JP2012010663A - Cell segregation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve therapeutic effect of a final product, while guaranteeing sterility.SOLUTION: There is provided a cell separation apparatus 1 comprising: a decomposition treatment unit 2 for producing a cell suspension by digesting living body tissue to release living body-derived cells from the living body tissue; a cell concentrating unit 3, comprising an arm 13a supporting a centrifugal separation container 10 receiving the cell suspension, to be swingable around the swinging axis, and a rotating part 13b rotating the arm 13a around a predetermined axis separate from the swinging axis, and for concentrating the cell suspension by centrifugation; and a yield-increasing unit for increasing in the cell concentrating unit 3 the yield of the living body-derived cells contained in the cell suspension, wherein the yield-increasing unit is set at the position adjacent to the centrifugal separation container 10 in the state that the arm 13a is stopped.

Description

  The present invention relates to a cell separation device.

  Conventionally, an apparatus for separating fat-derived cells including stem cells from adipose tissue collected from a living body is known (see, for example, Patent Document 1 and Patent Document 2).

International Publication No. 2005/012480 US Patent Publication No. 2009/0304644

  However, when the fat-derived cells are separated using the apparatus of Patent Document 1, the function of the fat-derived cells is reduced in the process of treatment. Therefore, there is a problem that it is difficult to quickly obtain a sufficient therapeutic effect even if the cell concentrate, which is the final product, is collected from the apparatus and used as it is for treatment. It is also conceivable that after the separation, the fat-derived cells are taken out of the apparatus and treated separately, such as activation. However, there is a possibility that foreign substances are mixed into the fat-derived cells that have been guaranteed sterilization in the apparatus. Furthermore, the fat-derived cells may rather be weakened by the increased number of centrifugation steps required to wash and concentrate the fat-derived cells prior to use in the treatment process, particularly therapy.

  In Patent Document 2, cell activation or yield increase is attempted by stimulating cells during the treatment process. However, depending on the timing of stimulation, there is a problem that the cell activity may decrease again during the treatment.

  This invention is made | formed in view of the situation mentioned above, Comprising: It aims at providing the cell separation apparatus which can maximize the therapeutic effect by a final product, ensuring sterility.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a decomposition processing unit that separates a living body-derived cell from a living tissue by digesting the living tissue to generate a cell suspension, and a centrifuge container that stores the cell suspension around an oscillation axis. An oscillatable arm, and a rotating unit that rotates the arm around a predetermined axis away from the oscillating axis, and a cell concentrating unit that concentrates the cell suspension by centrifugation, A yield increasing section for increasing the yield of the biologically-derived cells in the cell suspension in the cell concentrating section, and the yield increasing section is located at a position adjacent to the centrifuge container with the arm stationary. Provided is a cell separation device.

According to the present invention, the cell suspension containing the living body-derived cells generated in the decomposition processing unit is concentrated in the cell concentration unit, whereby the living cells can be extracted from the living tissue.
In this case, since the concentrated solution in which the yield of biological cells is increased by the yield increasing unit is collected as the final product, the therapeutic effect of the final product can be maximized.

In the above invention, based on the information on the number of living body-derived cells measured by the cell number measuring means and the number of living body-derived cells included in the cell suspension, the yield is measured. You may provide the control part which controls an increase part.
By doing in this way, a cell suspension can be processed by the yield increase part on the more suitable conditions according to the number of living body origin cells, and the yield of living body origin cells can be increased more effectively.

  According to the present invention, it is possible to maximize the therapeutic effect of the final product while ensuring sterility.

