JP2012095618A - Cell separation apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To recover cells from a living tissue in high concentration.SOLUTION: The cell separation apparatus 100 includes: a centrifugal separator that separates the cells C and other components contained in a cell suspension; and laser irradiation devices 18 for applying a laser beam having a wavelength band selectively absorbed in a hemoglobin in an erythrocyte E contained in the cell suspension, to the cell suspension before or during the separation of the cells C and other components by the centrifugal separator.

Description

  The present invention relates to a cell separation device.

  2. Description of the Related Art Conventionally, a cell separation device for separating a fat-derived stem cell group (hereinafter simply referred to as “cell”) from a living tissue is known (see, for example, Patent Document 1). In the cell separation device described in Patent Document 1, a centrifuge container containing a cell suspension containing a living tissue such as adipose tissue is rotated around an axis away from the centrifuge container, so that The contained cells are separated from other components, and the cells are concentrated and recovered.

JP 2009-189281 A

  However, since red blood cells are contained in the cell suspension and cells and red blood cells having substantially the same specific gravity cannot be separated by centrifugation, red blood cells are contained in the final product to be collected. Therefore, there is a problem that the necessary cell concentration in the final product is reduced by the amount of red blood cells.

  This invention is made | formed in view of the situation mentioned above, Comprising: It aims at providing the cell separation apparatus which can collect | recover a cell to high concentration.

In order to solve the above problems, the present invention employs the following means.
The present invention provides a separation unit that separates cells and other components contained in the cell suspension, and the cell suspension before or during separation of the cells and other components by the separation unit, Provided is a cell separation device including an irradiation unit that irradiates a laser beam having a wavelength band that is selectively absorbed by hemoglobin.

  According to the present invention, the cell suspension is irradiated with the laser light emitted from the irradiation unit, and the hemoglobin in the red blood cells contained in the cell suspension is absorbed, thereby heating the hemoglobin and Destroy selectively. By separating the cells in the cell suspension in which the red blood cells are destroyed and the other components by the separation unit, the concentration of the cells in the final product can be improved by the amount of the reduced red blood cell volume. Therefore, the cells can be collected at a high concentration.

  In the above invention, the separating section includes a rotary arm capable of supporting a bottomed cylindrical centrifuge container so as to be swingable about a predetermined swing axis, and about a rotation axis separated from the swing axis. It is good also as providing the rotation drive part which rotates a rotation arm.

  With this configuration, when the rotation arm that supports the centrifuge container is rotated by the operation of the rotation driving unit, the centrifuge container is rotated around the rotation axis, and the bottom part is directed radially outward. It is swung around the swing axis. If the cell suspension is accommodated in the centrifuge container and the rotation drive unit is operated, the centrifugal force acting radially outward acts on the cell suspension, and the cells and other components in the cell suspension are moved. Centrifuge. That is, cells having a large specific gravity are collected at the bottom of the centrifuge container, and other components having a small specific gravity are collected on the upper layer of the cells.

  In this case, the specific gravity of cells and erythrocytes is approximately equal. However, by destroying erythrocytes in advance by laser irradiation from the irradiation unit or by centrifuging them, the cells in the cell suspension can contain other erythrocytes. Can be easily and efficiently separated from these components.

  In the above invention, the measurement unit that measures the number of red blood cells contained in the cell suspension, and the laser light generated in the irradiation unit according to the number of red blood cells measured by the measurement unit. It is good also as providing the control part which controls intensity | strength.

  With this configuration, the cell suspension can be irradiated from the irradiation unit with laser light having an intensity corresponding to the number of red blood cells obtained by measurement for each specimen by the measurement unit by the operation of the control unit. Therefore, erythrocytes are destroyed by the minimum necessary laser irradiation, and damage to the cells around the erythrocytes in the cell suspension and damage to the cells due to temperature rise around the erythrocytes in the cell suspension are suppressed. Can do.

  Further, in the above invention, the container having the cell suspension before the cells and the other components are separated, and the cell in the container having a smaller cross-sectional area than the container. It is good also as providing the piping which distribute | circulates suspension outside, and the said irradiation part being provided in the said piping.

