JP2011004705A - Cell separation device and cell separation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To recover separated cells at a high yield by decreasing remaining cells attached on the inclined inside surface.SOLUTION: There is provided a cell separating device 1 comprising: centrifuge vessels 2 which have on the bottom 2a an inclined inside surface 2b inclined to be tapering toward the tip end and receives a cell suspension liquid A; a rotation mechanism 3 which rotates the centrifuge vessels 2 so that the bottom 2a heads toward the radial outside; a supernatant suction part 4 which soaks supernatant B separated inside the centrifuge vessels 2 as the result of rotation by the rotation mechanism 3; and a control part 6 which controls the rotation mechanism 3 and the supernatant suction part 4 to work the rotation mechanism 3 to centrifuge the cell suspension liquid A, to operate the supernatant suction part 4 to remove the supernatant B in the centrifuge vessels 2, and thereafter, further to work the rotation mechanism 3 to rotate the centrifuge vessels 2.

Description

  The present invention relates to a cell separation device and a cell separation method.

Conventionally, in order to wash and concentrate cells separated from living tissue, it was obtained by suspending living tissue in a biocompatible salt solution and adding digestive enzymes to digest living tissue. A processing apparatus using a centrifuge for cell suspension is known (for example, see Patent Document 1). In this treatment apparatus, the cell suspension is stored in a centrifuge container, and the bottom of the centrifuge container is rotated so that the bottom of the centrifuge container is directed radially outward. Can be separated into a cell group and a supernatant from the bottom side in order of increasing size.
The group of cells separated at the bottom of the centrifuge container can be collected by aspirating and removing the upper supernatant.

International Publication No. 2005/012480 Pamphlet

  However, the processing apparatus of Patent Document 1 has a tapered shape that tapers toward the center of the tip of the centrifuge container in order to collect the cells separated at the center of the tip of the centrifuge and to facilitate cell recovery. An inclined inner surface is provided. For this reason, when centrifuging, many cells move along the inclined inner surface and are collected at the center of the tip of the bottom of the centrifuge container, but some of them adhere to the inclined inner surface and are centrifuged. There is an inconvenience that it remains uncollected at the bottom end of the container and the recovery rate decreases.

  The present invention has been made in view of the above-described circumstances, and reduces the number of cells that remain attached to the inclined inner surface, and allows the separated cells to be recovered at a high recovery rate and the cell separation. It aims to provide a method.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a centrifuge container having a slanted inner surface inclined so as to taper toward the tip at the bottom and containing a cell suspension, and the centrifuge container so that the bottom is directed radially outward. A rotating mechanism for rotating, a supernatant suction section for sucking the supernatant separated in the centrifuge container as a result of being rotated by the rotating mechanism, and centrifuging the cell suspension by operating the rotating mechanism And operating the supernatant aspirating unit to remove the supernatant in the centrifuge container, and further operating the rotating mechanism to rotate the centrifuge container to rotate the centrifuge container and the supernatant aspirating unit. A cell separation device comprising a control unit for controlling is provided.

According to the present invention, in a state where the cell suspension is accommodated in the centrifuge container, the control unit operates the rotation mechanism to rotate the centrifuge container, so that the cell suspension accommodated in the centrifuge container is rotated. Each component of the suspension is separated by specific gravity, and cells having a large specific gravity move toward the tip of the bottom surface, while supernatant having a small specific gravity is separated in the upper layer of the cell group. In this process, since the cells are moved along the inclined inner surface provided at the bottom, a part of the cells adheres to the inclined inner surface.
Then, the control unit operates the supernatant aspirating unit to aspirate and remove the supernatant, thereby leaving a cell group in the centrifuge container.

  In this state, the control unit operates the rotation mechanism again to rotate the centrifuge container. Thereby, the cells remaining in the centrifuge container are subjected to centrifugal force again. In this case, since the supernatant is removed from the centrifuge container, the force applied to the cells attached to the inclined inner surface is the centrifugal force due to rotation and the frictional force between the inclined inner surface and the supernatant. Since the frictional force between them is lost, the force to resist against the centrifugal force is weak. Therefore, the cells adhering to the inclined inner surface are also collected toward the center tip of the bottom, and can be easily recovered at a high recovery rate.

  In the first aspect of the present invention, a centrifuge container having an inclined inner surface inclined so as to taper toward the tip at the bottom and containing a cell suspension is rotated so that the bottom is directed radially outward. A supernatant removing step for sucking the supernatant separated in the centrifugal container by the first rotating step, and the centrifugal container in which the supernatant has been removed by the supernatant removing step And a second rotation step in which the bottom portion is rotated so that the bottom portion is directed radially outward.