It is a whole block diagram of the cell separation device concerning one embodiment of the present invention. It is a figure which shows the structure of the centrifuge of the cell separation apparatus of FIG. It is a figure which shows the structure of the centrifuge container used for the centrifuge of FIG. It is a figure which shows the receiving part provided in the centrifuge of FIG. It is a figure explaining the structure of a cell number measurement part, and the method of measuring the number of fat origin cells by this cell number measurement part. It is a figure explaining the method of measuring the three-dimensional shape of a cell cluster by a cell number measurement part. It is a figure which shows the structure of a cell stimulation part, and has shown arrangement | positioning of a heater and a temperature sensor. It is a figure which shows the modification of arrangement | positioning of the heater and temperature sensor which were shown in FIG. It is a figure which shows another modification of arrangement | positioning of the heater and temperature sensor which were shown in FIG. It is a figure which shows the modification of a cell stimulation part. It is a figure which shows the modification of a receiving part. It is a figure which shows another modification of a receiving part. It is a figure which shows the modification of arrangement | positioning of a cell stimulation part. It is a figure which shows the modification of a cell number measurement part. It is a figure which shows another modification of the structure of a cell number measurement part. It is a figure which shows another modification of the structure of a cell number measurement part. It is a figure which shows the modification of arrangement | positioning of a cell number measurement part. It is a figure which shows another modification of arrangement | positioning of a cell number measurement part.

Below, cell separation device 1 concerning one embodiment of the present invention is explained with reference to drawings.
As shown in FIG. 1, the cell separation device 1 according to the present embodiment concentrates a decomposition processing unit 2 that digests adipose tissue (biological tissue) and a cell suspension generated in the decomposition processing unit 2. A cell concentration unit 3, a decomposition processing unit 2, a cell concentration unit 3, a transport path 6 that connects the washing liquid bag 4 and the waste liquid bag 5, and a control unit 7 that controls the operations of the units 2, 3 and the transport path 6. ing.

  The degradation processing unit 2 agitates the adipose tissue collected by aspiration from within the living body and the digestive enzyme solution that degrades the adipose tissue in the collection container 8 using an agitator (not shown). As a result, adipose-derived cells (living cells) bound to the adipose tissue are separated from the adipose tissue, and a cell suspension in which the adipose-derived cells are suspended in the digestive enzyme solution is generated. ing. The bottom surface 8a of the collection container 8 is inclined in one direction, and the cell suspension is discharged from the lowest position of the bottom surface 8a. Reference numeral 8b is a filter that prevents relatively large impurities such as digested adipose tissue residues from being discharged together with the cell suspension.

The cell concentration unit 3 includes a centrifuge 9 and, as will be described later, a cell number measurement unit (cell number measurement means) that measures the number of fat-derived cells in the centrifuge container 10 supported by the centrifuge 9. 11 and a cell stimulating part (yield increasing part) 12 for stimulating fat-derived cells.
As shown in FIG. 2, the centrifuge 9 includes a rotating shaft member 13 having an arm 13a, a bucket 14 supported by the arm 13a and provided with a centrifuge container 10, and a rotating shaft member 13 having a substantially central axis. The rotating shaft member 13 is integrally provided with a receiving portion 15 that receives the bottom of the bucket 14.

  The rotating shaft member 13 is provided substantially upright in the vertical direction. The bucket 14 is slightly larger than the centrifuge container 10 and has substantially the same shape as that of the centrifuge container 10, and the bottom part of the bucket 14 is fixed to the both ends of the arm 13 a fixed substantially orthogonal to the rotary shaft member 13. Is supported so as to be swingable in a direction away from the head. Thereby, when the rotating shaft member 13 is rotated in a state where the centrifuge container 10 containing the cell suspension is stored in the bucket 14, the bucket 14 and the centrifuge container 10 are swung so that the bottom portion is out of the radial direction. The fat-derived cells in the cell suspension are aggregated at the bottom of the pocket portion 10b described later.

  As shown in FIG. 3, the centrifuge container 10 has a pipe 10a connected to the conveyance path 6 inserted therein, and a cell suspension is supplied into the centrifuge container 10 through the pipe 10a. It has become. In addition, the pipe 10a is connected to the conveyance path 6 via a rotary joint (not shown) so that the pipe 10a is not twisted during centrifugation.

