JP5688001B2 - Centrifuge - Google Patents

Description

  The present invention relates to a centrifugal separator that rotates a container at high speed to precipitate or separate an internal sample, and more particularly, to a centrifugal apparatus capable of introducing a sample while keeping the inside of the container isolated from the outside.

  In recent years, regenerative medicine, in which cells are amplified and cultured outside the body to produce tissues and organs and applied to medicine, has attracted attention. Many of the cells used in regenerative medicine are adherent cells that adhere to a culture vessel and proliferate. In general, when adherent cells are cultured, there is gradually no room for growth in the container, and the cells cannot grow. For this reason, when the cells are filled in the container, an operation of peeling and diluting the cells and reseeding the cell suspension in a new container is required. This operation is called passaging.

  Centrifugation is an apparatus widely used for precipitation and separation, but is mainly used for passage in cell culture. After the cells are peeled off and diluted, the cells and the supernatant are separated using a centrifuge, so that impurities can be removed, enzymes for removing the cells can be removed, and the medium can be exchanged efficiently.

  When centrifuging, it was necessary to open the lid to transfer the cell suspension from the culture vessel to the centrifuge tube. In order to reduce the risk of being contaminated by external bacteria, it is required to sterilize as much as possible when opening the lid. This has led to complicated equipment and increased work time for sterilization. However, if centrifugation can be performed without opening the lid, the apparatus for maintaining the sterilization environment can be simplified, which helps to reduce the cost of the culture apparatus. Based on such a concept, Patent Document 1 discloses a method of performing centrifugation with a closed system. This method has a pipette for supplying and discharging the medium to and from the centrifuge tube, and is equipped with a mechanism for stopping the rotation of the centrifuge tube.

JP 2009-268825 A

  The method disclosed in Patent Document 1 is intended to prevent the liquid feeding tube from twisting even if centrifugation is performed with the liquid feeding tube attached. However, no consideration is given to a method of actively operating the liquid delivery tube.

  In general, in order to efficiently remove the supernatant after centrifugation, it is desirable to insert the pipette into the bottom as much as possible. However, if the depth is too deep, there is a risk that cells will be simultaneously aspirated when the supernatant is aspirated. Conversely, if the pipette is placed away from the bottom of the centrifuge tube, the efficiency of discharging the supernatant liquid will deteriorate.

  An object of the present invention is to provide a centrifugal separation means capable of realizing an efficient centrifugal separation without changing the depth of pipette insertion by constructing a pipette up-and-down movement mechanism on a rotating body and allowing cells to be exposed to the outside air.

  In order to achieve the above object, the main features of the centrifugal separator according to the present invention are as follows.

  In a centrifuge that rotates a container whose interior is isolated from the outside and precipitates or separates the sample liquid stored in the container, the sample liquid attached to the inside of the container is introduced into the container, or A pipette that sucks out the sample liquid to the outside of the container, a flexible tube that is attached to the outside of the container and delivers the sample liquid in the pipette to the outside of the container, and is placed on the container and rotated into a container by rotation. A rotating body that applies force, and a connecting portion that provides a fulcrum during rotation to the container placed on the rotating body, and the connecting section suppresses rotation about the central axis of the container when rotating the container. It comprises rotation suppression means and pipette tip moving means for adjusting the relative position between the inner wall surface of the container and the tip of the pipette.

  In addition, in a centrifuge for rotating or precipitating or separating the sample liquid stored in the container by rotating the container isolated from the outside, the sample liquid attached to the inside of the container is placed inside the container. A pipette that introduces or sucks the sample liquid to the outside of the container, a flexible tube that is attached to the outside of the container and delivers the sample liquid in the pipette to the outside of the container, and a centrifugal force is applied to the container by rotation. A rotating part to be applied, a container supporting part for supporting a container connected to the rotating part by a hinge, a wheel attached to the vicinity of the tip of the container supporting part for serving the rotation of the container, and supporting the wheel and rotating the wheel. A wheel support plate having a track, a connecting portion that gives a fulcrum during rotation to the container, a vertical movement mechanism that moves the container support portion up and down around the hinge by moving the wheel support plate up and down, and a connecting portion In Characterized in that it has a suppressing rotation suppressing means the central axis of rotation of the container when rotating the container.

