JP5078020B2 - Centrifuge container - Google Patents

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 the conventional centrifuge container as shown in Patent Document 1, since the apex angle of the conical bottom wall is relatively wide as 50 ° to 90 °, the cell layer accumulated in the center of the bottom wall is directed upward. It is formed into a conical shape that expands, and the interface with the supernatant becomes wider as the amount increases. For this reason, if the supernatant is aspirated from the centrifuge container after separation, some of the cells in the cell layer are likely to rise into the supernatant from the interface due to the flow of the supernatant accompanying the aspiration, and the cells are aspirated and discharged together with the supernatant. There is an inconvenience. As a result, there is a problem that the yield of cells is lowered and the number of cells finally collected is reduced.

  The present invention has been made in view of the above-described circumstances, and provides a centrifuge container capable of suppressing cell rising from a cell layer even by aspiration of a supernatant and improving cell yield. It is an object.

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 a centrifugal force, the first cylinder having a substantially cylindrical shape An inner surface, a tapered inner surface disposed at a tip of the inner surface of the first cylinder and gradually tapering toward the tip; a substantially cylindrical shape disposed at a tip of the tapered inner surface and closed at the tip; A second cylindrical inner surface having a length dimension that forms a boundary surface between the layer and the supernatant at an intermediate position in the axial direction, and a supernatant suction port for sucking the supernatant above the boundary surface A centrifuge container is provided.

  According to the present invention, when a cell suspension is stored and centrifugal force is applied, cells having a large specific gravity move toward the tip, and are collected at the center by the taper inner surface and arranged at the tip of the taper inner surface. Deposited on the inner surface portion of the second cylinder. As a result, the cell suspension forms a boundary surface in the middle of the second cylindrical inner surface in the axial direction and is separated into the cell layer and the supernatant above it.

In this case, the second cylindrical inner surface has a substantially cylindrical shape with a sufficiently smaller diameter than the first cylindrical inner surface by the tapered inner surface tapered from the first cylindrical inner surface. The area can be made sufficiently small. Moreover, since the second cylindrical inner surface has a substantially cylindrical shape, even if the number of cells varies, variation in the area of the boundary surface to be formed can be suppressed to a small value. As a result, even if the supernatant is aspirated in the vicinity of the boundary surface, it becomes difficult for cells to rise from the boundary surface, and it is possible to prevent inconvenience that the cells are discharged together with the supernatant. In addition, by providing a supernatant suction port for aspirating the supernatant above the boundary surface, the supernatant and the cell layer are separated by centrifugation, and then the supernatant is aspirated from the supernatant suction port. As a result, the supernatant can be removed by suction while suppressing the cell from rising from the boundary surface.

In the above invention, the first cylindrical inner surface and the second cylindrical inner surface may have an inclination angle so as to be gradually tapered toward the tip.
By doing in this way, draft angle can be formed in the 1st cylindrical inner surface and the 2nd cylindrical inner surface, and it can make it easy to manufacture by molding.

The present invention also provides a centrifuge container for storing a cell suspension and separating the cell suspension into a cell layer and a supernatant by applying a centrifugal force, and has a substantially cylindrical inner surface. And a first taper inner surface which is disposed at the tip of the inner surface of the cylinder and gradually tapers toward the tip; and a taper which is disposed at the tip of the first taper inner surface and gradually tapers toward the closed tip. A second tapered inner surface having a taper angle smaller than that of the first tapered inner surface and having a length dimension that forms a boundary surface between the cell layer and the supernatant at an intermediate position in the axial direction ; Provided is a centrifuge container provided with a supernatant suction port for aspirating the supernatant above the boundary surface .

According to the present invention, since the second tapered inner surface disposed at the tip of the first tapered inner surface has a smaller taper angle than the first tapered inner surface, the area of the boundary surface to be formed can be made sufficiently small. In addition, even if the number of cells varies, variation in the area of the boundary surface to be formed can be kept small. As a result, even if the supernatant is aspirated in the vicinity of the boundary surface, it becomes difficult for cells to rise from the boundary surface, and it is possible to prevent inconvenience that the cells are discharged together with the supernatant. In addition, by providing a supernatant suction port for aspirating the supernatant above the boundary surface, the supernatant and the cell layer are separated by centrifugation, and then the supernatant is aspirated from the supernatant suction port. As a result, the supernatant can be removed by suction while suppressing the cell from rising from the boundary surface.

