JP2010158203A - Agitator, and device for separating cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To agitate treating liquid while preventing the formation of air bubbles in the treating liquid and reducing damage given to a biological specimen. <P>SOLUTION: This agitator 1 includes a base stand 3 for loading a container 2 for housing the biological specimen A and the treating liquid C for treating the biological specimen A, a motor 5 for rotating the base stand 3 around an eccentric shaft rotating along with a prescribed circular orbital in a horizontal plane while keeping its posture, weight-measuring parts 6a, 6b, 6c for measuring the position of center of gravity in horizontal direction of the container 2 loaded on the base stand 3 over time while rotating the base stand 3, and a controlling part 7 for controlling the speed and phase of rotation of the base stand 3 caused by the motor 5 based on the changes over time of the position of center of gravity measured by the weight-measuring parts 6a, 6b, 6c. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

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

  2. Description of the Related Art Conventionally, there is known an apparatus that separates cells that have been decomposed with an enzyme and bound to the living tissue (see, for example, Patent Document 1 and Patent Document 2). In these apparatuses, the decomposition of the biological tissue by the enzyme is promoted by rotating or vibrating the container containing the biological tissue and the solution of the degrading enzyme at a low speed.

  In addition, a cell culture device that performs passage of cultured cells and medium exchange is known (see, for example, Patent Document 3). When cells adhering to the bottom surface of the culture container are peeled off with an enzyme, the culture container is swung so that the enzyme solution is evenly distributed over the bottom surface of the container.

International Publication No. 2004/027014 Pamphlet JP 2005-218376 A JP 2005-218413 A

  However, when the rotation, vibration, or swing of the container is accelerated or decelerated to stop stirring, the wave of the processing liquid breaks in the container and bubbles are generated in the processing liquid. As a result, there is a problem that cells and biological tissues contained in the treatment liquid are damaged by being brought into contact with air.

  The present invention has been made in view of the above-described circumstances, and provides a stirrer and a cell separation device that can prevent bubbles from being generated in a treatment liquid and can stir while reducing damage to a biological sample. The purpose is to do.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a base on which a biological sample and a container for storing a treatment liquid for processing the biological sample are placed, and an eccentric shaft along a predetermined circumferential orbit in a horizontal plane while maintaining the posture of the base. A motor that rotates around, a weight measuring unit that measures the position of the center of gravity in the horizontal direction of the container placed on the base over time during the rotation of the base, and the weight measuring unit A stirrer provided with a control part which controls the speed and phase of rotation of the base by the motor based on a time change of a gravity center position is provided.

  According to the present invention, when the container is placed on the base and the motor is operated, and the container is rotated around the eccentric axis in the horizontal plane simultaneously with the base, the rotation of the container becomes a driving force and the processing liquid is supplied. The biological sample can be rotated in the container and stirred in the treatment liquid.

  In this case, the treatment liquid is pulled radially outward by a centrifugal force centered on the center of the circumferential path around which the container is rotated, and is pressed against the inner wall on the radially outer side of the container in a close contact state. The inside of the container can be rotated. For this reason, the height of the surface of the processing liquid, that is, the weight of the processing liquid has a radial gradient of rotation, and in a container rotating around an eccentric axis, the weight at each position is the speed of rotation of the processing liquid. And periodically changes according to the phase. Thereby, the gravity center position of the container periodically moves in the circumferential direction in the horizontal plane, and this time change is measured by the weight measuring unit.

  The control unit controls the phase and speed of the rotation of the base so that the phase shift is always within the range of the predetermined rotation angle with respect to the periodic time change of the gravity center position of the container. That is, the rotation of the container is adjusted without the driving force applied to the processing liquid by the rotation of the container against the inertial force of the processing liquid rotating in the container. As a result, the rotation of the treatment liquid is disturbed and it is prevented from peeling off from the inner wall that has been in close contact until now, or the treatment liquid undulates, and bubbles are prevented from being generated in the treatment liquid, thereby causing damage to the biological sample. The biological sample can be agitated in the treatment liquid while reducing.

In the above invention, the control unit may cause the rotation phase of the base to advance by a predetermined angle with respect to the phase of temporal change in the center of gravity position.
By doing so, the container is rotated while being advanced by a predetermined rotation angle with respect to the rotation of the processing liquid in the container, and the rotation of the processing liquid is accelerated without generating bubbles in the processing liquid. Can do.

