JP2010115175A - Centrifugal separation device and centrifugal separation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To collect a cell suspension containing a cell group from which an unnecessary component is removed by efficiently washing the cell group. <P>SOLUTION: The centrifugal separation device includes a centrifugal separation container 6 for storing the cell suspension and to be rotated by directing the bottom part to the outer direction of the radial direction, a rotational driving mechanism 2 for rotating the centrifugal separation container 6, and a control part 7 for periodically changing the rotational speed by the rotational driving mechanism 2. The centrifugal separation container 6 has a cylindrical container body 6a for storing the cell suspension, and a movable body 6e arranged in the vicinity of the bottom part of the container body 6a, and installed so as to be relatively displaceable based on the container body 6a by the change of the rotational speed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a centrifuge device and a centrifuge method.

Conventionally, a centrifuge container containing a cell suspension obtained by decomposing adipose tissue and isolating adipose-derived cells is contained in the cell suspension by rotating it around an axis away from the centrifuge container. There is known a centrifugal separation device that separates components that are separated by specific gravity (see Patent Document 1).
The centrifuge container is formed in a substantially cylindrical shape with one end closed, and a component having a large specific gravity moves toward the closed end by rotating the closed end so that the closed end is directed radially outward. They are separated side by side in descending order of specific gravity.

International Publication No. 2005/012480 Pamphlet

  However, there is an inconvenience that the cell group separated at the bottom of the centrifuge container by centrifuging the cell suspension is formed into a solid pellet that is compressed by centrifugal force. That is, if the cell group is formed in a pellet shape, it becomes difficult to take out the cell group after centrifugation from the centrifuge container by suction. In addition, unnecessary components such as proteolytic enzymes and fats may be formed in the cell group during centrifugation, and may be formed into pellets, which makes it difficult to remove the unnecessary components.

  The present invention has been made in view of the above-described circumstances, and is a centrifugal separator that can efficiently recover a cell suspension containing a cell group from which unnecessary components have been removed by washing the cell group. The object is to provide a centrifugation method.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a centrifuge container that contains a cell suspension and is rotated with its bottom portion directed radially outward, a rotation drive mechanism that rotates the centrifuge container, and a rotation speed by the rotation drive mechanism. A control unit that fluctuates automatically, the centrifuge container is disposed in the vicinity of the bottom of the cylindrical container main body that contains the cell suspension, and the container main body due to fluctuations in rotational speed A centrifuge provided with a movable body that is relatively displaceable.

  According to the present invention, the rotation drive mechanism is operated in a state where the cell suspension is accommodated in the container body of the centrifuge container, and the cell suspension is rotated at a constant speed so that the bottom of the centrifuge container is directed radially outward. Thus, centrifugal force can be applied to the cell suspension, and a cell group having a large specific gravity can be moved to the bottom of the container body and centrifuged. In this case, since a constant centrifugal force also acts on the movable body arranged in the container body, the movable body is maintained stationary in the container body, and the cell group is stably centrifuged. .

  On the other hand, when washing the cell group, the rotation speed by the rotation drive mechanism is periodically changed by the control unit. Thereby, since the centrifugal force which acts on the movable body arrange | positioned in a container main body also fluctuates, a movable body is moved relatively with respect to a container main body. As a result, the pelleted cell group centrifuged at the bottom of the container is agitated, and the cell group can be easily and efficiently washed to remove unnecessary components.

In the above invention, the movable body may be an elastic member provided in the shape of a cantilever provided such that the tip is movable in a direction along the bottom surface of the container body.
By doing so, when the rotational speed of the rotational drive mechanism is periodically changed by the control unit, the cantilever-like elastic member is moved in the direction along the bottom surface of the container body. The pellet-like cell group that has been centrifuged at the bottom of the main body can be efficiently stirred.

Moreover, in the said invention, the mass part may be provided in the front-end | tip of the said elastic member.
By doing this, the natural frequency is reduced by the mass part at the tip of the cantilever-like elastic member, and the elastic member is displaced by the fluctuation of the rotational speed due to the lower frequency without damaging the cell group. Can be stirred.

Moreover, in the said invention, the said movable body provided the mass part provided so that the movement to the direction which approaches or leaves | separates with respect to the bottom face of the said container main body, and the spring provided so that expansion / contraction was possible in the movable direction of this mass part And a member.
In this way, when the rotational speed of the rotational drive mechanism is periodically changed by the control unit, the spring member expands and contracts, causing the mass unit to move in the direction approaching or separating from the bottom surface of the container body. It is done. Thereby, it can stir efficiently so that the pellet-shaped cell group currently centrifuged at the bottom part of the container main body may be hit.

  In the present invention, the cell suspension is housed in a container in which a movable body relatively displaceable with respect to the container is disposed near the bottom of the container, and the bottom of the container is radially outward. A centrifugal separation method is provided in which the rotation speed of the container is periodically changed so that the movable body is displaced inside the container.