  According to the present invention, in the first rotation step, the cell suspension in the centrifuge container is separated into a cell group and a supernatant, and in the supernatant removal step, the separated supernatant is removed from the centrifuge container. Removed. In the second rotation step, since the centrifugal force is applied to the cell group in the absence of the supernatant, the cells are separated from the bottom of the centrifuge container by the centrifugal force because there is no frictional force with the supernatant. The cells attached to the inclined inner surface are also collected toward the center tip of the bottom and can be easily recovered at a high recovery rate.

In the said invention, it is preferable to perform the said 1st rotation step for 3 to 7 minutes with the acceleration of 400-500G.
In the above invention, the second rotation step may be performed at an acceleration of 400 to 500 G for 1 to 7 minutes.
By doing in this way, most of the cells attached to the inclined inner surface can be gathered together at the center tip of the bottom of the centrifuge container, the cell recovery rate can be improved, and excessive Since the centrifugal force is not applied to the cells, damage to the cells can be reduced, and the cells can be separated and recovered in a healthy state.

  According to the present invention, it is possible to reduce the number of cells that remain attached to the inclined inner surface and recover the separated cells at a high recovery rate.

It is a typical lineblock diagram showing the cell separation device concerning one embodiment of the present invention. It is a longitudinal cross-sectional view which shows an example of the centrifuge container with which the cell separation apparatus of FIG. 1 is equipped. It is a flowchart which shows the cell separation method which concerns on one Embodiment of this invention. FIG. 2 is a graph showing a comparison between the number of cells in a cell group collected by an example of a cell separation method using the cell separation apparatus of FIG. 1 and the number of cells in a cell group collected by a comparative example by a conventional method. .

A cell separation 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 separation device 1 according to the present embodiment includes a centrifuge container 2 that contains a cell suspension A, and the centrifuge container 2 so that its bottom portion 2a faces outward in the radial direction. A centrifugal separator 3 that is rotated into the centrifugal container 2, a supernatant suction part 4 that sucks and removes the supernatant B separated in the centrifugal container 2, a cleaning liquid supply part 5 that supplies the cleaning liquid C into the centrifugal container 2, and a centrifugal separator A control unit 6 that controls the separator 3, the supernatant suction unit 4, and the cleaning liquid supply unit 5 is provided.

As shown in FIG. 2, the centrifuge container 2 has an inclined inner surface 2b having a tapered inner surface that tapers toward the center of the tip near the bottom.
The centrifuge 3 includes a motor 7 and an arm 8 that is horizontally rotated by the motor 7, and the centrifuge container 2 is swingably attached around a horizontal axis 9 provided at both ends of the arm 8. ing. A rotary joint 10 is attached to the arm 8, and a movable part 10a that is rotated integrally with the arm 8 and a fixed part 10b that is fixed to the base (not shown) are rotated relative to each other while being relatively rotated. The connection of the flow path is maintained.

  As shown in FIG. 2, the supernatant aspirating unit 4 includes a suction pipe 11 having a suction port 11 a disposed in the middle of the centrifuge container 2 in the depth direction, and a pipe 12 connected to the suction pipe 11. And a pump 13 for feeding the supernatant B in the pipe 12, for example, a peristaltic pump. The pipe 12 includes a movable pipe 12 a that connects between the movable part 10 a of the rotary joint 10 and the suction pipe 11, and a fixed pipe 12 b that is connected to the outside from the fixed part 10 b of the rotary joint 10. By operating the pump 13, the pressure in the fixed pipe 12b is reduced, and the supernatant B separated in the centrifuge container 2 can be sucked and discharged to the outside.

  The cleaning liquid supply unit 5 is a pump that feeds the cleaning liquid C in the pipe 15, a supply pipe 14 having a discharge port 14 a disposed near the bottom 2 a of the centrifuge container 2, a pipe 15 connected to the supply pipe 14. 16, for example, a peristaltic pump. The pipe 15 includes a movable pipe 15 a that connects the movable part 10 a of the rotary joint 10 and the supply pipe 14, and a fixed pipe 15 b that is connected to the outside from the fixed part 10 b of the rotary joint 10. By operating the pump 16, the pressure in the fixed pipe 15b is reduced, and the cleaning liquid C supplied from an external cleaning liquid container (not shown) can be discharged into the centrifuge container 2 from the discharge port 14a. Yes.

  The control unit 6 operates the centrifuge 3 to rotate the centrifuge container 2, thereby centrifuging the internal cell suspension A into the supernatant B and the cell group D, and the supernatant suction unit. 4 to discharge the supernatant B in the centrifuge container 2, and to operate the cleaning liquid supply unit 5 to supply the cleaning liquid C into the centrifuge container 2 to resuspend the cell group D. After repeating the above, the supernatant suction unit 4 is operated to discharge the supernatant B in the centrifuge container 2, and then the centrifuge 3 is operated again to control the centrifuge container 2 to rotate. It is like that.

