Elutriation chamber for an elutriator system

Abstract

The invention relates to an elutriation chamber for an elutriator system for washing and/or isolating cells, in particular thrombocytes, which elutriation chamber comprises a feed line (1) for an aqueous medium containing the cells to be washed and/or to be isolated in suspended form, and a discharge line (2) for the washed and/or isolated cells, wherein the chamber (5) is rotationally symmetrical to the axis (a), characterized in that the ratio of the area of the section through the lumen of the chamber (5) perpendicular to the axis (a) at the widest point (5a-5b) to the area of the section (1a) through the feed line (1) is in the range of 1,000 to 250,000.

Claims

1. An elutriation device for an elutriator system, the elutriation device having a chamber for washing and/or isolating cells, in particular thrombocytes, which elutriation device comprises: a feed line for an aqueous medium containing the cells to be washed and/or to be isolated in suspended form, a chamber lumen, a discharge line for the washed and/or isolated cells, and an axis extending from the feed line to the discharge line, wherein a ratio of an area of a section through the chamber lumen perpendicular to the axis at a widest point to the area of a section through the feed line is in a range of 1,000 to 250,000.

2. An elutriation device according to claim 1, further comprising a further feed line for a gaseous or liquid medium.

3. An elutriation device for an elutriator system, the elutriation device having a chamber for washing and/or isolating cells, in particular thrombocytes, which elutriation device comprises: a chamber entrance, a feed line coupled to the chamber at the chamber entrance, the feed line having a circular cross-section for passing an aqueous medium containing the cells to be washed and/or to be isolated in suspended form through the narrowing of the chamber entrance and into the chamber, a lumen of the chamber, a discharge line for the washed and/or isolated cells, and an axis extending from the feed line to the discharge line, wherein, in the feed line at the chamber entrance, a narrowing is provided in such a way that a ratio between an area of a section through the lumen of the chamber perpendicular to the axis at a widest point to an area of a section through the narrowing is in a range of 1,000 to 250,000.

4. An elutriation device according to claim 1, further comprising a chamber entrance provided with a narrowing, the feed line being coupled to the chamber at the chamber entrance such that the aqueous medium containing the cells is passed through the narrowing of the chamber entrance and into the chamber.

5. An elutriation device according to claim 3, further comprising a further feed line for a gaseous or liquid medium.

6. An elutriation device according to claim 1, wherein the chamber is rotationally symmetrical about the axis.

7. An elutriation device according to claim 1, wherein the feed line has a circular cross-section.

8. An elutriation device according to claim 3, wherein the narrowing at the chamber entrance is conical.

9. An elutriation device according to claim 4, wherein the narrowing at the chamber entrance is conical.

10. An elutriation device according to claim 3, wherein the chamber is rotationally symmetrical about the axis.

Description

(1) Preferred embodiments are described in further detail on the basis of the accompanying drawing.

(2) FIG. 1 shows an elutriation chamber (having a lumen 6 and a chamber entrance 7) contained in a commercially available elutriator system.

(3) Said chamber is anchored on the centrifuge rotor with its axis of symmetry along the radius so that the fluid supply (1) is located close to the periphery of the rotor and, correspondingly, the fluid discharge (2) is located close to the axis. During the operation of the centrifuge, the respective fluid streams can now be introduced and discharged, respectively, via the rotor axis and generate in the chamber the stream directed against the centrifugal acceleration. Due to expansion of the chamber cross-section in the centripetal direction and the continuously decreasing flow speed associated therewith, an equilibrium between the centrifugal and resisting forces acting on a particle may be achieved within certain limits. By that, particles of an appropriate range of sedimentation speeds in the system are held in suspense, while the remaining portions of the fluid are washed out continuously.

(4) The constructive connection of the supply and the discharge of the elutriation chamber via the axis of rotation necessitates a feed channel which, coming from the centre of the rotor, must be deflected directly in front of the separation chamber at the periphery of the rotor into a direction extending radially to the rotor axis. Since thereby the wall of the channel must extend in one point normally to the particle motion resulting from the sedimentation and flow speeds, this area is prone to massive formation of pellets, especially in case of particles which tend to agglutinate, such as, e.g., thrombocytes, which operates detrimental to the intended application.

(5) FIG. 2 shows an elutriation chamber (having a lumen 6 and a chamber entrance 7) according to the invention, wherein the feed line (1) has a cross-section which is so narrow that, with the applied flow rate, a transmission rate of more than 2 cm/s, preferably >10 cm/s (7 to 16 cm/s), is achieved. By increasing the flow rate via a reduced conduit cross-section, the pellet formation in the area of the deflection of the supplied fluid stream into the centripetal direction is counteracted directly in front of the separation chamber. It is thereby crucial that the ratio of the area of the section through the lumen of the chamber (5) perpendicular to the axis (a) at the widest point (5a-5b) to the area of the section (1a) through the feed line (1) is in the range of 1,000 to 250,000.

(6) FIG. 3 shows a further elutriation chamber (having a lumen 6 and a chamber entrance 7) according to the invention comprising a further direct inlet (3) which is independent of the normal feed line (1) which serves for loading and the counterflow. At the end of the process, said inlet serves as a clean supply for extracting the content of the chamber without any material that has sedimented in the normal supply being dragged into the chamber and mixed with its contents.