  In addition, a substantially hemispherical pocket portion 10b having a recessed inner surface is provided at the bottom of the centrifuge container 10. The suction / discharge port 10c opened at the distal end surface of the pipe 10a is disposed at a position away from the bottom surface in the pocket portion 10b. Thus, when the supernatant is discharged from the suction / discharge port 10c after centrifugation of the cell suspension, the supernatant is discharged while leaving the cell mass precipitated on the bottom surface of the pocket portion 10b, and the supernatant is discharged after discharging the supernatant. When the cleaning liquid is supplied from the outlet 10c, the cell mass is efficiently stirred in the cleaning liquid by the momentum of the inflow of the cleaning liquid. Further, by repeating suction and discharge from the suction / discharge port 10c, the cell suspension can be pipetted and stirred.

  The receiving portion 15 has a receiving surface 15 a formed at a position adjacent to the bottom portion of the bucket 14 in a state where the rotation shaft member 13 stops rotating. As shown in FIG. 4, the receiving surface 15 a has a substantially concave hemispherical shape whose outer side in the radial direction is open with respect to the rotating shaft member 13, and has a shape complementary to the bottom of the bucket 14. As a result, the bucket 14 can be smoothly separated from the receiving surface 15a without being hindered from swinging at the start and end of the centrifugation, and can be brought close to the receiving surface 15a.

  At this time, a magnet 16 that generates an attractive force on the bottom of the bucket 14 and the receiving surface 15a is provided so that the bottom of the bucket whose position has been lowered at the end of the centrifugal separation is surely adhered to the receiving surface 15a. The magnet 16 has a magnetic force sufficiently smaller than the centrifugal force that acts on the bottom of the bucket 14 during centrifugation. The magnet 16 is an electromagnet, and may be activated at the end of centrifugation to generate a magnetic force.

Both the cell number measuring unit 11 and the cell stimulating unit 12 are provided on the receiving surface 15a.
As shown in FIG. 5, the cell number counting unit 11 emits the detection light L in a substantially horizontal direction toward the pocket portion 10b received by the receiving surface 15a, and the detection light L is reflected or scattered backward. Detects return light. Here, when the cell number counting unit 11 detects the return light in a state where the cell mass A is precipitated at the bottom of the pocket portion 10b, the intensity of the return light changes at the interface between the cell mass A and the supernatant B. The cell number measuring unit 11 detects the upper end position of the cell mass A from the change in the intensity of the return light, calculates the volume of the cell mass A from the detected upper end position, and calculates the number of fat-derived cells from the calculated volume. It is supposed to be.

  At this time, as shown in FIG. 6, it is preferable that the detection light L is emitted from at least three positions so that the upper end position of the cell mass A is detected from at least three directions. By doing in this way, the shape of the upper end surface of the cell mass A is detected in three dimensions, the volume of the cell mass A is obtained with higher accuracy, and the number of fat-derived cells contained in the cell mass A is more accurately calculated. can do.

  It should be noted that the detection light L and the bottom of the bucket 14 are arranged so that the detection light L and the return light are not blocked at an intermediate position between the cell number measurement unit 11 and the centrifuge container 10 installed in the bucket 14. A window (not shown) that transmits the light is provided at a position where the return light passes. In addition, when the centrifuge container 10 is installed in the bucket 14, the bottom of the bucket 14 may be cut and opened so that the bottom of the centrifuge container 10 protrudes from the bucket.

  As shown in FIG. 7, the cell stimulating unit 12 is a heater (hereinafter also referred to as heater 12) provided on the receiving surface 15a. A temperature sensor 17 is also provided on the receiving surface 15a. Thereby, the inside of the pocket part 10b received by the receiving surface 15a is heated efficiently, and the temperature in the pocket part 10b is detected more accurately. Information on the temperature detected by the temperature sensor 17 is transmitted to the control unit 7, and the control unit 7 controls the temperature of the heater 12 to be kept constant at a predetermined temperature.

  In addition, arrangement | positioning of the heater 12 and the temperature sensor 17 which were shown in FIG. 7 is an example, and can be changed suitably. For example, as shown in FIG. 8, the heater 12 may be provided on the bottom surface, and the temperature sensor 17 may be provided on the side surface, or both the heater 12 and the temperature sensor 17 may be provided on the side surface as shown in FIG. Good.

