber module; and the cell suspension inside the hollow fiber membrane of the first hollow fiber module. Cell suspension supply means for supplying a suspension and pressurizing the cell suspension in the first hollow fiber module, and the piping from the first hollow fiber module to the second hollow fiber module And a pressurizing means for moving the cell suspension through the cell, and the second hollow fiber module has a membrane volume smaller than that of the first hollow fiber module.

  According to the present invention, the cell suspension is supplied into the hollow fiber membrane of the first hollow fiber module by the cell suspension supply means, so that the cells in the cell suspension While remaining inside, red blood cells and enzymes in the cell suspension are discharged out of the hollow fiber membrane together with water. Thereby, the cells in the cell suspension are washed. And the cell suspension in a 1st hollow fiber module is moved by a pressurization means to a 2nd hollow fiber module via piping. In the second hollow fiber module, cells in the cell suspension remain inside the hollow fiber membrane, while water and the like in the cell suspension are discharged to the outside of the hollow fiber membrane. Here, since the second hollow fiber module has a smaller membrane volume than the first hollow fiber module, the cell suspension remaining inside the hollow fiber membrane of the second hollow fiber module is concentrated. Is done.

  As described above, according to the present invention, the cell suspension is washed to remove red blood cells and enzymes that are not necessary for cell therapy, and the cell suspension can be increased without providing a centrifuge or the like. It can be concentrated to a concentration.

The said invention WHEREIN: It is good also as providing the washing | cleaning liquid supply means which supplies a washing | cleaning liquid inside the hollow fiber membrane of a said 1st hollow fiber module.
In this way, the cell suspension in the hollow fiber membrane of the first hollow fiber module can be washed with a washing solution to remove impurities such as red blood cells and enzymes, and the final concentrated solution obtained. The purity of the cells in the cell suspension can be increased.

The said invention WHEREIN: It is good also as providing the washing | cleaning liquid supply means which supplies a washing | cleaning liquid between the hollow fiber membrane of an said 1st hollow fiber module, and an outer cylinder.
By doing so, the pressure between the hollow fiber membrane of the first hollow fiber module and the outer cylinder is increased, and the cell suspension from the first hollow fiber module to the second hollow fiber module by the pressurizing means is performed. The movement of the suspension can be assisted.

In the above invention, the cleaning liquid supply means may supply the cleaning liquid by pulsing it.
By doing in this way, even when cells or the like have adhered to the holes of the hollow fiber membrane of the first hollow fiber module, the cells or the like can be peeled from the holes by pulsing the cleaning liquid. . Thereby, while improving the washability of a cell suspension, the yield of the cell finally obtained (cell suspension) can be raised.

  According to the present invention, there is an effect that cells can be concentrated at a high concentration while downsizing the device.

1 is an overall configuration diagram of a cell concentration device according to an embodiment of the present invention. It is a figure which shows the supply operation | movement of the cell suspension in the cell concentration apparatus of FIG. It is a figure which shows washing | cleaning operation | movement of the cell suspension in the cell concentration apparatus of FIG. It is a figure which shows the washing | cleaning operation | movement (backwashing) of the cell suspension in the cell concentration apparatus of FIG. It is a figure which shows the concentration operation | movement of the cell suspension in the cell concentration apparatus of FIG. It is a figure which shows the waste liquid operation | movement of the cell suspension in the cell concentration apparatus of FIG. It is a figure which shows the collection | recovery operation | movement of the cell suspension in the cell concentration apparatus of FIG. It is a whole block diagram of the cell concentration apparatus which concerns on the modification of FIG.

A cell concentration device 1 according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the cell concentration apparatus 1 according to the present embodiment includes a cell suspension container 5 that stores a cell suspension A, a cleaning liquid container 6 that stores a cleaning liquid B, and a waste liquid C (FIG. 2). A waste liquid container 7 for storing a reference), a first hollow fiber module 10 and a second hollow fiber module 20 having a plurality of hollow fibers, and tubes 31 to 37 for connecting them.

  The cell suspension container 5 is a container for containing the cell suspension A, and a vent nozzle 15 for taking outside air into and out of the container as the cell suspension A increases or decreases, and a vent nozzle are provided at the top of the container. 15 and a vent filter 16 provided in the vehicle. Here, the cell suspension A is a suspension in which, for example, fat-derived cells constituting the adipose tissue are isolated and suspended by mixing and stirring a biological tissue such as adipose tissue with a digestive enzyme solution. That is.