A cell separation method using the cell separation device 1 according to the present embodiment configured as described above will be described below.
In order to separate and collect the cell group D concentrated from the cell suspension A using the cell separation device 1 according to the present embodiment, the cell suspension A is placed in the centrifuge container 2 as shown in FIG. , The control unit 6 operates the centrifuge 3 to rotate the centrifuge container 2, and the cell suspension A stored in the centrifuge container 2 is transferred to the cell group D and the supernatant B. Is centrifuged (centrifugation step S1).

  As a result, the cells floating in the cell suspension A move radially outward due to the difference in specific gravity with the liquid component due to centrifugal force, while the liquid component moves radially inward. Thereby, the cell group D is isolate | separated to the bottom part 2a side of the centrifuge container 2, and supernatant B is isolate | separated into the upper layer. When the cells move in the centrifuge container 2, the cells move along the inclined inner surface 2 b provided in the centrifuge container 2, so that the cells are collected at the center of the tip, thereby forming a pellet-shaped cell group D.

At this time, some cells adhere to the inclined inner surface 2b and remain there.
Next, the control unit 6 operates the supernatant suction unit 4 to suck the supernatant B from the suction port 11a and discharge it to the outside (supernatant removal step S2). Thereby, the cell group D remains in the centrifuge container 2 together with some supernatant. At this time, a part of the cell group D is collected at the center of the tip of the centrifuge container 2, and the other part is attached to the inclined inner surface 2b.

  In this state, the control unit 6 operates the cleaning liquid supply unit 5 to supply the cleaning liquid C from the discharge port 14a into the centrifuge container 2 (cleaning liquid supply step S3). Since the discharge port 14a is disposed in the vicinity of the bottom 2a of the centrifuge container 2, the cell group D separated into pellets by the cleaning liquid C discharged from the discharge port 14a is released and resuspended. Thereby, chemicals such as digestive enzymes and dust adhering to the cells are washed away from the cells.

Thereafter, the control unit 6 repeats the first rotation step S1 to the cleaning liquid supply step S3 (repetition step S4).
And the control part 6 operates the centrifuge 3, rotates the centrifuge container 2, and centrifuges the cell suspension A accommodated in the centrifuge container 2 into the cell group D and the supernatant B. (First rotation step S5).

  Next, the control unit 6 operates the supernatant suction unit 4 to suck the supernatant B from the suction port 11a and discharge it to the outside (supernatant removal step S6). As a result, the washed cell group D remains in the centrifuge container 2 together with some supernatant B. At this time, a part of the cell group D is collected at the center of the tip of the centrifuge container 2, and the other part is attached to the inclined inner surface 2b.

In the cell separation method according to the present embodiment, from this state, the control unit 6 operates the centrifuge 3 again to rotate the centrifuge container 2 (second rotation step S7).
In the second rotation step S7, since the supernatant B is hardly present in the centrifuge container 2, unlike the centrifuge step S1, the cell group D receiving the centrifugal force is subjected to the inner surface of the centrifuge container 2. It can be moved toward the bottom 2a against the friction between the two.

  That is, in the centrifugal separation step S1 and the first rotation step S5, the cell group D must move along the inclined inner surface 2b in the presence of the liquid component, and therefore friction between the inclined inner surface 2b and the inclined inner surface 2b. Not only is there friction between the liquid components. For this reason, in order to move the cells adhering to the inclined inner surface 2b to the distal end side of the centrifuge container 2, a force exceeding the frictional force between the inclined inner surface 2b and the frictional force between the liquid component is exceeded. It is necessary to apply centrifugal force. On the other hand, in the second rotation step S7, since no liquid component is present, the cell group D is only subjected to centrifugal force exceeding the frictional force between the inclined inner surface 2b and the centrifuge container 2 It can move easily toward the tip side of the.

  As a result, according to the cell separation device 1 and the cell separation method according to the present embodiment, in the centrifugation step S1 and the first rotation step S5, cells adhere to the inclined inner surface 2b without applying excessive centrifugal force. In a state, the cell group D and the supernatant B can be separated. Further, the cell group D attached to the inclined inner surface 2b in the second rotation step S7 can be moved toward the center of the tip of the centrifuge container 2 without applying excessive centrifugal force. Therefore, according to the cell separation device 1 and the cell separation method according to the present embodiment, there is an advantage that the concentrated cell group D can be recovered at a high recovery rate.

Here, an example of a cell separation method using the cell separation device 1 according to the present embodiment will be described.
In this example, cell suspensions A1 and A2 were prepared by the following method.