In the above invention, the taper angle of the first tapered inner surface may be 25 ° to 45 °, and the taper angle of the second tapered inner surface may be 5 ° to 25 °.
In this way, by applying a centrifugal force, cells can be easily collected along the inner surface of the first taper, and an increase in the area of the boundary surface is suppressed, and the yield of the cells is increased. Can be made.

Moreover, in the said invention, the said supernatant suction port may open in the radial direction.
By doing in this way, since a supernatant is attracted | sucked to radial direction, the influence of the suction exerted on the boundary surface distribute | arranged to the downward direction can be suppressed.

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, there is an effect that the yield of cells can be improved by suppressing the rising of cells from the cell layer even by aspiration of the supernatant.

The centrifugation container 1 which concerns on one Embodiment of this invention is demonstrated 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 2 are provided.

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

  The axial length of the second cylindrical inner surface 2d is such that when the cell suspension A is separated into the cell layer B and the supernatant C, the boundary surface D between the cell layer B and the supernatant C is the second cylinder. It is set to the length dimension formed in the middle position of the inner surface 2d in the axial direction.

  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 is provided with engagement protrusions 3a that engage with the flange 2e of the container body 2 and maintain the lid 3 in a fitted state with 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 opening 4a includes a first conduit 4b disposed in the vicinity of the closed end 2f at the tip of the second cylindrical inner surface 2d, and a second conduit 4c disposed concentrically outward in the radial direction. Yes.
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, all the contents can be sucked from the vicinity of the closed end 2f of the container body 2.

  The second pipe 4c has a gap between the first pipe 4b at the tip thereof and a radial direction at a height position near the boundary between the tapered inner surface 2c and the second cylindrical inner surface 2d. A through-hole 4f that penetrates the container body 2 opens into the space inside the container body 2. An external pipe (not shown) is connected to the gap between the second pipe line 4 c and the first pipe line 4 b outside the lid 3.

  Thereby, the cell suspension A is introduced into the container body 2 through the cylindrical space between the first pipe 4b and the second pipe 4c, or the supernatant C of the container body 2 is A cleaning liquid such as a saline solution is supplied or discharged. 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 openings 4a and 4f at the tips of the first pipe 4b and the second pipe 4c are formed. The positioning state is maintained.

  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. 3, the closed end 2 f of the container body 2 is radially outward by a centrifuge not shown in the 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 2f.

In the middle of the movement, the cells are collected toward the second cylindrical inner surface 2d by rolling on the tapered inner surface 2c, and are deposited on the closed end 2f of the second cylindrical inner surface 2d. When the centrifugation proceeds sufficiently, as shown in FIG. 4, the cell layer B and the supernatant C are clearly separated, and a boundary surface D is formed between them.
In the present embodiment, the second cylindrical inner surface 2d is formed in such a length dimension that the boundary surface D is formed at an intermediate position in the axial direction. Therefore, as shown in FIG. D is formed, and the through hole 4f provided in the second pipe 4c opens into the supernatant C above the boundary surface D.

In this state, when the cylindrical space between the first pipe 4b and the second pipe 4c is sucked to a negative pressure, the supernatant in the container body 2 through the through hole 4f as shown in FIG. C is aspirated and aspirated until some supernatant C remains above the cell layer B as shown in FIG.
Subsequently, the washing liquid is supplied to the cylindrical space between the first pipe 4b and the second pipe 4c, and the suspension is resuspended by mixing with the cell layer B remaining in the container body 2 and the slight supernatant C. It becomes cloudy. Then, the digestive fluid adhering to the cells is washed by repeating centrifugation, aspiration of the supernatant C, and supply / resuspension of the washing solution at least once, and the concentration of the digestive enzyme in the cell suspension A is adjusted. Can be reduced.

  Then, in a state in which the concentration of the digestive enzyme is sufficiently reduced, a small amount of physiological saline is supplied to the separated cell layer B and some of the supernatant C to resuspend the resuspension A. ′ And sucked by a syringe (not shown) attached to the end of the first pipe 4b outside the container body 2 as shown in FIG. It can be sucked into the syringe without leaving it in the main body 2.