  The first hollow fiber module 10 has a plurality of hollow fiber membranes 11 that allow a substance smaller than the cells in the cell suspension A to permeate, and an outer cylinder 12 that houses these hollow fiber membranes 11. In addition, in the figure, since the first hollow fiber module 10 is schematically shown, only one hollow fiber membrane 11 is illustrated in the outer cylinder 12, but a plurality of hollow fibers are included in the outer cylinder 12. The thread membrane 11 is accommodated.

  The hollow fiber membrane 11 is a membrane having a straw shape and formed hollow inside, and has many pores smaller than the cells in the cell suspension A on the surface. By having such a configuration, by passing the cell suspension A through the hollow fiber membrane 11, the cells in the cell suspension A remain inside the hollow fiber membrane 11. The red blood cells and enzymes in the cell suspension A are discharged together with water to the outside. Thereby, the separation of the cells in the cell suspension A from unnecessary substances such as red blood cells and enzymes, that is, the cell suspension A can be washed.

The pore diameter of the hollow fiber membrane 11 is preferably 8 to 10 μm because it is desirable to pass red blood cells (φ about 7.8 μm) but not fat-derived stem cells (φ about 10 μm or more). As the hollow fiber material, for example, polyethylene terephthalate, cellulose, cellulose triacetate, polysulfone, polyacrylonitrile, polyvinylidene fluoride, and polypropylene are preferable in consideration of biocompatibility.
The outer cylinder 12 is a cylindrical casing, for example, and accommodates a plurality of hollow fiber membranes 11 therein.

  The cell suspension container 5 and the first hollow fiber module 10 are connected by a tube 31. The tube 31 is connected to the inside of the hollow fiber membrane 11 of the first hollow fiber module 10, and a pump (cell suspension supply means) P1 is provided at an intermediate position. The pump P1 is, for example, a tube pump, and compresses and crushes the tube 31 with a roller (not shown) and moves the compressed position in the discharge direction, thereby extruding and conveying the cell suspension A in the tube 31. It is like that. Thereby, the pump P1 supplies the cell suspension A in the cell suspension container 5 to the inside of the hollow fiber membrane 11 of the first hollow fiber module 10 through the tube 31. .

  The cleaning liquid container 6 and the first hollow fiber module 10 are connected by a tube 33. The tube 33 is connected to the outside of the hollow fiber membrane 11 of the first hollow fiber module 10 (between the hollow fiber membrane 11 and the outer cylinder 12), and a valve V1 is provided in the middle of the tube 33. . By opening the valve V1, the cleaning liquid B can be supplied to the outside of the hollow fiber membrane 11 of the first hollow fiber module 10.

  A tube 32 is connected between the pump P <b> 1 in the tube 31 and the first hollow fiber module 10. The other end of the tube 32 is connected to the cleaning liquid container 6, and a pump (pressurizing means, cleaning liquid supply means) P <b> 2 is provided in the middle of the tube 32. The pump P2 has the same configuration as the pump P1, and pushes and conveys the cleaning liquid B in the tube 32. Accordingly, the pump P2 supplies the cleaning liquid B in the cleaning liquid container 6 to the inside of the hollow fiber membrane 11 of the first hollow fiber module 10 via the tubes 31 and 32.

  The waste liquid container 7 is a container for storing the waste liquid C from the first hollow fiber module 10 and the second hollow fiber module 20, and outside air is put into and out of the container as the waste liquid C increases and decreases in the upper part of the container. And a vent filter 18 provided on the vent nozzle 17.

  The first hollow fiber module 10 and the waste liquid container 7 are connected by a tube 34. The tube 34 is connected to the outside of the hollow fiber membrane 11 of the first hollow fiber module 10 (between the hollow fiber membrane 11 and the outer cylinder 12), and a valve V2 is provided in the middle thereof. . By opening the valve V2, the waste liquid C outside the hollow fiber membrane 11 of the first hollow fiber module 10 can be discharged to the waste liquid container 7.

  The second hollow fiber module 20 and the waste liquid container 7 are connected by a tube 35. The tube 35 is connected to the outside of the hollow fiber membrane 21 of the second hollow fiber module 20 (between the hollow fiber membrane 21 and the outer cylinder 22), and a valve V5 is provided in the middle thereof. . By opening the valve V5, the waste liquid C outside the hollow fiber membrane 21 of the second hollow fiber module 20 can be discharged to the waste liquid container 7.

