JP2010115142A - Container for centrifugal separation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable washing a separated cell layer sufficiently by a simple constitution and efficiently recovering cells by making the cell layer hardly attachable to an inner surface. <P>SOLUTION: The centrifugal separation container 1 storing cell suspension and separating the cell suspension into a cell layer B and supernatant C by effecting a centrifugal force includes: a cylindrical container body 2 of which one end in its axial direction is closed, having an opening at another end; a lid body 3 for closing the opening 2a of the container body 2; and a feeding/discharging pipe 4c extending from the lid body 3 toward the closed end 2e of the container body 2, feeding cleaning liquid from its tip end opening 4f arranged in the upper direction of a boundary surface D of the cell layer B and supernatant C to the inside of the container body 2 and on the other hand sucking the supernatant C from the tip end opening 4f; wherein the centrifugal separation container 1 is formed with the tip end opening 4f so as to eject the cleaning liquid in the direction facing the closed end 2e side by inclining against the axial line of the container body 2. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a centrifuge container.

Conventionally, as a container for washing cells contained in a cell suspension, it has a substantially cylindrical side wall and a conical bottom wall, and the apex angle of the conical bottom wall is 50 ° to 90 °. An elongated centrifuge tube is known (for example, refer to Patent Document 1).
According to this elongate centrifuge tube, by storing and centrifuging the cell suspension, a cell having a large specific gravity moves toward the bottom wall by the action of centrifugal force, and near the center along the conical bottom wall. To be collected. As a result, the cell suspension can be separated into a cell layer collected at the center of the conical bottom wall and a supernatant above the cell layer.

Japanese Patent No. 3615226

  However, in a conventional centrifuge container as shown in Patent Document 1, the supernatant and the cell layer are separated by centrifugation, the separated supernatant is aspirated and discarded, and instead the washing solution is supplied to remove the cell layer. The cells are washed by repeating the agitation work, but simply supplying the washing solution into the centrifuge container cannot sufficiently agitate the cell layer, and the degrading enzyme remains in the cell layer. Or the cell layer may adhere to the centrifuge container.

  The present invention has been made in view of the above-described circumstances, and it is possible to recover cells efficiently by sufficiently washing a cell layer separated with a simple configuration and making it difficult to adhere to the inner surface. An object of the present invention is to provide a centrifuge container.

In order to achieve the above object, the present invention provides the following means.
The present invention is a centrifuge container for storing a cell suspension and separating the cell suspension into a cell layer and a supernatant by applying centrifugal force, wherein one end in an axial direction is closed and the other end A cylindrical container body having an opening in the container, a lid body for closing the opening of the container body, and extending from the lid body toward the closed end of the container body, and a boundary surface between the cell layer and the supernatant. A cleaning liquid is supplied into the container main body from the front end opening disposed above, and a supply / discharge tube for sucking the supernatant from the front end opening is provided, and the front end opening is inclined with respect to the axis of the container main body. And a centrifuge container configured to discharge the cleaning liquid in a direction toward the closed end.

  According to the present invention, the cell suspension is applied to the cell layer and the supernatant by applying a centrifugal force in a state where the cell suspension is stored in the container body sealed by closing the opening. To be separated. In this state, the supernatant is sucked from the tip opening of the supply / exhaust tube extending from the lid toward the closed end, the washing solution is supplied to the cell layer remaining at the closed end, stirred, and centrifuged. By repeating, the cells can be washed.

  In this case, since the tip opening discharges the cleaning liquid obliquely downward, a flow that causes a vortex is formed in the vicinity of the closed end where the cell layer is formed, and the cell layer is efficiently stirred. As a result, the cell layer is sufficiently washed, or the cell layer attached to the inner surface of the container body is sufficiently peeled off. Accordingly, it is possible to sufficiently dilute the degrading enzyme or the like adhering to the cells, and to prevent the inconvenience that the cells adhere and remain in the container body.

In the said invention, it is preferable that the said front-end | tip opening is provided with two or more at intervals in the circumferential direction.
By doing so, the flow of the cleaning liquid is formed in a plurality of directions, and a more turbulent flow is formed, so that the stirring efficiency of the cell layer can be improved.