  The conveyance path 6 includes a tube 18 that connects the collection container 8, the centrifuge container 10, the cleaning liquid bag 4 and the waste liquid bag 5, valves V <b> 1 to V <b> 10 provided in the middle of the tube 18, and a liquid feed pump 19. ing. Reference numeral 6 a indicates a joint that branches the tube 18. The cleaning solution bag 4 contains, for example, a lactated Ringer's buffer as a cleaning solution.

  The control unit 7 is connected to the decomposition processing unit 2, the cell concentration unit 3, the valves V1 to V10, and the liquid feeding pump 19 through a wiring (not shown), and controls these operations according to a preset processing procedure. It has become.

  Further, the control unit 7 causes the cell number measurement unit 11 to count the number of fat-derived cells after the last centrifugation and the discharge of the supernatant are completed in the cell concentration unit 3. And the control part 7 determines the processing time of the heating process performed next based on the number of fat origin cells measured by the cell number measurement part 11, and is detected by the temperature sensor 17 during the determined processing time. The temperature of the heater 12 is adjusted so that the set temperature is maintained at the set temperature. Here, the set temperature is set to a temperature at which moderate stimulation can be given to the fat-derived cells, specifically, 38 to 42 ° C, preferably 40 ° C.

  In addition, the control unit 7 slowly repeats suction and discharge through the pipe 10a during the heating process, so that the cell concentrate is a mixture of the fat-derived cells and the supernatant in the centrifuge container 10. Pipetting.

The operation of the cell separation device 1 configured as described above will be described below.
The cell separation device 1 according to the present embodiment digests adipose tissue in the decomposition processing unit 2 and then transports the generated cell suspension to the cell concentration unit 3 through the transport path 6. Next, the cell separator 1 centrifuges the cell suspension in the cell concentration unit 3, and then discharges the supernatant from the centrifuge container 10 to the waste liquid bag 5. At this time, the cell separation device 1 discharges other supernatant while leaving a small amount of a certain amount of supernatant in the pocket portion 10b. Next, the cell separation device 1 conveys the washing liquid from the washing liquid bag 4 to the centrifuge container 10, centrifuges, and discharges the fat-derived cells by discharging the centrifuge container 10 leaving a certain amount of supernatant from the centrifuge container 10. To do. Washing may be performed a plurality of times as necessary.

After washing the adipose-derived cells, the cell separation device 1 measures the number of adipose-derived cells precipitated in the pocket portion 10b and determines the heating treatment time. Then, the cell separation device 1 raises the temperature of the heater 12 and keeps the inside of the pocket portion 10b at a set temperature, preferably 40 ° C., while keeping the inside of the pocket portion 10b and a small amount of fat-derived cells in the pocket portion 10b. Wait while pipetting the supernatant.
By the above procedure, a cell concentrate in which fat-derived cells are suspended in a small amount of washing liquid as a final product is obtained in the pocket portion 10b.

  Thus, according to the present embodiment, the fat-derived cells are activated by being stimulated by the heating process immediately before the completion of all the processes. This has the advantage that the yield of adipose-derived cells with high activity that have just been activated can be increased and the therapeutic effect of the final product can be improved. In addition, by pipetting during the heating treatment, there is an advantage that the fat-derived cells can be more efficiently activated by uniformly transferring heat to the fat-derived cells in the cell concentrate.

  In addition, in order to activate the fat-derived cells in this way, the inside of the centrifuge container 10 only needs to be heated by the heater 12 from the outside, so that the sterility of the final product can be ensured and the centrifuge can be ensured. Since the number of treatments such as separation does not increase, there is an advantage that fat-derived cells can be recovered while being kept in a healthy state.

  Furthermore, in the conventional cell separation apparatus, since the position where the cells in the centrifuge container 10 are aggregated is not known, the position where the cells are not aggregated is stimulated, and the inactivation efficiency may be reduced. There was sex. In response to this problem, according to the cell separation device 1 according to the present embodiment, by providing the cell number measuring unit 11, it is possible to detect the position where the cells are aggregated and reliably stimulate the cells. The activation efficiency can be improved.