  Similar to the first hollow fiber module 10, the second hollow fiber module 20 contains a plurality of hollow fiber membranes 22 that allow a substance smaller than the cells in the cell suspension A to pass through, and these hollow fiber membranes 22. And an outer cylinder 22 to be used. In addition, in the figure, since the second hollow fiber module 20 is schematically shown, only one hollow fiber membrane 22 is illustrated in the outer cylinder 22, but a plurality of hollow fibers are included in the outer cylinder 22. The yarn film 22 is accommodated.

  Here, the second hollow fiber module 20 has a smaller membrane volume than the first hollow fiber module 10. That is, the hollow fiber membrane 22 of the second hollow fiber module 20 has a smaller inner volume than the hollow fiber membrane 12 of the first hollow fiber module 10. Thereby, by moving the cell suspension A from the hollow fiber membrane 12 of the first hollow fiber module 10 into the hollow fiber membrane 22 of the second hollow fiber module 20, the cell suspension A Concentration can be performed.

  The first hollow fiber module 10 and the second hollow fiber module 20 are connected by a tube (piping) 36. The tube 36 connects the inside of the hollow fiber membrane 11 of the first hollow fiber module 10 and the inside of the hollow fiber membrane 22 of the second hollow fiber module 20. Is provided. By opening the valve V3, operating the pump P2, and pushing the cleaning liquid B into the hollow fiber membrane 11 of the first hollow fiber module 10, from within the hollow fiber membrane 12 of the first hollow fiber module 10, The cell suspension A can be moved into the hollow fiber membrane 22 of the second hollow fiber module 20.

  A tube 37 is connected to the other end side of the second hollow fiber module 20. One end of the tube 37 is connected to the inside of the hollow fiber membrane 22 of the second hollow fiber module 20, a luer cap 38 is provided at the other end, and a valve V4 is provided at an intermediate position. The cell suspension A inside the hollow fiber membrane 22 of the second hollow fiber module 20 can be recovered by opening the valve V4, removing the luer cap 38, and sucking the tube 37 with a syringe or the like. ing.

The operation of the cell concentrator 1 having the above configuration will be described below with specific numerical values as examples.
As a prerequisite, the goal is to remove red blood cells from 375 ml of cell suspension A and concentrate to 3 ml. The volume of the first hollow fiber module 10 is 50 ml on the outside of the hollow fiber membrane 11, 50 ml on the inside of the hollow fiber membrane 11, and the surface area of the hollow fiber membrane 11 is 1.5 m ² . The volume of the second hollow fiber module 20 is 1.5 ml on the outside of the hollow fiber membrane 21, 1.5 ml on the inside of the hollow fiber membrane 11, and the surface area of the hollow fiber membrane 21 is 0.1 m ² . In addition, as for the 1st hollow fiber module 10 and the 2nd hollow fiber module 20, the hole diameter of a hollow fiber membrane shall be 8-10 micrometers as mentioned above. The capacity of both pumps P1 and P2 is 100 ml / min.

  First, as shown in FIG. 2, the pump P <b> 1 is operated to supply the cell suspension A in the cell suspension container 5 to the inside of the hollow fiber membrane 11 of the first hollow fiber module 10. At this time, the valves V1 and V3 are closed and the pump P2 is stopped. As a result, the cells in the cell suspension A remain inside the hollow fiber membrane 11, while the red blood cells and enzymes in the cell suspension A are discharged out of the hollow fiber membrane 11 together with water. Specifically, for example, by supplying 125 ml of the cell suspension A into the hollow fiber membrane 11 of the first hollow fiber module 10, 50 ml of the cell suspension A remains in the hollow fiber membrane 11. 50 ml of water and impurities (red blood cells, enzymes) are discharged to the outside of the hollow fiber membrane 11. Then, by opening the valve V2 in this state, impurities in the 25 mL cell suspension A discharged to the outside of the hollow fiber membrane 11 are discharged together with water to the waste liquid container 7 as the waste liquid C.

  Next, as shown in FIG. 3, the pump P <b> 2 is operated to supply the cleaning liquid B in the cleaning liquid container 6 into the hollow fiber membrane 11 of the first hollow fiber module 10. Specifically, about 67 ml of the cleaning liquid B is supplied into the hollow fiber membrane 11 of the first hollow fiber module 10 by operating the pump P2 (capacity 100 ml / min) for 40 seconds. At this time, the valve V3 is closed and the pump P1 is stopped.