  In this case, according to the centrifuge container 1 according to the present embodiment, since the second cylindrical inner surface 2d having a sufficiently small diameter is provided at the tip of the tapered inner surface 2c, it is separated from the supernatant C. The area of the boundary surface D with the supernatant C of the deposited cell layer B can be kept sufficiently small. In addition, since the through hole 4f provided in the second pipe 4c for sucking the supernatant C is formed to penetrate in the radial direction, the flow of the sucked supernatant C does not directly affect the boundary surface D. Formed as follows.

As a result, the flow of the supernatant C when the supernatant C is aspirated can effectively prevent cells from rising from the boundary surface D into the supernatant C. It is possible to prevent the occurrence of inconvenience that is discharged.
That is, there is an advantage that the inconvenience that a part of the cells is discharged from the separated cell layer B can be prevented, and the yield of the cells can be improved.

  Moreover, according to the centrifuge container 1 according to the present embodiment, since the second cylindrical inner surface 2d has a substantially cylindrical shape, the number of cells to be separated varies for each different cell suspension A. Thus, even if the height position of the boundary surface D varies, the variation of the area of the boundary surface D can be suppressed as much as possible. As a result, even in such a case, it is possible to effectively prevent cells from rising from the boundary surface D into the supernatant C.

  Moreover, according to the centrifuge container 1 according to the present embodiment, as described above, the cell force from the boundary surface D is prevented by suppressing the area of the boundary surface D. There is an advantage that the damage to the cells can be reduced and the integrity of the cells can be maintained.

  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 substantially cylindrical second cylindrical inner surface 2d is provided at the tip of the tapered inner surface 2c. Instead of this, as shown in FIG. It is good also as having the 2nd taper inner surface 2g which has a small taper angle.
In this case, for example, the taper angle of the taper inner surface 2c may be set to 25 ° to 45 °, and the taper angle of the second taper inner surface 2g may be set to 5 ° to 20 °.

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 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 which applied the centrifugal force to the centrifuge container of FIG. 3, and isolate | separated into the cell layer and the supernatant liquid. FIG. 5 is a longitudinal sectional view showing a step of sucking and discharging the separated supernatant in the centrifuge container of FIG. 4. FIG. 6 is a longitudinal sectional view showing a state in which the supernatant is sucked and discharged in the centrifuge container of FIG. 5. FIG. 7 is a longitudinal sectional view showing a step of sucking and collecting the resuspended cell suspension in the centrifuge container of FIG. 6. It is a longitudinal cross-sectional view which shows the modification of the centrifuge container of FIG.

Explanation of symbols

A Cell suspension B Cell layer C Supernatant D Boundary surface 1 Centrifugal container 2b First cylindrical inner surface 2c Tapered inner surface (first tapered inner surface)
2d 2nd cylindrical inner surface 2g 2nd taper inner surface 4a Opening part (cell suction port)
4f Through hole (Supernatant suction port)

Claims (6)

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 first cylindrical inner surface having a substantially cylindrical shape;
A tapered inner surface disposed at the tip of the inner surface of the first cylinder and gradually tapering toward the tip;
A second cylinder having a substantially cylindrical shape disposed at the tip of the tapered inner surface and closed at the tip and having a length dimension that forms a boundary surface between the cell layer and the supernatant at an intermediate position in the axial direction. A cylindrical inner surface ;
A centrifuge container comprising a supernatant suction port for sucking the supernatant above the boundary surface .   The centrifuge container according to claim 1, wherein the first cylindrical inner surface and the second cylindrical inner surface have an inclination angle so as to be gradually tapered toward the tip. 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 inner surface having a substantially cylindrical shape;
A first tapered inner surface disposed at the tip of the cylindrical inner surface and gradually tapering toward the tip;
A boundary between the cell layer and the supernatant, which is disposed at the tip of the inner surface of the first taper, is gradually tapered toward the closed tip, has a smaller taper angle than the inner surface of the first taper. A second taper inner surface having a length dimension that forms the surface at an intermediate position in the axial direction ;
A centrifuge container comprising a supernatant suction port for sucking the supernatant above the boundary surface .   The centrifuge container according to claim 3, wherein a taper angle of the first taper inner surface is 25 ° to 45 °, and a taper angle of the second taper inner surface is 5 ° to 20 °. The centrifuge container according to any one of claims 1 to 4, wherein the supernatant suction port is opened in a radial direction. The cell separation container according to any one of claims 1 to 5 , further comprising a cell suction port that is disposed near the closed end and sucks the cell layer.

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