  According to the present invention, it is possible to recover a cell suspension containing a cell group from which unnecessary components have been removed by efficiently washing the cell group.

A centrifugal separator 1 according to an embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the centrifugal separator 1 according to this embodiment includes a motor 2, a rotary shaft 3 that is arranged in the vertical direction and is driven to rotate by the motor 2, and is fixed to the rotary shaft 3. A beam-like arm 4 extending outward in the direction, two holding portions 5 supported at both ends of the arm 4 so as to be swingable about an axis 5a perpendicular to the longitudinal axis of the arm 4, and the holding portion 5 Are provided with two centrifuge containers 6 that are detachably supported, and a control unit 7 that controls the rotational speed of the motor 2.

  As shown in FIG. 2, the centrifuge container 6 has a substantially cylindrical container body 6a having one end opened and the other end gradually tapered and closed, and an opening of the container body 6a. A lid 6c that closes 6b, a pipe 6d that extends from the center of the lid 6c along the axis of the container body 6a, supplies cleaning liquid, and sucks the supernatant, and is provided at the tip of the pipe 6d. And a cantilevered elastic member 6e.

  The elastic member 6e is opposed to the bottom surface of the container main body 6a with a gap between the elastic member 6e and the holding portion 5 holding the container main body 6a is swung by the action of an external force as shown in FIG. The tip can be displaced in the same direction as the direction. The elastic member 6e is made of, for example, polycarbonate and has a diameter of 0.3 mm and a length of 10 mm. In this case, the natural frequency in the bending direction of the elastic member 6e is about 6 Hz. Since the elastic member 6e is disposed inside the cell group B having strong viscosity in the cell suspension A, it can be excited by an external force at a frequency lower than the natural frequency.

  The control unit 7 drives the motor 2 to rotate in a first mode in which the motor 2 is rotated at a predetermined rotation speed and a second mode in which the rotation speed is periodically changed as shown in FIG. Be able to. That is, when centrifuging the cell suspension A, the motor 2 is driven at a predetermined rotational speed in the first mode so that a predetermined centrifugal force is applied to the cells and the cells are separated by specific gravity. When the obtained cells are stirred together with the washing liquid, the rotation speed of the motor 2 is periodically changed in the second mode.

The operation of the centrifugal separator 1 according to this embodiment configured as described above will be described.
As shown in FIG. 5, the method for centrifuging cells in the cell suspension A using the centrifuge 1 according to the present embodiment is first centrifuged in the holding portions 5 provided at both ends of the arm 4. The separation container 6 is held, and the cell suspension A is accommodated in the centrifugation container 6 (step S1). In this state, the control unit 7 rotationally drives the motor 2 in the first mode (step S2). In the first mode, the motor 2 is rotationally driven at one or more predetermined rotation speeds and rotation times.

  Since the holding unit 5 holding the centrifuge container 6 is swingably attached to both ends of the arm 4, when the rotary shaft 3 and the arm 4 are rotated by the rotation drive of the motor 2, the arm 4 On the other hand, the holding portion 5 swings and the bottom portion of the container main body 6a changes into a posture toward the radially outward direction and is rotated. As a result, centrifugal force acts on the cell suspension A in the centrifuge container 6 in the direction toward the bottom of the container body 6a, and the containers are arranged in descending order of specific gravity according to the specific gravity of the cells in the cell suspension A. The layers are sequentially deposited from the bottom side of the main body 6a.

In this first mode, since the motor 2 is rotated at a predetermined rotational speed, the elastic member 6e provided at the tip of the pipe 6d is not excited, and the cell group B is not stirred and has a clear boundary surface. From the supernatant.
Thereafter, the motor 2 is temporarily stopped, and the supernatant is sucked through the pipe 6d (step S3). Thereafter, the cleaning solution is supplied to the cell group B remaining in the vessel body 6a through the pipe 6d (step S4). Even if the cell group B is aggregated in a pellet form, it is stirred to some extent by the flow energy of the supplied cleaning liquid.

In this state, the control unit 7 rotationally drives the motor 2 in the second mode (step S5).
In the second mode, since the rotational speed of the motor 2 is periodically changed, as shown in FIG. 6, the swing angle of the holding portion 5 varies with respect to the arm 4, and the tip of the pipe 6d The cantilever-like elastic member 6e is excited in the swinging direction of the holding portion 5, and its tip is reciprocated in a direction along the bottom surface of the container body 6a.

  As a result, the cell group B is directly agitated by the elastic member 6e disposed in the cell group B deposited on the bottom surface of the container body 6a. Therefore, even if the cell group B is aggregated in a pellet form, it can be efficiently stirred and loosened.