(7) FIG. 4 shows an additional elutriation chamber (having a lumen 6 and a chamber entrance 7) according to the invention, wherein a strong narrowing of the inflow cross-section (4) is provided in the feed line (1). Through a simple interruption of the influx after elutriation has taken place, this enables an almost complete and effective separation of the chamber lumen from the normal supply line and thus prevents carry-over of material sedimented therein by diffusion or minor pressure fluctuations. During elutriation, the narrowing of the supply ensures a reduction in the extent of the creeping backflow from the chamber into the supply line along the wall facing away from the direction of rotation, which is caused by the Coriolis force. Furthermore, a dispersion of the inflowing fluid across the chamber cross-section is caused by the jet entering at a high speed and the turbulence associated therewith in the inflow area, which, on the one hand, contributes to an effective washing and, on the other hand, counteracts the formation of a channel flow along the chamber wall located in the direction of rotation as a result of the Coriolis force.

(8) A preferred embodiment of the elutriation chamber according to the invention is described in the following example and consists in a combination of all three variants illustrated in FIGS. 2, 3 and 4.

EXAMPLE

(9) The elutriation chamber manufactured from a transparent synthetic material is essentially composed of two straight hollow cones connected at their bases so that a chamber in the shape of a double cone emerges. It has its widest point (5a-5b) with an inner diameter of 50 mm on the common circular section, from where the two hollow cones extend 51 mm and 8 mm, respectively, toward their tips in each case in opposite directions.

(10) This base body is oriented with its axis of symmetry (a) in the centrifuge rotor along the radius in such a way that the tip of the low cone points in the direction of the rotor axis and, accordingly, that of the high cone points toward the periphery of the rotor. For the supply (1) during normal operation, a narrowing (4) to a diameter of 0.3 mm is provided at the tip of the high cone, which tip is located peripherally to the rotor axis, the narrowing expanding conically toward the outside to the inner diameter of the attached supply tube of 1.2 mm. The ratio of the area of the section through the lumen of the chamber (5) perpendicular to the axis (a) at the widest point (5a-5b) perpendicular to the axis (a) to the area of the section (1a) through the feed line (1) thus amounts to 1736 (1962 mm.sup.2/1.13 mm.sup.2). The ratio of the cross-sectional areas increases to 27778 (1962 mm.sup.2/0.071 mm.sup.2), if a narrowing to 0.3 mm on average is provided.

(11) At the cone tip which is situated opposite thereto and in close proximity to the rotor axis, the outlet port having a diameter of 2.9 mm is located, which opens into the discharge tube having the same inner diameter. For emptying out the contents of the chamber independently of the normal supply, the generated surface of the flat hollow cone additionally includes 14 mm acentrically of the axis of symmetry, a further inlet port (3) which has a diameter of 2.9 mm and is connected to the corresponding supply tube.

(12) That centrifuge insert is installed on the rotor of a commercially available elutriation system which provides both peristaltic pumps for ensuring the required liquid streams and a connection to the corresponding supply and discharge lines of the rotating chamber.

(13) The two supply lines are brought together outside of the rotor via a three-way cock and subsequently are connected to a multiple-way cock for supplying the respective liquid via a drip chamber and a pump. The discharge line leaving the rotor leads to two mutually switchable containers, on the one hand, for collecting the processed fraction and, on the other hand, for accommodating all the remaining liquids.

(14) At the start of the process, the chamber and the entire piping is filled with a suitable solution, the centrifuge is brought to a number of revolutions of 2600 min.sup.1, and a continuous stream of 5 ml/min is established across the chamber supply which, for normal operation, is located at the periphery of the rotor, wherein the second supply line intended for evacuation is closed at the three-way cock.

(15) Upon establishment of a steady flow through the system, the chamber is loaded through a continuous supply of the entire platelet-rich plasma, which is diluted in an appropriate buffer solution. Subsequently, the flow continues to be maintained with a suitable washing solution. If the required extent of washing has been achieved, the flow and the centrifuge are stopped at the same time, and an interconnection between only two chamber supplies is established via the three-way cock. After the elutriation chamber has been removed from the anchorage of the rotor and has been oriented with the discharge opening downward, as well as after the switching of the discharge line, the contents of the chamber are now pressed with air into the collecting vessel for the processed fraction exclusively via the second, additional supply line.

(16) The elutriation chamber according to the invention permits the extraction of cells and cell components, whereby the supplying tube being dimensioned such that no thrombocyte-rich pellet can be formed during the elutriation process.

(17) Furthermore, the elutriation chamber according to the invention allows high inflow speeds of the cell suspension or the suspension of cell fragments, which is to be cleaned, ranging between 1 and 10 m/sec, preferably of 5 m/sec.

(18) If two discharge openings are provided in the upper funnel part of the elutriation chamber, that embodiment enables the extraction of elutriated cells or cell components without contamination with particles which are still present in the feed tube to the elutriation chamber.

(19) In addition, the narrowing of the liquid entry into the elutriation chamber allows to achieve a high flow speed of the cell suspensions or the suspensions of cell components and to minimize the consequences of the Coriolis effect.