  According to the present invention, the accessory is not twisted even if it is rotated while the accessory such as a tube or an electric wire is attached to the centrifuge tube. Cell suspension transfer to a centrifuge is possible.

  As a result, an apparatus for sterilizing the external environment of the cell culture apparatus can be simplified, and the culture cost can be reduced.

  In addition, the pipette tip can be brought close to the lowermost part of the centrifuge tube and can be separated from the lower part of the centrifuge tube as much as possible so that the pipette tip does not interfere with cell precipitation during centrifugation.

  In this way, it is easy to construct a culture apparatus that does not require tube cutting or reconnection. Therefore, there is an effect that automation of the cell culture apparatus is easy to realize.

It is a figure which shows one Example of the centrifuge which uses a universal joint. It is a figure which shows one Example of a pipette moving mechanism. It is a figure which shows one Example of the electric power supply method to a pipette moving mechanism. It is a figure which shows one Example of the centrifuge mechanism by a culture container. It is a figure which shows one Example of a culture container. It is a figure which shows another Example of a culture container. It is a block diagram of a wheel. It is a schematic diagram of a hollow disk guide. It is a figure which shows another Example of the centrifuge tube used for a centrifuge mechanism. FIG. 3 is a diagram illustrating an example of a flowchart illustrating the operation of the centrifugal separator 10. FIG. 3 is a diagram illustrating an example of a flowchart illustrating the operation of the centrifugal separator 10.

  In order to realize the purpose of constructing an automatic culture apparatus that has a low risk of contamination and is easy to automate, a centrifugal separator suitable for the purpose was realized.

FIG. 1 shows an embodiment of a centrifugal separator according to the present invention using a universal joint.
Here, 10 is a centrifugal separator, and 11 is a motor that generates rotational motion. Reference numeral 12 denotes a centrifuge tube into which a cell suspension is introduced in the centrifugal separator 10. Reference numeral 13 denotes a pipette, and reference numeral 14 denotes a liquid feeding tube attached to the pipette 13. The liquid feeding tube is a flexible tube made of a flexible material that can be freely bent. The pipette 13 is attached to the centrifuge tube 12, and the cell suspension is introduced or sucked into the centrifuge tube 12 by the pipette 13. A pipette moving mechanism 15 is attached to the centrifuge tube 12. The pipette moving mechanism 15 has a mechanism for changing the relative positions of the centrifuge tube 12 and the pipette 13. Reference numeral 16 denotes an electric cable which supplies the pipette moving mechanism 15 with electric energy necessary for movement.

  In general, when a rotary motion is applied by the motor 11 with the liquid feeding tube 14 attached, the centrifuge tube 12 is pressed against the rotary arm 17 by centrifugal force, and therefore tends to rotate integrally with the rotary arm 17 by frictional force. Accordingly, the centrifuge tube 12 itself rotates, and the liquid feeding tube 14 is likely to be twisted. Therefore, a means for suppressing the rotation of the centrifuge tube 12 is required. Reference numeral 18 denotes a universal joint, and the present invention solves the above problem by the universal joint 18. The universal joint 18 is disposed on the rotating shaft of the motor 11 and is fixed to the casing of the centrifugal separator 10. As a result, the pipette moving mechanism 15 and the centrifuge tube 12 can rotate around the rotation axis of the motor 11, but the rotation axis is constrained. For this reason, the twist of the liquid feeding tube 14 and the electric cable 16 is suppressed.