In the cell group B aggregated by centrifugation, unnecessary tissue fragments, digestive fluid components, and the like may be retained so as to be involved. Therefore, by stirring the cell group B to loosen it, Such unnecessary tissue pieces and washing liquid components can be released from the cell group B and released.
Then, the motor 2 is rotationally driven again in the first mode (step S6). Thereby, the cell group B which released the unnecessary component can be centrifuged.

  If necessary, a clean cell group B in which the concentration of unnecessary components contained in the cell group B is sufficiently reduced can be obtained by repeating steps S3 to S6 (step S7). Then, before collecting the cell group B obtained in the container main body 6a, the motor 2 is rotationally driven again in the second mode (step S8). Thereby, it is possible to generate a cell suspension in which the collected cell group B is loosened by stirring from a state where the cells are aggregated in a pellet shape and is uniformly dispersed in the washing solution.

  The cell suspension thus generated can be aspirated and collected without leaving the cell group B in the container body 6a by inserting a pipette or syringe (not shown) through the opening 6b and aspirating it. (Step S9). Therefore, according to the centrifugal separator 1 according to the present embodiment, there is an advantage that the yield of cells can be improved.

In the centrifugal separator 1 according to the present embodiment, a rod-shaped member provided at the tip of the pipe 6d is adopted as the elastic member 6e. Instead, the elastic member 6e is configured by a tubular member as shown in FIG. May be. In this case, since the tip of the elastic member 6e is disposed in the vicinity of the bottom surface of the container body 6a, the suction tube 10 for sucking and collecting the cell suspension in which the cell group B after centrifugation, which is the final product, is suspended. It is preferable that the front end portion of each is constituted by the elastic member 6e.
In addition, as shown in FIG. 8, by providing a weight (mass part) 11 at the tip of the elastic member 6 e, the natural frequency may be lowered to facilitate excitation at a lower frequency.

In the present embodiment, the elastic member 6e is exemplified as a cantilever, but instead, it is constituted by a weight 11 and a coil spring (spring member) 12 as shown in FIG. May be.
By doing in this way, if the control part 7 changes the rotational speed of the motor 2 periodically, since the centrifugal force which acts on the weight 11 will fluctuate, the coil spring 12 will approach or separate from the bottom face of the container main body 6a. Stretch in the direction. Thereby, the cell group B can be stirred so that the cell group B deposited on the bottom surface of the container body 6a is hit by the weight 11 in the deposition direction.

It is a whole lineblock diagram showing the centrifuge concerning one embodiment of the present invention. It is a longitudinal cross-sectional view which shows the centrifuge container of the state which accommodated the cell suspension with which the centrifuge of FIG. 1 is equipped. It is an expanded sectional view which shows the bottom face vicinity of the centrifuge container of FIG. It is a graph which shows the rotational speed change of the motor by the 2nd mode of the centrifuge of FIG. It is a flowchart explaining the centrifugation method by the centrifuge of FIG. It is a whole block diagram explaining operation | movement of the centrifuge container in the 2nd mode of the centrifuge of FIG. It is a longitudinal cross-sectional view which shows the modification of the centrifuge container of FIG. It is a longitudinal cross-sectional view which shows the other modification of the centrifuge container of FIG. It is a longitudinal cross-sectional view which shows the other modification of the centrifuge container of FIG.

Explanation of symbols

A cell suspension B cell group 1 centrifuge 2 motor (rotation drive mechanism)
6 Centrifuge container 6a Container body 6e Elastic member (movable body)
7 Control part 11 Weight (mass part, movable body)
12 Coil spring (spring member)

Claims (5)

A centrifuge container containing the cell suspension and rotated with the bottom facing radially outward;
A rotation drive mechanism for rotating the centrifuge container;
A controller that periodically varies the rotational speed of the rotational drive mechanism,
The centrifuge container is disposed in the vicinity of the bottom of the cylindrical container main body for storing the cell suspension and the container main body, and is provided so as to be relatively displaceable with respect to the container main body due to fluctuations in rotational speed. A centrifuge comprising the body.   The centrifuge according to claim 1, wherein the movable body is an elastic member provided in a cantilever shape provided such that a tip thereof is movable in a direction along a bottom surface of the container main body.   The centrifuge according to claim 2, wherein a mass part is provided at a tip of the elastic member.   The said movable body is provided with the mass part provided so that the movement to the direction which approaches or leaves | separates with respect to the bottom face of the said container main body, and the spring member provided so that it can expand-contract in the movable direction of this mass part. The centrifuge described in 1. A cell suspension is accommodated in a container in which a movable body that is displaceable relative to the container is disposed near the bottom of the container,
Rotating the bottom of the container radially outward,
A centrifugation method in which the rotational speed of the container is periodically changed so that the movable body is displaced inside the container.

Images