Moreover, in the said invention, the said control part is good also as controlling the said motor so that the phase of the rotation of the said base may be delayed only a predetermined rotation angle with respect to the phase of the time change of the said gravity center position.
In this way, the rotation of the processing liquid is reduced without causing bubbles in the processing liquid by rotating the container while retreating by a predetermined rotation angle with respect to the rotation of the processing liquid in the container. Can do.

Moreover, in the said invention, it is good also as a plurality of the said weight measurement parts being provided in the circumferential direction at intervals.
By doing in this way, the time change of the gravity center position of the container during rotation is measured more precisely, and the rotation of the base can be controlled more appropriately.

  In addition, the present invention includes any one of the agitators described above, and contains in the container a biological tissue containing biological cells and a degrading enzyme solution that decomposes the biological tissue, and the biological cells are extracted from the biological tissue. A separation processing unit for separating and generating a cell suspension; a cell concentration unit for concentrating the biological cells by centrifuging the cell suspension generated in the decomposition processing unit; and Provided is a cell separation device including a liquid feeding path for feeding the cell suspension to a cell concentration unit.

  According to the present invention, when the container is rotated around the eccentric axis by the stirrer in the decomposition processing unit, the biological tissue is stirred in the decomposing enzyme solution and decomposed by the decomposing enzyme. And a living body origin cell isolate | separates from a biological tissue, and the cell suspension in which a living body origin cell floats is produced | generated. When the cell suspension is sent to the cell concentrating portion through the liquid sending route and centrifuged, and the precipitated living body-derived cells are collected together with a small amount of supernatant, a cell concentrated solution of the living body-derived cells is obtained.

  In this case, even if the rotation of the container is accelerated or decelerated by the stirrer, the decomposing enzyme solution is rotated in the container without peeling off or undulating from the inner wall that has been in close contact therewith. As a result, no bubbles are generated in the decomposing enzyme solution, so that the living cells can be recovered in a healthy state by stirring in the decomposing enzyme solution while suppressing damage to living tissue and living cells. .

In the above invention, the living tissue may be adipose tissue collected from within the living body, and the living body-derived cells may be fat-derived cells encapsulated in the adipose tissue.
By doing in this way, a fat-derived cell can be collect | recovered in a healthy state from an adipose tissue.

  According to the present invention, it is possible to prevent bubbles from being generated in the processing liquid and to stir while reducing damage to the biological sample.

It is (a) top view and (b) front view showing the whole agitator composition concerning one embodiment of the present invention. It is a figure explaining operation | movement of (a) processing container and (b) decomposing enzyme liquid when the stirrer of FIG. 1 is rotated. It is a figure explaining the control at the time of (a) accelerating the rotation of the stirrer of FIG. 1, (b) operating at a constant speed, and (c) decelerating. It is a whole block diagram of the cell separation device concerning one embodiment of the present invention.

An embodiment of the present invention will be described below with reference to FIGS.
In the present embodiment, a process of separating fat-derived cells (biologically derived cells) B from adipose tissue (biological sample, biological tissue) A will be described as an example.
As shown in FIG. 1, the stirrer 1 according to the present embodiment includes a base 3 on which a cylindrical processing container (container) 2 is placed, and three shafts 4 provided on the bottom surface of the base 3. , A load cell (weight measuring unit) 6a, 6b, 6c provided on each shaft 4, and a control unit 7 connected to each motor 5 and each load cell 6a, 6b, 6c. Yes.

In the processing container 2, adipose tissue A collected by a method such as suction from a living body and a decomposing enzyme solution (processing solution) C for decomposing the adipose tissue A are accommodated.
The base 3 has a cylindrical shape in which the front side surface and the ceiling surface are open, and the bottom surface is disposed substantially horizontally. A seat heater 3 a is provided on the bottom surface of the base 3, and the bottom and side surfaces are covered with a heat insulating material 3 b so that the inside of the processing container 2 placed on the base 3 is kept at about 37 ° C. It has become.

The shaft 4 is fixed by inserting one end vertically into a through-hole provided in the bottom surface of the base 3 and being divided into three equal parts in the circumferential direction with respect to the center of the bottom surface of the base 3. Has been placed. The other end of each shaft 4 is connected to a motor 5 and is rotated by the motor 5 in the same direction while synchronizing on a circumferential track having the same diameter. As a result, when the motor 5 is operated, the base 3 can be rotated on a predetermined circular orbit around the eccentric shaft in the vertical direction while maintaining its posture.
The speed and phase of rotation of the shaft 4 by the motor 5 are controlled by the control unit 7.