  Reference numeral 19 denotes a pressure plate, which is provided so that the liquid feeding tube 14 and the electric cable 16 do not get entangled with the universal joint 18 when the rotary arm 17 rotates. For this purpose, as the material of the pressing plate 19, a material that is more difficult to deform than the liquid feeding tube 14, for example, a material having high rigidity is used. Reference numeral 20 denotes a counterweight, which is provided to eliminate the occurrence of vibration during centrifugation.

  As illustrated in the figure, the casing of the centrifugal separator 10 may include a temperature control device such as a heater or a cooler realized by a Peltier element or a heat exchanger. As a result, the cell can be manipulated while maintaining a temperature suitable for the cell.

FIG. 2 is a diagram for explaining an embodiment of the pipette moving mechanism.
A centrifuge tube 21 is screwed or frictionally coupled to the centrifuge tube 12. A sliding seal 22 is provided at the center of the centrifuge tube lid 21, and the pipette 13 penetrates the sliding seal 22. The pipette 13 can move up and down in the centrifuge tube 12 while contacting the sliding seal 22. The cell suspension inside the centrifuge tube 12 is sealed by the sliding seal 22 and does not leak out.

  The centrifuge tube lid 21 desirably has a gas permeable membrane in part. This allows air having the same volume as the liquid volume to be released to the outside of the centrifuge tube 12 when the cell suspension is introduced, and the pressure in the centrifuge tube 12 can be kept substantially constant. In this figure, the gas permeable membrane is not shown for simplicity.

  Reference numeral 23 denotes a bellows, which is formed of, for example, a fluororesin or silicon. The bottom of the bellows 23 is coupled to the centrifuge tube lid 21, and the top of the bellows 23 is coupled to a bellows stopper 24 provided on the pipette 13, and a portion of the pipette 13 inside the bellows 23 is exposed to the outside. There is nothing to do. The centrifuge tube 12, the pipette 13, the liquid feeding tube 14, the centrifuge tube lid 21, the sliding seal 22 and the bellows 23 are assembled and sterilized by γ-rays or ultraviolet rays, and assembled into the pipette moving mechanism 15 by the centrifuge user. It is done.

  25 is a solenoid, and 26 is a pipette vertical movement link, both of which are arranged in the pipette moving mechanism 15. The solenoid 25 is supplied with electric power by an electric cable 16 (not shown here) and operates the pipette vertical movement link 26. The pipette vertical movement link 25 is fitted into the protrusion 27 of the pipette 13 and transmits the operation of the solenoid 25 to the pipette 13.

  FIG. 3 shows another embodiment of a method for supplying power to the pipette moving mechanism 15. Here, reference numeral 30 denotes a printed wiring provided on the rotary arm 17. 31 is a slip ring, 32 is a brush. The printed wiring 30 is electrically coupled to the slip ring 32, and the slip ring 31 rotates while being in contact with the brush 32. The brush 32 is electrically coupled to the electric cable 16. Thereby, the electric energy supplied by the electric cable 16 is supplied to the pipette moving mechanism 15 via the brush 32, the slip ring 31, and the printed wiring 30. With such a configuration, the electric cable 16 does not move violently due to the rotation of the rotating arm 17, and as a result, there is an effect of extending the life of the centrifuge.

  FIG. 4 shows another embodiment of a centrifugal mechanism using a culture vessel. Reference numeral 41 denotes a culture container. In this embodiment, the culture container 41 is rotated to eliminate the liquid feeding step from the culture container 41 to the centrifuge tube 12. In addition, illustration of the counterweight was abbreviate | omitted in this figure.

  Reference numeral 42 denotes a container support arm, which is attached to the rotary arm 17. The angle formed between the container support arm 42 and the rotary arm 17 is coupled by a hinge or the like so that the angle is variable. The culture container 41 is fixed on the container support arm 42. Reference numeral 43 denotes a wheel, which is a wheel-like mechanism attached to the outer periphery of the culture vessel 41 and the vessel support arm 42. The configuration of the wheel 43 will be described later. 44 is a hollow disk guide, and the wheel 43 moves on this while contacting. This allows the culture vessel to revolve around the rotation axis of the motor 11.