In the above invention, the container body has a tapered inner surface that gradually tapers toward the closed end, and the tip opening is at a shallower angle than 90 ° in the direction toward the closed end with respect to the tapered inner surface. It is preferable that the cleaning liquid is provided so as to enter.
By doing so, the cleaning liquid discharged from the tip opening of the supply / exhaust pipe enters the tapered inner surface at an angle shallower than 90 °, thereby suppressing branching occurring at the incident position and increasing the flow momentum. Can be directed to the cell layer near the closed end. Thereby, the cell layer can be more efficiently stirred and washed.

Moreover, in the said invention, the said front-end | tip opening may be provided so that a washing | cleaning liquid may inject along the eccentric direction with respect to the said taper inner surface.
By doing so, the cleaning liquid discharged from the opening at the end of the supply / discharge pipe enters the tapered inner surface, thereby forming a flow having a tangential component of the tapered inner surface, and a spiral flow is formed along the circumferential direction of the tapered inner surface. The Thereby, the cell layer can be more efficiently stirred and washed.

Moreover, in the said invention, you may provide the cell suction port which is arrange | positioned in the obstruction | occlusion end vicinity and aspirates a cell layer.
By doing in this way, by sucking from the cell suction port, it is possible to suck and take out without leaving the cell layer arranged near the closed end. At the time of aspiration, a resuspended cell suspension may be aspirated by supplying physiological saline or the like.

  According to the present invention, the cell layer separated with a simple structure is sufficiently washed, and it is difficult to adhere to the inner surface, so that the cells can be efficiently recovered.

A centrifuge container 1 according to an embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the centrifuge container 1 according to the present embodiment includes a container body 2, a lid body 3 that closes the upper opening 2 a of the container body 2, and a supply / discharge unit provided in the lid body 3. Means 4 and a filter 5 provided on the lid 3 are provided.

  As shown in FIG. 2, the container body 2 has a substantially cylindrical inner surface 2b extending from the upper opening 2a, and is smoothly connected to the cylindrical inner surface 2b and gradually tapers at a predetermined taper angle toward the tip. And a tapered inner surface 2c with the tip closed. The cylindrical inner surface 2b is formed to be slightly tapered toward the tip. A flange 2d is provided on the outer side in the radial direction of the upper opening 2a.

  The lid 3 is fitted into the upper opening 2 a of the container body 2, and the upper opening 2 a is sealed by the O-ring 6. Further, the lid 3 has an engagement protrusion 3a that engages with the flange 2d of the container body 2 and keeps the lid 3 fitted in the upper opening 2a of the container body 2 in the circumferential direction. A plurality are provided with a gap.

  The supply / discharge means 4 is formed in a double tubular shape penetrating the center of the lid 3 in the thickness direction, and the distal end of the supply / discharge means 4 is fitted in a position closing the upper opening 2 a of the container body 2. The first pipe line 4b in which the opening (cell suction port) 4a is disposed in the vicinity of the closed end 2e at the tip of the tapered inner surface 2c, and the second pipe line (supply / discharge) that is concentrically arranged radially outward. Tube) 4c.

  In the first pipe line 4b, a port 4d to which a suction means such as a syringe can be connected is provided at an end portion arranged outside the lid body 3, for example. In the figure, the port 4d is closed by a cap 4e. By connecting a syringe to the port 4d and sucking it, the entire contents can be sucked from the vicinity of the closed end 2e of the container body 2.

The second conduit 4c is provided with a through hole 4f that is closed at the tip of the second conduit 4c and that opens in a direction inclined toward the closed end 2e while being inclined with respect to the axis. ing. The through hole 4f is formed so that the cleaning liquid is incident on the tapered inner surface 2c at an angle shallower than 90 ° toward the closed end. An external pipe (not shown) is connected to the outside of the lid 3 in the cylindrical gap between the second pipe 4c and the first pipe 4b.
Further, as shown in FIG. 3, the through holes 4 f are provided in a plurality of places, for example, four places radially at intervals in the circumferential direction.

  Thereby, the cell suspension A is introduced into the container body 2 from the through hole 4f through the cylindrical space between the first pipe 4b and the second pipe 4c, or the container body 2 The supernatant C is sucked from the through hole 4f, or a cleaning solution such as physiological saline is supplied and discharged from the through hole 4f. The supply / discharge means 4 formed in a double tubular shape is supported by a cylindrical portion 3 b extending from the lid 3 in the axial direction. Thereby, even if a centrifugal force acts on the centrifuge container 1, the position of the supply / discharge means 4 is held so as not to fluctuate, and the opening 4a and the through hole at the tips of the first pipe 4b and the second pipe 4c are retained. 4f is maintained in the positioning state.