  Load cells 6 a, 6 b, 6 c are arranged on the upper end surface of each shaft 4, and the vertical load applied to the motor 5 during the operation of the motor 5 is measured over time. Thereby, when the processing container 2 is mounted on the base 3, the time change of the weight of the processing container 2 in the position of each load cell 6a, 6b, 6c is measured. Information on the magnitude of the load measured by each load cell 6a, 6b, 6c is sequentially sent to the control unit 7.

  The controller 7 determines the load applied to the bottom surface of the base 3 from the magnitude of the load input from each load cell 6a, 6b, 6c and the position within the bottom surface of the base 3 of each load cell 6a, 6b, 6c. The position of the center of gravity, that is, the position of the center of gravity in the horizontal direction of the processing container 2 is calculated, and the change with time is stored.

  When the processing container 2 is placed on the base 3 and the motor 5 is operated and the base 3 is rotated as shown in FIG. 2A, the processing container 2 is also integrated with the predetermined axis around the eccentric axis. Can be rotated on the circumference orbit. The rotation of the processing container 2 becomes a driving force, and the decomposing enzyme solution C is rotated in the processing container 2.

  At this time, the decomposing enzyme solution C is pulled outward in the radial direction with respect to the center of the circumferential orbit by centrifugal force, and the liquid level increases in the direction. Then, the processing container 2 is rotated in a state in which the position of the center of gravity of the processing container 2 is shifted radially outward from the center of the processing container 2 with respect to the center of the circumferential track. As a result, as shown in FIG. 2 (b), the temporal change in the gravity center position of the processing container 2 that periodically changes in the circumferential direction is stored in the control unit 7.

  In addition, the control part 7 memorize | stores the time change of the gravity center position of a horizontal direction when rotating the empty processing container 2 previously as a background. The net barycentric position obtained by subtracting the background from the barycentric position actually measured is stored.

  In addition, when the controller 7 accelerates the rotation of the shaft 4, as shown in FIG. 3A, the phase of the rotation of the shaft 4 is a predetermined rotation angle with respect to the time change of the center of gravity position of the processing container 2. The motor 5 is controlled so as to move forward only. When the rotation of the shaft 4 reaches a desired rotation speed, the phase of the rotation of the shaft 4 matches the phase of the temporal change in the center of gravity position of the processing container 2 as shown in FIG. The motor 5 is controlled. Further, when the rotation of the shaft 4 is decelerated, as shown in FIG. 3C, the phase of the rotation of the shaft 4 is delayed by a predetermined rotation angle with respect to the time change of the center of gravity position of the processing container 2. The motor 5 is controlled.

  At this time, the predetermined rotation angle with respect to the time change of the gravity center position of the processing container 2 is sufficiently small with respect to the inertial force of the decomposing enzyme C that the rotation driving force that the rotation of the processing container 2 gives to the decomposing enzyme solution C. Set within the range. Specifically, the optimum value is determined by the inner diameter and rotation speed of the processing container 2, the volume of the decomposing enzyme solution C, the viscosity, and the like.

The configuration and operation of the cell separation device 100 including the stirrer 1 configured as described above will be described below.
As shown in FIG. 4, the cell separation device 100 according to the present embodiment includes a decomposition processing unit 20 including a stirrer 1, a cell concentration unit 30 including a centrifuge 8, a decomposition processing unit 20, and a cell concentration unit. 30 is provided.

  The degradation processing unit 20 agitates the adipose tissue A with the agitator 1 in the degrading enzyme solution C to decompose the adipose tissue A, and separates the adipose-derived cells B included in the adipose tissue A. Then, by allowing the treatment container 2 to stand after stirring, the fat content generated by decomposing the adipose tissue A is in the upper layer, the degradation enzyme solution C containing the fat-derived cells B, that is, the cell suspension is in the lower layer, The layers are separated by the difference in specific gravity.

  The liquid feeding path 40 is provided in the vicinity of the tube 10 connected to the centrifuge container 9 attached to the centrifuge 8 from the discharge port 2a opened on the bottom surface of the processing container 2, and in the vicinity of the discharge port 2a of the tube 10. A valve 11 and a liquid feed pump 12 provided in the middle of the tube 10 are provided. When the valve 11 is opened after the layers are separated in the processing container 2 and the liquid feed pump 12 is operated, the cell suspension in the lower layer of the processing container 2 is sent to the centrifuge container 9.

  The cell concentrating unit 30 centrifuges the sent cell suspension to separate the fat-derived cell B pellet and supernatant into layers. And if a pellet is collect | recovered, the fat origin cell B isolate | separated from the fat tissue A can be obtained.