  By comprising in this way, a laser beam is irradiated to the cell suspension currently distribute | circulating piping by an irradiation part, and a red blood cell is destroyed. The cell suspension in circulation is limited in density according to the cross-sectional area of the pipe, so as to irradiate the cell suspension stored in the container with a large cross-sectional area with laser light, It is not necessary to have a laser intensity enough to reach the deep part or a laser intensity for irradiating a large amount of red blood cells at once. Therefore, the intensity of the laser beam can be relatively reduced, and damage to cells around the red blood cells in the cell suspension can be suppressed.

  According to the present invention, there is an effect that cells can be collected at a high concentration.

It is a schematic block diagram of the pre-processing unit which concerns on one Embodiment of this invention. It is a schematic block diagram of the centrifuge unit which concerns on one Embodiment of this invention. It is a schematic block diagram of the pre-processing unit which concerns on the modification of one Embodiment of this invention.

A cell separation device according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIGS. 1 and 2, the cell separation device 100 according to the present embodiment destroys red blood cells E in the cell suspension before separating the cells C and other components contained in the cell suspension. And a centrifuge unit 30 that separates the cells C in the cell suspension in which the red blood cells E have been destroyed by the pretreatment unit 10 and other components.

  As shown in FIG. 1, the pretreatment unit 10 includes a cell collection container (container) 11 that contains a cell suspension before pretreatment (before destruction of red blood cells E), and a cell suspension after pretreatment. The cell separation container 13 which accommodates, and the piping 15 which connects the cell collection container 11 and the cell separation container 13 and distribute | circulates a cell suspension are provided.

  The pipe 15 has a cross-sectional area perpendicular to the flow direction and smaller than a cross-sectional area perpendicular to the depth direction of the cell collection container 11, and is formed of a shape and a material with stable optical characteristics. The pipe 15 includes a measuring instrument (measuring unit) 17 for measuring the number of red blood cells E contained in a cell suspension sent from the cell collection container 11 to the cell separation container 13 and a cell suspension circulating in the pipe. A laser irradiation device (irradiation unit) 18 that irradiates the liquid with laser light and a control unit 19 that controls the intensity of the laser light emitted from the laser irradiation device 18 are provided.

The measuring instrument 17 includes a sensor (not shown) that discriminates the red blood cells E in the cell suspension and optically measures the number thereof.
The laser irradiation device 18 emits laser light having a wavelength band (for example, a range of about 400 nm to about 600 nm) that is selectively absorbed by hemoglobin in red blood cells.
Two measuring devices 17 and two laser irradiation devices 18 are provided, and the measuring device 17 and the laser irradiation device 18 are alternately arranged in this order from the cell collection container 11 side to the cell separation container 13 side.

  The control unit 19 controls the intensity of laser light generated from the laser irradiation device 18 according to the number of red blood cells E measured by the measuring instrument 17. That is, the control unit 19 increases the laser intensity when the number of red blood cells E is large in the cell suspension, and decreases the laser intensity when the number of red blood cells E is small in the cell suspension.

  As shown in FIG. 2, the centrifuge unit 30 includes two bottomed cylindrical centrifuge containers 1 that store cell suspensions, and the centrifuge containers 1 around the rotation axis A that is separated from the centrifuge containers 1. A centrifugal separator (separator) 20 is provided.

  The centrifuge container 1 includes a container body 7 having a bottom 3 that closes one end and an opening 5 that opens to the other end, and a lid 9 that closes the opening 5 of the container body 7. The container main body 7 is formed in a substantially conical shape that gradually tapers from an intermediate position in the depth direction to the bottom 3. Further, the centrifuge container 1 has a common tube (not shown) that extends from the center of the lid 9 along the axis of the container body 7 and supplies a cleaning liquid and sucks the supernatant, and cells as a final product. A suction tube (not shown) for sucking a cell suspension containing C is provided.

  The centrifuge 20 includes a base 22 that houses a motor (rotation drive unit) 21 therein, and a rotary shaft that is supported in the vertical direction by the base 22 and is driven to rotate around the rotation axis A by the motor 21. 23, a beam-like rotary arm 25 fixed to the rotary shaft 23 and extending in a direction orthogonal to the rotary axis A, and provided at both ends of the rotary arm 25 and horizontally supporting the centrifuge container 1 so as to be swingable. Two oscillating shafts (oscillating axis) 27 are provided.