  45 is an actuator that moves the hollow disk guide 44 up and down, 46 is an encoder, and 47 is a rotation stop position control means. When the rotation direction when the pipette 13 is viewed as a rotation axis is defined as a roll angle, and the relative rotation direction between the rotation arm 17 and the container support arm 42 is defined as a pitch angle, in the configuration of FIG. 41 roll angle changes. In order to make the culture surface of the culture vessel 41 horizontal, it is necessary to grasp the rotation angle of the motor 11 and set it to a predetermined position during culture. The rotation stop position control means 47 always sets the stop position of the motor to the same place, sets the roll angle to 0, and stops the culture vessel. The rotation stop position control means 47 acquires the signal of the encoder 46, grasps the rotation angle of the motor 11, and controls the position of the motor 11. Thereby, it becomes possible to centrifuge in the culture vessel. As the encoder 46, an absolute type capable of measuring an absolute angle, or one capable of generating a reference position signal (Z signal) at least once per rotation is used.

  When the actuator 45 is raised, the culture vessel 41 is arranged in the horizontal direction and is a suitable position for cell culture. On the other hand, when the actuator 45 is lowered, the culture vessel 41 rotates in the pitch direction, and the pipette tip approaches the deepest part of the culture vessel 41. In the present embodiment, the position of the tip of the pipette 13 is moved by inclining the culture vessel 41 by the actuator as described above.

  In addition, as shown in FIG. 1, the casing of the centrifugal separator may include a temperature control device (not shown) such as a heater or a cooler realized by a Peltier element or a heat exchanger. As a result, the cell can be manipulated while maintaining a temperature suitable for the cell.

  FIG. 5 is a diagram showing the shape of the culture vessel 41. FIG. 5A is a diagram showing the overall shape, FIG. 5B is a diagram showing a state during aspiration, and FIG. 5C is a diagram showing a state during stationary culture. The shape of the culture vessel 41 is such that the height of the tip of the pipette 13 from the culture vessel 41 changes depending on the pitch angle. 51 is a flat shape to which adherent cells adhere, and 52 is a medium recovery site. Assuming that the distance between the tip of the pipette 13 at the time of suction and the bottom of the container is h1, and the distance between the tip of the pipette 13 and the bottom of the container at stationary culture is h2, the culture container 41 tilts to increase the size of h1 and h2. It changes. As a result, the tip of the pipette 13 can be brought closer to the bottom of the container at the time of aspiration, and there is an effect that it is possible to reduce the unsucked supernatant.

  In addition, the culture container 41 shown to Fig.5 (a) has two lids. One is provided for gas transmission and reception, and the other is for liquid transmission and reception. This makes it possible to adjust the air and medium necessary for the culture according to the growth of the cells. As another example, the culture vessel 41 may have a structure with an air permeable membrane that can exchange gas with the outside by using one lid.

  FIG. 6 is a view showing another form of the culture vessel 41. FIG. 6A is a diagram showing a state at the time of aspiration, and FIG. 6B is a diagram showing a state at the time of centrifugation and stationary culture.

  The pipette 13 is arranged at an angle with the flat shape 51. 52 is a medium collection site, and 53 is a cell accumulation site during centrifugation. In the state of FIG. 6A, since the gravity works in the vertical direction when not rotating, the culture medium collects at the culture medium recovery site 52. On the other hand, the state shown in FIG. At this time, the cells receive the resultant force of gravity and centrifugal force and gather at the cell accumulation portion 53 during centrifugation. Therefore, according to this configuration, since the tip of the pipette 13 is arranged at a location different from the cell accumulation location when the supernatant is aspirated, the number of cells sucked when the supernatant is aspirated is small, which is convenient in terms of culture efficiency. In the cell culture, the state shown in FIG. 6B is used, and the cell culture is performed with adherent cells attached to the flat portion.