In addition, in the said embodiment, although it decided to stimulate by heating a fat-derived cell, it may replace with this and may stimulate by irradiating an ultrasonic wave or infrared light.
In this case, as shown in FIG. 10, an ultrasonic transducer or an infrared light source 20 is provided on the receiving surface 15a in place of the heater 12. Even in this way, the fat-derived cells in the cell concentrate C can be stimulated simply and effectively to activate the fat-derived cells.

  At this time, the control unit 7 controls the intensity of the ultrasonic wave or infrared light output from the ultrasonic vibrator or the infrared light source 20 so as to be proportional to the number of fat-derived cells measured by the cell number measuring unit 12. Or adjust the output time. In addition, when the temperature detected by the temperature sensor 17 exceeds a predetermined threshold, the control unit 7 temporarily stops the irradiation of the ultrasonic wave or the infrared light, and the temperature detected by the temperature sensor 17 is the predetermined threshold. When it becomes lower, the irradiation of ultrasonic waves or infrared rays is resumed. Thereby, it can prevent that the temperature of the cell concentrate C rises excessively by irradiation of an ultrasonic wave or infrared rays.

  Alternatively, a liquid volume measuring unit (not shown) for measuring the liquid volume of the cell suspension in the pocket portion 10b is provided, and the control unit 7 performs an operation based on the liquid volume of the cell suspension measured by the liquid volume measuring unit. The irradiation conditions of sound waves or infrared light may be controlled. As the liquid amount measuring means, for example, an optical sensor for detecting the liquid surface position of the cell suspension or a weighing scale for measuring the weight of the cell suspension can be used.

  Further, when an ultrasonic transducer is employed as the cell stimulating unit 12, an optical sensor (not shown) that detects the density of fat-derived cells at each position of the cell concentrate C or the presence or absence of bubbles in the cell concentrate. ) May be provided. By doing in this way, it can be detected whether the cell concentrate C is not irradiated with the ultrasonic wave of excessive intensity | strength, and it can prevent that an excessive stimulation is given to a fat origin cell.

  Moreover, in the said embodiment, although the cell stimulation part was employ | adopted as a yield increase part, the structure which removes an unnecessary cell from a cell suspension may be sufficient as a yield increase part. For example, a bead having an antibody that specifically binds to cells other than fat-derived cells in the cell suspension bound to the surface can be used as the yield increasing portion. In this case, the control unit 7 adjusts the amount of beads supplied into the centrifuge container 10 based on the amount of the cell suspension, for example.

  Moreover, in the said embodiment, although the cell stimulation part 12 was provided integrally with the rotating shaft member 13 so that the pocket part 10b might be covered, arrangement | positioning of the cell stimulation part 12 is limited to this. Instead, it may be arranged so that the cell concentrate in the pocket portion 10b can be efficiently stimulated.

  For example, the receiving portion 15 may have a U-shape in which a groove into which the pocket portion 10b is fitted is formed as shown in FIG. 11, and the surface adjacent to the pocket portion is formed as shown in FIG. It may be formed on a gently curved surface. Further, as shown in FIG. 13, a panel 21 surrounding the centrifuge 9 is provided, and the cell stimulating unit 12 is provided on the entire bottom surface of the panel 21 separately from the arm 13 a and the rotating shaft member 13. Good.

Moreover, in the said embodiment, although it was decided that the cell number measurement part 11 measured the number of fat origin cells based on the volume of the settled cell mass A, it replaced with this and the scattered light of the cell concentrate C is replaced with this. The number of fat-derived cells may be measured based on the intensity of the.
In this case, after washing the fat-derived cells and discharging the supernatant, pipetting and suspending the fat-derived cells and a small amount of the supernatant, the cell number counting unit 11 is as shown in FIG. The detection light L ′ is irradiated from the receiving surface 15a toward the pocket portion 10b, and the scattered light of the detection light L ′ is detected. Since the intensity of the scattered light at this time is proportional to the density of the fat-derived cells in the cell concentrate C, the cell number measurement unit 11 calculates the number of fat-derived cells based on the detected intensity of the scattered light. Can do.