  The rotating arm 25 rotates the centrifuge container 1 around the rotation axis A by rotating the rotating shaft 23. In this case, the centrifuge container 1 is swung around the swing shaft 27 by the action of the centrifugal force, and from the posture in which the bottom portion 3 is directed downward in the rotation axis A direction as shown in FIG. The posture can be changed and rotated so that the bottom 3 is directed.

Next, the operation of the cell separation device 100 according to the present embodiment configured as described above will be described below.
In order to recover the cells C from the cell suspension using the cell separation device 100 according to the present embodiment, first, the pretreatment unit 10 destroys the red blood cells E contained in the cell suspension before the cell separation treatment. Perform pre-processing.

  In the pretreatment, when the cell suspension accommodated in the cell collection container 11 is sent to the cell separation container 13 via the pipe 15, the cell suspension is circulated from the laser irradiation device 18 through the pipe 15. Red blood cells E in the cell suspension are selectively destroyed by irradiation with laser light.

  Specifically, by the operation of the measuring instrument 17, the number of red blood cells E in the cell suspension passing in front of the measuring instrument 17 is measured, and the measured value is sent to the control unit 19. The control unit 19 sets the laser intensity of the laser irradiation device 18 that is emitted when the cell suspension passes in front of the laser irradiation device 18 according to the number of red blood cells E measured by the measuring instrument 17.

  Subsequently, the laser irradiation device 18 operates to irradiate the cell suspension that passes in front of the laser irradiation device 18 with a laser beam having a wavelength band that is selectively absorbed by hemoglobin. When the cell suspension is irradiated with laser light, the laser energy is absorbed by hemoglobin in red blood cells E contained in the cell suspension. Thereby, hemoglobin is heated and red blood cells E are destroyed.

  In this case, the laser irradiation device 18 emits laser light having an intensity corresponding to the number of red blood cells E contained in the cell suspension passing in front of the laser irradiation device 18 by the operation of the control unit 19. Thus, the red blood cells E can be selectively destroyed by the minimum necessary laser irradiation. Thereby, the damage by the laser irradiation to the cell C around the red blood cell E in the cell suspension and the damage to the cell C due to the temperature rise around the red blood cell E in the cell suspension can be suppressed.

  Further, since the density of the cell suspension flowing through the pipe 15 is limited according to the cross-sectional area of the pipe 15, the cell suspension accommodated in the cell collection container 11 having a large cross-sectional area is irradiated with laser light. As in the case of the case, it is not necessary to have a laser intensity enough to reach the deep part or a laser intensity for irradiating a large amount of red blood cells at once. Therefore, the intensity of the laser beam can be relatively reduced, and damage to cells around the red blood cells E in the cell suspension can be suppressed.

  Subsequently, the second measuring instrument 17 measures the number of red blood cells E remaining in the cell suspension, and the operation of the control unit 19 causes laser light having an intensity corresponding to the number of red blood cells E to be emitted from the laser irradiation device. The cell suspension is irradiated from 18.

  In the pretreatment, the cell suspension is reciprocated between the cell collection container 11 and the cell separation container 13 until the number of red blood cells E in the cell suspension becomes a predetermined value or less, and the red blood cells E by the measuring instrument 17 are reciprocated. And the laser irradiation by the laser irradiation device 18 are repeated. Then, when the number of red blood cells E becomes equal to or less than a predetermined value, the cell suspension is completely fed to the cell separation container 13.

Next, the cell suspension in which the red blood cells E in the cell separation container 13 are destroyed is centrifuged by the centrifuge unit 30.
First, the cell suspension accommodated in the cell separation container 13 is injected into the centrifuge container 1 using a peristaltic pump, a syringe pump, or the like. At this stage, as shown in FIG. 2, the centrifuge container 1 is kept stationary with the bottom 3 facing downward in the direction of the rotation axis A.