  The filter 5 serves to block dust, bacteria, and the like contained in the outside air G from entering the inside of the container body 2 when the outside air G from the outside is introduced into the container body 2. By introducing the outside air G through the filter 5, it is possible to prevent the space inside the container body 2 from becoming negative pressure when the supernatant C, cells, etc. are sucked, and the suction operation can be performed smoothly. ing.

The operation of the centrifuge container 1 according to this embodiment configured as described above will be described below.
In order to separate and recover cells from the cell suspension A using the centrifuge container 1 according to the present embodiment, the first opening 2a of the container body 2 is sealed via the external pipe while the upper opening 2a of the container body 2 is sealed by the lid 3. The cell suspension A is introduced into the container body 2 from the cylindrical space between the pipe 4b and the second pipe 4c.

The cell suspension A is obtained by, for example, decomposing a living tissue such as adipose tissue with a digestive fluid and suspending isolated cells in the digestive fluid.
In this way, as shown in FIG. 4, the closed end 2 e of the container body 2 is radially outward by a centrifuge not shown in a state where a predetermined amount of the cell suspension A is stored in the container body 2. Is rotated so that a centrifugal force acts on the cell suspension A in the container body 2, and cells having a large specific gravity move toward the closed end 2e.

In the middle of movement, the cells are collected by rolling on the tapered inner surface 2c, and are deposited on the closed end 2e. When the centrifugation proceeds sufficiently, as shown in FIG. 5, the cell layer B and the supernatant C are clearly separated, and a boundary surface D is formed between them.
In the present embodiment, a through hole 4f provided in the second pipe 4c opens in the supernatant C above the boundary surface D between the cell layer B and the supernatant C.

  In this state, when the cylindrical space between the first pipe 4b and the second pipe 4c is sucked to a negative pressure, as shown in FIG. Clear C is aspirated and aspirated until some supernatant C remains above the cell layer B as shown in FIG.

Next, a cleaning solution F such as physiological saline is supplied to the cylindrical space between the first pipe 4b and the second pipe 4c, and the cell layer B and some supernatant C remaining in the container body 2 are supplied. Resuspend by mixing.
In this case, according to the centrifuge container 1 according to the present embodiment, the through hole 4f is inclined with respect to the axis and opens in the direction toward the closed end 2e, and thus is discharged from the through hole 4f. As shown in FIG. 8, the cleaning liquid F enters the tapered inner surface 2c at an angle shallower than 90 °. Thus, most of the fluid flows in the same direction toward the closed end 2e without being dispersed in various directions when colliding with the tapered inner surface 2c.

  Then, the cleaning liquid F that has flowed in the direction toward the closed end 2e by the tapered inner surface 2c is caused to flow in a vortex in the vicinity of the closed end 2e, and the cell layer B deposited in the vicinity thereof is stirred. It becomes like this. That is, in the state where the closed end 2e is arranged vertically downward, the cleaning liquid F is discharged obliquely downward, the direction of flow is changed by the tapered inner surface 2c, and flows while forming a vortex that entrains the cell layer B. The cell layer B can be washed with sufficient agitation.

  By repeating centrifugation, aspiration of supernatant C, and supply / resuspension of washing solution F one or more times, the digestive fluid adhering to the cells is washed, and the concentration of digestive enzyme in cell suspension A is reduced. can do. Further, adipose tissue debris mixed in the cell layer B can be expelled to the supernatant C side and efficiently removed.

  Then, in a state where the concentration of the digestive enzyme is sufficiently reduced, a small amount of physiological saline F is supplied to the separated cell layer B and some of the supernatant C to resuspend, thereby resuspension. A 'is prepared. At this time, the cell layer B adhering to the tapered inner surface 2c in the vicinity of the closed end 2e can be completely separated by forming a turbulent flow due to the cleaning liquid again in the vicinity of the cell layer B.

  In this state, as shown in FIG. 9, suction is performed by a syringe (not shown) attached to the end of the first pipe 4 b outside the container body 2. Thereby, the obtained resuspension A ′ can be sucked into the syringe through the first pipe 4 b without leaving it in the container body 2.

  Moreover, according to the centrifuge container 1 according to the present embodiment, the first cylindrical inner surface 2b and the second cylindrical inner surface 2d of the container main body 2 both have an inclination that is slightly tapered toward the tip. Therefore, this can be used as a draft and easily manufactured by a method such as resin injection molding.