  In this case, in the present embodiment, when the adipose tissue A and the degrading enzyme solution C are agitated in the processing container 2 in the decomposition processing unit 20, the decomposition that tries to maintain stillness or rotation in the processing container 2 is performed. The rotation of the processing container 2 is accelerated and decelerated while the driving force given by the rotation of the processing container 2 around the eccentric axis is always changed within a sufficiently small range with respect to the inertial force of the enzyme solution C.

  That is, the rotation of the decomposing enzyme solution C is disturbed, and the adipose tissue A and the decomposing enzyme solution C are not peeled off from the inner wall of the processing vessel 2 that has been in close contact with each other, or the liquid surface is waved and crushed. Is gently stirred throughout. Thereby, since air bubbles are not mixed in the degradation enzyme solution C, the fat-derived cells B in a healthy state can be obtained without being damaged by exposure of the fat-derived cells B to the air. There is.

Moreover, the time change of the gravity center position of the processing container 2 is measured more correctly by arranging the plurality of load cells 6a, 6b, 6c at intervals on the bottom surface of the base 3 along the circumferential direction. Thereby, there exists an advantage that rotation of the base 3 can be controlled more appropriately with respect to rotation of the decomposing enzyme liquid C.
Further, by keeping the inside of the processing container 2 at about 37 ° C., the activity of the degrading enzyme C can be increased and the adipose tissue A can be efficiently decomposed.

In the above embodiment, the case where the agitator 1 stirs the adipose tissue A and the degrading enzyme solution C has been described as an example, but a solution containing other biological samples, for example, a medium containing cells, blood, protein, etc. It is good also as stirring the solution of these.
Even in such a case, the solution is gently stirred from beginning to end, and bubbles are not mixed in the solution, so that cells, blood cells, proteins, etc. do not come into contact with air. Thereby, it can stir, preventing damage, such as modification | denaturation and deformation | transformation of these biological samples.

The adipose-derived cells B include vascular endothelial cells, fibroblasts, hematopoietic stem cells, blood cells, adipose-derived stem cells, and the like. Here, the stem cell is a cell collected from adipose tissue for the purpose of cell therapy or the like, and refers to a cell having multipotency and self-renewal ability.
The adipose tissue A used may be a human-derived subcutaneous adipose tissue, visceral adipose tissue, white adipose tissue, brown adipose tissue, or the like, and the adipose region of a human or the like is collected with a sharp instrument such as scissors. There may be.

1 Stirrer 2 Processing container (container)
3 base 3a seat heater 3b heat insulating material 4 shaft 5 motor 6a, 6b, 6c load cell (weight measuring section)
DESCRIPTION OF SYMBOLS 7 Control part 8 Centrifuge 9 Centrifugation container 10 Tube 11 Valve 12 Liquid feed pump 20 Decomposition processing part 30 Cell concentration part 40 Liquid feed path 100 Cell separation apparatus A Adipose tissue (biological sample, biological tissue)
B Fat-derived cells (living cells)
C Degradation enzyme solution (treatment solution)

Claims (6)

A base on which a biological sample and a container for storing a treatment liquid for processing the biological sample are placed;
A motor for rotating the base around an eccentric axis along a predetermined circumferential orbit in a horizontal plane while maintaining the posture;
During the rotation of the base, a weight measuring unit that measures the position of the center of gravity in the horizontal direction of the container placed on the base over time;
A stirrer provided with a control part which controls the speed and phase of rotation of the base by the motor based on the time change of the gravity center position measured by the weight measurement part.   The stirrer according to claim 1, wherein the control unit controls the motor so that a phase of rotation of the base advances by a predetermined rotation angle with respect to a phase of temporal change of the gravity center position.   The stirrer according to claim 1 or 2, wherein the control unit controls the motor so that a phase of rotation of the base is delayed by a predetermined rotation angle with respect to a phase of temporal change of the gravity center position.   The stirrer according to any one of claims 1 to 3, wherein a plurality of the weight measuring units are provided at intervals in the circumferential direction. A stirrer according to any one of claims 1 to 4, comprising: a living tissue containing living body-derived cells and a degrading enzyme solution for decomposing the living tissue in the container; A decomposition processing unit that separates the derived cells to generate a cell suspension;
A cell concentrating unit for concentrating the biologically-derived cells by centrifuging the cell suspension generated in the decomposition processing unit;
A cell separation apparatus comprising: a liquid supply path for supplying the cell suspension from the decomposition processing unit to the cell concentration unit. The living tissue is adipose tissue collected from within the living body;
The cell separation device according to claim 5, wherein the living cell is a fat-derived cell encapsulated in the adipose tissue.

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