  Next, the motor 21 is operated to rotate the rotation shaft 23 around the rotation axis A. When the rotary arm 25 rotates together with the rotary shaft 23, the centrifuge container 1 swings around the swing shaft 27 by centrifugal force, changes the posture of the bottom portion 3 radially outward and rotates around the rotation axis A. It is done.

  Thereby, a centrifugal force acts on the cell suspension in the centrifuge container 1, and the cells C and other components in the cell suspension are separated in a stacked state in the depth direction of the centrifuge container 1 due to the difference in specific gravity. . Specifically, cells C having a large specific gravity are collected at the bottom 3 of the centrifuge container 1, and digestive enzymes and the like having a small specific gravity are collected on the upper layer of the cells C. When the centrifugation is completed, the centrifuge container 1 is returned to a stationary state, and the cells C deposited on the bottom 3 are collected.

  As described above, according to the cell separation device 100 according to the present embodiment, the red blood cells E having the same specific gravity as the cells C contained in the cell suspension are selectively destroyed in advance by laser irradiation. In this separation process, the cells C in the cell suspension can be easily and efficiently separated from other components including the red blood cells E. As a result, the cell C as the final product can be recovered at a high concentration by the amount that the volume of the red blood cell E is reduced.

  In the present embodiment, the centrifuge 20 has been described as an example of the separation unit. However, it is sufficient that the cell C and other components contained in the cell suspension can be separated. For example, the cell suspension It is good also as employ | adopting the filtering apparatus which isolate | separates the cell C contained in by filtering.

  In the present embodiment, the laser beam is irradiated to the cell suspension in circulation through the pipe 15. Instead, for example, the laser is applied to the cell suspension accommodated in the centrifuge container 1. It is good also as irradiating light. Although the cell suspension is irradiated with laser before the separation treatment, the cell suspension may be irradiated with laser during centrifugation.

This embodiment can be modified as follows.
For example, in the present embodiment, two measuring instruments 17 and two laser irradiation devices 18 are provided in the pipe 15 respectively. However, as shown in FIG. 3, the measuring instrument 17 and the laser irradiation apparatus 18 are respectively provided in the pipe 15. It is good also as providing one by one.

In this case, the cell suspension is reciprocated between the cell collection container 11 and the cell separation container 13, and the measurement of the number of red blood cells E and the laser are performed until the number of red blood cells E in the cell suspension becomes a predetermined value or less. What is necessary is just to repeat irradiation. By doing in this way, the cell separation apparatus 100 can be reduced in size and cheaply manufactured compared with the case where the piping 15 is provided with two or more measuring instruments 17 and the laser irradiation apparatuses 18.
Three or more measuring instruments 17 and three laser irradiation devices 18 may be provided in the pipe 15. By doing in this way, the time required for pre-processing can be shortened.

1 Centrifugal container 3 Bottom 11 Cell collection container (container)
15 Piping 17 Measuring instrument (measurement part)
18 Laser irradiation device (irradiation part)
19 Control device (control unit)
20 Centrifuge (separator)
21 Motor (rotary drive)
25 Rotating arm 27 Oscillating axis (oscillating axis)
100 Cell separator A Rotation axis

Claims (4)

A separation unit for separating cells and other components contained in the cell suspension;
A cell separation apparatus comprising: an irradiation unit that irradiates a laser beam having a wavelength band that is selectively absorbed by hemoglobin with respect to the cell suspension before or during separation of cells and other components by the separation unit . A rotating arm capable of swinging a bottomed cylindrical centrifuge container around a predetermined swing axis;
The cell separation device according to claim 1, further comprising: a rotation driving unit configured to rotate the rotating arm about a rotation axis separated from the swing axis. A measurement unit for measuring the number of red blood cells contained in the cell suspension;
The cell separation device according to claim 1, further comprising: a control unit that controls intensity of laser light generated in the irradiation unit according to the number of red blood cells measured by the measurement unit. A storage container for storing the cell suspension before the cells and the other components are separated;
Having a smaller cross-sectional area than the container, and a pipe for circulating the cell suspension in the container to the outside,
The cell separation device according to claim 1, wherein the irradiation unit is provided in the pipe.

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