  In the present embodiment, the four through holes 4f are provided at the distal end of the second pipe line at intervals in the circumferential direction. However, instead of this, one through hole 4f may be provided. You may decide to provide the above arbitrary through-holes 4f. Further, a plurality of rows of through holes 4f may be provided in the axial direction.

  Further, in the present embodiment, the through holes 4f are provided radially, but instead, as shown in FIG. 10, each through hole 4f is located with respect to the central axis of the second conduit 4c. It may be arranged eccentrically. By doing in this way, since the cleaning liquid F discharged from each through hole 4f does not enter the taper inner surface 2c perpendicularly, the flow after colliding with the taper inner surface 2c flows along the circumferential direction of the taper inner surface 2c. It is possible to have the component of. As a result, a spiral flow along the circumferential direction of the tapered inner surface 2c is formed in the vicinity of the closed end 2e, and the cell layer B can be more efficiently stirred.

  Further, as shown in FIG. 11, the first and second pipes 4b and 4c themselves can be connected without decentering the through hole 4f with respect to the central axis of the second pipe 4c as shown in FIG. The container body 2 may be eccentric. By doing in this way, the spiral flow along the circumferential direction of the taper inner surface 2c can be formed by the cleaning liquid F discharged from the through hole 4f extending in the radial direction from the center of the second conduit 4c.

It is a longitudinal section showing a centrifuge container concerning one embodiment of the present invention. It is a longitudinal cross-sectional view which shows the container main body of the centrifuge container of FIG. It is a cross-sectional view of the through-hole part of the supply / discharge means of the centrifugal container of FIG. It is a longitudinal cross-sectional view which shows the state which stored the cell suspension in the centrifuge container of FIG. It is a longitudinal cross-sectional view which shows the state isolate | separated into the cell layer and the supernatant liquid by making centrifugal force act on the centrifuge container of FIG. FIG. 6 is a longitudinal sectional view showing a step of sucking and discharging the separated supernatant in the centrifuge container of FIG. 5. FIG. 7 is a longitudinal sectional view showing a state in which the supernatant is sucked and discharged in the centrifuge container of FIG. 6. FIG. 8 is an enlarged longitudinal sectional view showing a state in which the cleaning liquid is discharged from the through hole of the second pipe line in the centrifuge container of FIG. FIG. 9 is a longitudinal sectional view showing a step of sucking and collecting the resuspended cell suspension in the centrifuge container of FIG. 8. It is a cross-sectional view which shows the modification of the centrifuge container of FIG. It is a cross-sectional view which shows the other modification of the centrifuge container of FIG.

Explanation of symbols

A Cell suspension B Cell layer C Supernatant D Interface F Washing liquid 1 Centrifugal container 2 Container body 2a Opening 2b First cylindrical inner surface 2c Tapered inner surface (first tapered inner surface)
2e Closed end 3 Lid 4a Opening (cell suction port)
4c Supply / discharge pipe (second pipe)
4f Through hole (tip opening)

Claims (5)

A centrifuge container that stores a cell suspension and separates the cell suspension into a cell layer and a supernatant by applying centrifugal force,
A cylindrical container body having one end closed in the axial direction and having an opening at the other end;
A lid for closing the opening of the container body;
While supplying the cleaning liquid into the container main body from the front end opening extending from the lid body toward the closed end of the container main body and disposed above the boundary surface between the cell layer and the supernatant, A supply and discharge tube for sucking the supernatant,
A centrifuge container in which the tip opening is formed so as to incline with respect to the axis of the container body and discharge the cleaning liquid in a direction toward the closed end.   The centrifuge container according to claim 1, wherein a plurality of the tip openings are provided at intervals in the circumferential direction. The container body has a tapered inner surface that tapers gradually toward its closed end;
The centrifuge container according to claim 1, wherein the tip opening is provided so that the cleaning liquid is incident at an angle shallower than 90 ° in a direction toward the closed end with respect to the tapered inner surface.   The centrifuge container according to any one of claims 1 to 3, wherein the tip opening is provided so that a cleaning liquid is incident along an eccentric direction with respect to the tapered inner surface.   The centrifuge container according to any one of claims 1 to 4, further comprising a cell suction port that is disposed near the closed end and sucks the cell layer.

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