  Thereby, cells in the cell suspension A remain inside the hollow fiber membrane 11, while red blood cells and enzymes in the cell suspension A are discharged together with the washing liquid B to the outside of the hollow fiber membrane 11. Then, by opening the valve V2, red blood cells and enzymes in the cell suspension A discharged to the outside of the hollow fiber membrane 11 are discharged together with the cleaning liquid B to the waste liquid container 7 as the waste liquid C. Thereby, the cell suspension A inside the hollow fiber membrane 11 is washed.

  Further, as shown in FIG. 4, the cell suspension A inside the hollow fiber membrane 11 of the first hollow fiber module 10 is temporarily placed in the cell suspension container 5 by rotating the pump P1 in the reverse direction. It is good also as returning to. Specifically, about 33 ml of cell suspension A is temporarily returned to the cell suspension container 5 by operating the pump P1 (capacity 100 ml / min) for 20 seconds. In this case, the cleaning liquid B in the cleaning liquid container 6 is supplied to the outside of the hollow fiber membrane 11 of the first hollow fiber module 10 by opening the valve V1, and passes through the hole of the hollow fiber membrane 11. Into the hollow fiber membrane 11.

  Thereafter, as shown in FIG. 2, the pump P <b> 1 is operated again, and the cell suspension A in the cell suspension container 5 is returned to the inside of the hollow fiber membrane 11 of the first hollow fiber module 10. By repeating these operations, the cells adhering to the pores of the hollow fiber membrane 11 can be peeled off, and impurities (erythrocytes, enzymes) in the cell suspension A can be removed. Can be improved.

  Next, as shown in FIG. 5, the pump P <b> 2 is operated to supply the cleaning liquid B in the cleaning liquid container 6 into the hollow fiber membrane 11 of the first hollow fiber module 10. At this time, the valves V1, V2, and V4 are closed and the valve V3 is opened. As a result, the cell suspension A inside the hollow fiber membrane 11 of the first hollow fiber module 10 is pressurized by the pump P2, and the inside of the hollow fiber membrane 21 of the second hollow fiber module 20 via the tube 36. Moved to.

  In the second hollow fiber module 20, as in the first hollow fiber module 10, the cells in the cell suspension A remain inside the hollow fiber membrane 21, while the water in the cell suspension A etc. Is discharged outside the hollow fiber membrane 21. Here, since the second hollow fiber module 20 has a smaller membrane volume than the first hollow fiber module 10, the cell suspension remaining inside the hollow fiber membrane 21 of the second hollow fiber module 20. The suspension A is concentrated. Specifically, out of the 50 ml of cell suspension A sent from the first hollow fiber module 10, 1.5 ml of the cell suspension A is left inside the hollow fiber membrane 21, and 1.5 ml of water Etc. are discharged outside the hollow fiber membrane 21. In this state, by opening the valve V5, 46 mL of water or the like discharged to the outside of the hollow fiber membrane 21 is discharged as the waste liquid C into the waste liquid container 7.

  Next, as shown in FIG. 6, the red blood cells and enzymes in the cell suspension A remaining outside the hollow fiber membrane 11 of the first hollow fiber module 10 are discharged together with the cleaning liquid B by opening the valve V2. C is discharged to the waste liquid container 7.

  Next, as shown in FIG. 7, the luer cap 38 is removed, and the syringe 30 is connected to the open end of the tube 37. Then, by opening the valve V4 and sucking with the syringe 30, 1.5 ml of the cell suspension A concentrated in the hollow fiber membrane 22 of the second hollow fiber module 20 is recovered.

  As described above, according to the cell concentration device 1 according to the present embodiment, the cell suspension A is supplied to the inside of the hollow fiber membrane 11 of the first hollow fiber module 10 by the pump P1, so that the cell While cells in the suspension A remain inside the hollow fiber membrane 11, red blood cells and enzymes in the cell suspension A are discharged out of the hollow fiber membrane 11 together with water. Thereby, the cells in the cell suspension A are washed. And the cell suspension A in the 1st hollow fiber module 10 is moved to the 2nd hollow fiber module 20 via the tube 36 by being pressurized by the pump P2.