  FIG. 7 is a configuration diagram of the wheel 43. 61 is an inner ring that holds the culture vessel 41 and the container support arm 42, and 62 is an outer ring that rotates while contacting the hollow disk guide 44. A bearing 63 is disposed between the inner ring 61 and the outer ring 62. Thereby, the inner ring 61 and the outer ring 62 can rotate with each other.

  FIG. 8 is an overall view of the hollow disk guide 44 portion. The hollow disc guide 44 has a disc shape in which a central portion where the motor 11 is disposed is hollowed out, and moves concentrically on the hollow disc guide 44 while the wheel 43 contacts and rotates. An actuator 45 is attached to the hollow disk guide 44 and moves the hollow disk guide 44 up and down while maintaining the horizontal surface of the hollow disk guide 44. As a result, a pitch angle can be generated in the container support arm 42, and the pitch angle can be controlled in each step of culture, centrifugation, and suction.

  FIG. 9 shows another embodiment of a centrifuge tube used for the centrifugal mechanism. Here, 71 is a pipette for cell suspension, and 72 is a pipette for supernatant. The cell suspension pipette 71 and the supernatant pipette 72 are fixed to the centrifuge tube lid 21 so that the pipette tip depth is different. The cell suspension pipette 71 is a pipette used for pipetting or extracting all cells, and the supernatant pipette 72 is a pipette used for extracting the supernatant. By arranging them at different depths in this way, it is possible to reduce the risk of removing cells when removing the supernatant, and to remove the cells without loss, and the effect intended by the present invention can be expected. .

  FIG. 10 is a flowchart showing the operation of the centrifugal separator 10 in FIG. When introducing the cell suspension into the centrifuge 10 for the first time, the pipette is raised for the purpose of suppressing the generation of bubbles. When the cell suspension is introduced while the pipette is lowered, if air is mixed in the tube, the air in the tube may enter the cell suspension as bubbles. In general, it is said that bubbles are physically damaged when the bubbles are repelled, and the present invention takes the adverse effects into consideration.

  When sucking out the supernatant, lower the pipette as much as possible so as not to suck out the cells. If the cell concentration is known, the amount of precipitation can also be estimated, so the amount of decrease can be set in advance. Of course, there may be a configuration in which the cell concentration is received from a host controller (not shown) and the descending amount is sequentially calculated.

  In addition, “pipetting” in the figure refers to an operation of making a cell suspension by repeatedly sucking and discharging a small amount of liquid. By this sequence, dead cells and debris (debris) can be removed, the cell suspension can be concentrated, and the cells can be washed.

  FIG. 11 is a flowchart showing the operation of the centrifugal separator 10 in FIG. In FIG. 4, culture and centrifugation are performed in the same container. After culturing, washing with PBS (physiological saline), and peeling off with trypsin (one of digestive enzymes) to prepare a cell suspension, the sequence is the same as in FIG.

Below, the technical thought which concerns on the centrifuge other than the claim which can be grasped | ascertained from the said embodiment of this invention is described.
(1) In a centrifuge that rotates a container whose inside is isolated from the outside and precipitates or separates the sample liquid stored in the container,
A pipette attached inside the container for introducing the sample liquid into the container, or sucking the sample liquid out of the container;
A flexible tube attached to the outside of the container and delivering the sample liquid in the pipette to the outside of the container;
A rotating unit that applies centrifugal force to the container by rotation;
A container support for supporting the container connected to the rotating part by a hinge;
A roller wheel attached to the vicinity of the tip of the container support portion for rotation of the container;
A wheel support plate that supports the wheel and includes a rotating track of the wheel;
A connecting portion for providing a fulcrum during rotation to the container;
A vertical movement mechanism that moves the container support portion up and down around the hinge by moving the roller support plate up and down;
The connection portion has a rotation suppression means for suppressing rotation around the central axis of the container when rotating the container,
The pipette has a first angle formed between one end of the bottom surface of the container and one side surface contacting the one end, and a second angle formed by the other side surface contacting the other end of the bottom surface and the other end. The centrifuge is characterized in that the tip of the centrifuge is disposed on the bottom surface on the second angle side.
(2) The centrifugal separator according to (1), wherein at least two types of pipettes having different distances between the bottom of the container and the tip of the pipette are mounted inside the container.
(3) In the above (2), the two kinds of pipettes are selectively used according to the object to be sucked out.