  In addition, the configuration of the cell number measurement unit 11 employed in the above embodiment is an example, and is not limited to this configuration. For example, as shown in FIG. 15, the cell number measuring unit 11 includes a camera 11 a provided on the receiving surface 15 a and a memory 11 b provided on the bucket 14, and a cell mass in an image captured by the camera 11 a. The height position of A may be measured by the memory 11b, and the number of cells may be calculated from the measured height position. At this time, a part of the bucket 14 can be appropriately made of a transparent member or a hole can be formed in a part of the bucket 14 so that the camera 11a can photograph the inside of the centrifuge container 10 and the memory 11b.

  Alternatively, as shown in FIG. 16, a lattice-shaped line 11c attached to the bucket 14 in a state where the fat-derived cells are suspended may be photographed by the camera 11a. In this case, since the appearance of the line 11c varies depending on the concentration of the cell suspension, the number of fat-derived cells can be measured.

  In addition, as shown in FIG. 17, the cell number measuring unit 11 may be provided on the panel 21 and the receiving surface 15a, or as shown in FIG. 18, two cells 12 may be provided on the panel 12. When the cell number measurement unit 11 is provided on the panel 21, the position of the cell number measurement unit 11 and the centrifuge container 10 may be shifted when the rotation of the centrifuge 9 is stopped. Therefore, the motor 13b may be provided with a brake so that the arm 13a stops at a predetermined position with respect to the cell number measuring unit 11 when the rotation is stopped, or the stop position is adjusted by rotating the arm 13a in the reverse direction. Good.

Moreover, in the said embodiment, arrangement | positioning with the cell stimulation part 12 and the cell number measurement part 11 can be changed suitably. Specifically, both the cell stimulating unit 12 and the cell number measuring unit 11 may be provided integrally with the arm 13a, or the cell stimulating unit 12 may be provided integrally with the arm 13a and the cell number measuring unit 11 may be provided on the panel 21. Alternatively, the cell stimulation unit 12 may be provided on the panel 21 and the cell number measurement unit 11 may be provided integrally with the arm 13a.
Further, as the cell number measuring unit 11, a flow cytometry, a particle size distribution measuring device, or a turbidimeter can be used.

DESCRIPTION OF SYMBOLS 1 Cell separator 2 Decomposition processing part 3 Cell concentration part 4 Washing liquid bag 5 Waste liquid bag 6 Conveyance path 7 Control part 8 Collection container 8a Bottom surface 8b Filter 9 Centrifuge 10 Centrifugation container 10a Piping 10b Pocket part 10c Suction discharge port 11 Cell Number counting unit (cell number counting means)
11a camera 11b memory 11c grid-like line 12 cell stimulating part (yield increasing part), heater 13 rotating shaft member 13a arm 13b motor (rotating part)
14 bucket 15 receiving part 15a receiving surface 16 magnet 17 temperature sensor 18 tube 19 liquid feed pump 20 ultrasonic vibrator or infrared light source 21 panel A cell mass B supernatant C cell concentrate

Claims (2)

A digestion processing unit that separates living body-derived cells from the living tissue by digesting the living tissue to generate a cell suspension;
An arm that supports the centrifuge container containing the cell suspension so as to be swingable about a swing axis, and a rotating unit that rotates the arm about a predetermined axis away from the swing axis; A cell concentration part for concentrating the cell suspension by centrifugation;
A yield increasing part for increasing the yield of the living body-derived cells in the cell suspension in the cell concentration part,
The yield increasing unit is a cell separation apparatus provided at a position adjacent to the centrifuge container in a state where the arm is stationary. Cell number measuring means for measuring the number of living body-derived cells contained in the cell suspension;
The cell separation device according to claim 1, further comprising: a control unit that controls the yield increasing unit based on information on the number of the living body-derived cells measured by the cell number measuring unit.

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