(1) 200 g of adipose tissue and 200 mL of lactated Ringer's solution were placed in a digestion vessel and stirred.
(2) The mixture was allowed to stand until the adipose tissue and the lactated Ringer solution were separated, and only the lactated Ringer solution was discarded.
(3) Steps (1) and (2) were repeated three times.
(4) 100 mL of lactated Ringer's solution was placed in a digestion vessel containing adipose tissue.
(5) 0.5 WU / mL of collagenase and 214 caseinase U / mL of thermolysin were added to the digestion vessel.
(6) Digested with stirring for 15 minutes.
(7) After digestion, the mixture was allowed to stand until the fat layer and the liquid layer were separated, and 50 mL of the liquid layer was recovered to obtain a cell suspension A1.
(8) 200 mL of lactated Ringer's solution was placed in the digestion vessel and stirred.
(9) The mixture was allowed to stand until the fat layer and the liquid layer were separated, and 100 mL of the liquid layer was recovered to obtain a cell suspension A2.

Next, cell group D was recovered according to the following steps.
(1) 25 mL each of cell suspension A1 was put into two centrifuge containers 2.
(2) The cell suspension A1 was centrifuged by rotating the centrifuge container 2 at 400 G for 5 minutes.
(3) 23.5 mL of the supernatant B was discarded so as not to aspirate the precipitated cell group D.
(4) 50 mL of cell suspension A2 was placed in each of two centrifuge containers 2.
(5) The cell suspension A2 was centrifuged by rotating the centrifuge container 2 at 400G for 5 minutes.
(6) 50 mL of the supernatant B was discarded so as not to aspirate the precipitated cell group D.
(7) 35 mL of new lactated Ringer's solution was put in each centrifuge container 2.
(8) Each centrifuge container 2 was centrifuged at 400 G for 3 minutes.
(9) 35 mL of the supernatant B was discarded so as not to aspirate the precipitated cell group D.
(10) Steps (7) to (9) were repeated three times.
(11) Each centrifugation container 2 was rotated at 400 G for 3 minutes.
(12) 1.5 mL of the cell group D remaining in each centrifuge container 2 was collected.
(13) The number of cells contained in the collected cell group D was counted.

A similar cell separation method not including the above step (11) is performed as a comparative example, and the comparison result with this example is shown in FIG.
According to the cell separation method according to the present example, the number of cells contained in the collected cell group D is twice or more that of the collected cell group D in the comparative example, and the number of cells remaining on the inclined inner surface 2b is It turns out that there is hardly any. On the other hand, in the comparative example, it was found that more than half of the centrifuged cell group D remained on the inclined inner surface 2b without being collected.

  Thus, according to the cell separation device 1 and the cell separation method according to the present embodiment, the cells can be separated without applying an excessive centrifugal force, and the soundness of the cells can be maintained. Then, there is an advantage that the number of cells remaining on the inclined inner surface 2b of the centrifuge container 2 can be greatly reduced, and the washed and concentrated cell group D can be recovered at a high recovery rate.

  In the present embodiment, 400 G and 3 to 5 minutes are exemplified as the centrifugal force for separating the cell group D without damaging the cells. However, the centrifugal force is not limited to this. , 400 to 500 G, 1 to 7 minutes, even if selected arbitrarily, the same effect can be obtained.

A, A1, A2 Cell suspension B Supernatant C Wash solution D Cell group S5 First rotation step S6 Supernatant removal step S7 Second rotation step 1 Cell separation device 2 Centrifugal container 2a Bottom 2b Inclined inner surface 3 Centrifugation Machine (rotating mechanism)
4 Supernatant suction part 6 Control part

Claims (4)

A centrifuge container containing a cell suspension having an inclined inner surface inclined at the bottom so as to taper toward the tip; and
A rotation mechanism for rotating the centrifuge container so that the bottom portion is directed radially outward;
As a result of being rotated by the rotating mechanism, a supernatant aspirating part for aspirating the supernatant separated into the centrifuge container;
The rotation mechanism is operated to centrifuge the cell suspension, the supernatant suction part is operated to remove the supernatant in the centrifuge container, and then the rotation mechanism is further operated to operate the centrifuge container. A cell separation device comprising: the rotation mechanism and a control unit that controls the supernatant suction unit so as to rotate the cell. A first rotation step of rotating a centrifuge container having a slanted inner surface that is slanted toward the tip at the bottom and containing a cell suspension so that the bottom is directed radially outward;
A supernatant removal step of aspirating the supernatant separated into the centrifuge container by the first rotation step;
A cell separation method comprising: a second rotation step of rotating the centrifuge container from which the supernatant has been removed by the supernatant removal step so that the bottom portion is directed radially outward.   The cell separation method according to claim 2, wherein the first rotation step is performed at an acceleration of 400 to 500 G for 3 to 7 minutes.   The cell separation method according to claim 2 or 3, wherein the second rotation step is performed at an acceleration of 400 to 500 G for 1 to 7 minutes.

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