10 ... centrifuge,
11 ... motor,
12 ... centrifuge tube,
13 ... Pipette,
14 ... liquid feeding tube,
15 ... Pipette moving mechanism,
16 ... electric cable,
17 ... Rotating arm,
18 ... Universal joint,
19 ... Presser plate,
20 ... counterweight,
21 ... centrifuge tube lid,
22 ... sliding seal,
23 ... Bellows,
24 ... Stop bellows,
25 ... Solenoid,
26 ... Pipette up / down link,
27 ... protruding part,
30 ... printed wiring,
31 ... Slip ring,
32 ... Brush,
41 ... culture container,
42 ... container support arm,
43 ... wheels,
44 ... Hollow disk guide,
45 ... Actuator,
46. Encoder,
47. Rotation stop position control means,
51. Flat shape,
52 ... Medium recovery site,
53 ... Cell accumulation site during centrifugation,
61 ... Inner ring,
62 ... Outer ring,
63 ... Bearing,
71 ... Pipette for cell suspension,
72 ... Supernatant pipette.

Claims (8)

In a centrifuge that rotates a container whose inside is isolated from the outside and precipitates or separates the sample liquid stored in the container,
Mounted within said container, a pipette suction introducing the sample liquid into the interior of said container, or a liquid for the sample to the outside of the container,
A flexible tube connected to the pipette, supplying the sample liquid to the pipette, or delivering the sample liquid to the outside of the pipette;
A rotating body for revolving the container with respect to a first rotation axis in order to place the container and apply centrifugal force to the container by rotation;
Pipette tip moving means for adjusting the relative position between the inner wall surface of the container and the tip of the pipette;
Some a communication contact has been the pipette tip moving hand stage of the container, connected to the casing of the centrifugal separator, have a connecting portion to provide a fulcrum at the time of rotation to the container,
The connecting portion,
Said first rotary shaft which is mounted in front Symbol casing,
Mounted in front Symbol pipette tip moving means, a second rotary shaft which is rotating axis of the vessel their respective a junction joint can be,
In a direction opposite to the rotation direction of the container so as to return the twist of the flexible tube due to the rotation of the container, which occurs when the rotating body revolves the container with respect to the first rotation axis. A centrifugal separator having a rotation suppression function of rotating the second rotation shaft . The centrifuge according to claim 1,
The centrifuge is characterized in that the connecting part is constituted by a universal joint. The centrifuge according to claim 1,
The container includes a portion having a flat shape for attaching cells to be cultured when culturing cells separated from the sample liquid, and the sample liquid when aspirating the sample liquid. And a culture medium recovery site for recovery. The centrifuge according to claim 3 ,
The medium collecting part is a corner part formed by one end of the side surface of the container constituting the part having the flat shape and one end of the bottom surface of the container in contact with the one end. . The centrifuge according to claim 1,
The pipette tip moving means is supplied with electric energy required for the vertical movement of the pipette tip by means of electrical contact with an electric cable through a slip ring attached to the rotating body. Separation device. The centrifuge according to claim 1,
A centrifugal separator, comprising: a member attached to the casing so as to partition the first rotating shaft of the connecting portion and the flexible tube and having rigidity higher than that of the flexible tube. The centrifuge according to claim 1,
An encoder that detects a rotational position of a motor that supplies rotational force to the rotating body, and a rotation stop position control means that controls a rotation stop position of the rotating body;
The rotation stop position control means controls the rotation stop position of the container so that the output of the encoder becomes a predetermined stationary position. The centrifuge according to claim 1,
A centrifugal separator further comprising temperature control means for holding at least the container at a predetermined temperature.

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