  In the second hollow fiber module 20, cells in the cell suspension A remain inside the hollow fiber membrane 21, while water and the like in the cell suspension A are discharged to the outside of the hollow fiber membrane 21. . Here, since the second hollow fiber module 20 has a smaller membrane volume than the first hollow fiber module 10, the cell suspension remaining inside the hollow fiber membrane 21 of the second hollow fiber module 20. The suspension A is concentrated.

  Thus, according to the cell concentration apparatus 1 according to the present embodiment, the cell suspension A is washed to remove erythrocytes and enzymes that are not necessary for cell therapy, and without providing equipment such as a centrifuge. The cell suspension A can be concentrated to a high concentration.

  In addition, the cleaning liquid B is supplied to the inside of the hollow fiber membrane 11 of the first hollow fiber module 10 by the pump P2, so that the cell suspension A in the hollow fiber membrane 11 of the first hollow fiber module 10 is washed with the cleaning liquid. By washing with B, impurities such as red blood cells and enzymes can be removed, and the purity of the cells in the concentrated cell suspension A finally obtained can be increased.

  When the tube 33 is provided with a pump (cleaning liquid supply means) and the cell suspension A is moved from the first hollow fiber module 10 to the second hollow fiber module 20, the pump uses the first hollow fiber. The cleaning liquid B may be supplied between the hollow fiber membrane 11 and the outer cylinder 12 of the module 10.

  By doing so, the pressure between the hollow fiber membrane 11 and the outer cylinder 12 of the first hollow fiber module 10 is increased, and the cell suspension A leaks to the outside of the hollow fiber membrane 11. And the movement of the cell suspension A from the first hollow fiber module 10 to the second hollow fiber module 20 by the pump P2 can be assisted.

The pump may supply the cleaning liquid B by pulsing it.
By doing in this way, even when a cell etc. have adhered to the hole of the hollow fiber membrane 11 of the 1st hollow fiber module 10, the hollow fiber membrane 11 is supplied by pulsing and supplying the washing | cleaning liquid B. Cells and the like can be peeled from the pores. Thereby, while improving the washability of the cell suspension A, the yield of the cell finally obtained (cell suspension A) can be improved.

[Modification]
Further, as a modification of the cell concentration device 1 according to the present embodiment, as shown in FIG. 8, the first hollow fiber module 10 is inclined and arranged, and the tube 31 is placed below the first hollow fiber module 10. While connecting, the tube 34 may be connected to the upper position of the first hollow fiber module 10.

  According to the cell concentration device 2 according to this modification, cells having a large specific gravity in the cell suspension A are located in the hollow fiber membrane 11 below the hollow fiber membrane 11 of the first hollow fiber module 10. Settling. That is, the cells in the cell suspension A and impurities such as the washing liquid B can be separated by the gravity and the hollow fiber membrane 11, and the detergency of the cell suspension A can be improved.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included.

DESCRIPTION OF SYMBOLS 1, 2 Cell concentration apparatus 5 Cell suspension container 6 Washing liquid container 7 Waste liquid container 10 1st hollow fiber module 11 Hollow fiber membrane 12 Outer cylinder 20 Second hollow fiber module 21 Hollow fiber membrane 22 Outer cylinder 31-37 Tube A cell suspension B washing liquid C waste liquid P1 pump (cell suspension supply means)
P2 pump (pressurization means, cleaning liquid supply means)
V1-V5 valve

Claims (4)

A first hollow fiber module and a second hollow fiber module having a hollow fiber membrane that allows a substance smaller than cells in a cell suspension to permeate and an outer cylinder that accommodates the hollow fiber membrane;
A pipe connecting the inside of the hollow fiber membrane of the first hollow fiber module and the inside of the hollow fiber membrane of the second hollow fiber module;
Cell suspension supply means for supplying the cell suspension into the hollow fiber membrane of the first hollow fiber module;
Pressurizing the cell suspension in the first hollow fiber module to move the cell suspension from the first hollow fiber module to the second hollow fiber module via the pipe Means and
The cell concentrating device, wherein the second hollow fiber module has a smaller membrane volume than the first hollow fiber module.   The cell concentrator according to claim 1, further comprising a cleaning liquid supply unit configured to supply a cleaning liquid into the hollow fiber membrane of the first hollow fiber module.   The cell concentrating device according to claim 1, further comprising a cleaning liquid supply unit configured to supply a cleaning liquid between the hollow fiber membrane and the outer cylinder of the first hollow fiber module.   The cell concentration apparatus according to claim 3, wherein the cleaning liquid supply means supplies the cleaning liquid by pulsating.

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