GB2149324A - Compartmentalized centrifugation chamber - Google Patents

Abstract

The stability of buoyant density gradients in centrifuge tubes decreases rapidly when the volume of the tubes is increased beyond 50-100 ml. This is because vortices form in large tubes on acceleration and deceleration of the centrifuge. For large-scale density gradient separation of particles, e.g. biological cells, using centrifuge tubes it is therefore necessary to distribute the material into a number of small tubes. This time consuming procedure is simplified by using a compartmentalized centrifugation chamber, which is subdivided by radially and vertically oriented walls or lamellae into a number of hydrostatically communicating compartments. The walls stabilize the large volume density gradient without interfering with sedimentation of the particles. As the compartments communicate, it is possible to layer and fractionate the density gradient in all compartments simultaneously. This results in a considerable reduction of time and labour required to separate large volumes of material by density gradient centrifugation.

Description

SPECIFICATION Compartmentalized centrifugation chamber This invention is a centrifugation chamber for the separation of suspended particles by buoyant density gradient centrifugation.

Separation of biological particles such as cells, organelles and macromolecules by utilizing either differences in their buoyant densities or in particle sizes is a well-established biomedical method.

Buoyant density gradient centrifugation is used to separate particles of different densities. Centrifuge tubes containing volumes of about 10 through 50 ml are commonly used for this purpose.

Since density gradients become increasingly unstable in larger tubes and swirling occurs on acceleration and deceleration of the centrifuge, tubes of larger volumes can hardly be used even for preparative purposes.

To separate large volumes of material it is necessary to distribute aliquots into a number of small gradient tubes, which is time consuming.

Different types of zonal rotors can be used for large-scale density gradient centrifugation, but these are expensive and additional equipment such as special pumping devices are required.

Receptacles and devices for centrifugation which are subdivided into two or more compartments are disclosed in U.S. Pat. Off.

3456876 and DE-OS 1648844 (Fed.Rep.of Germany). The subdivisions are thought to reduce the pathlenght of sedimentation for pelleting very small particles, particularly of particles with sedimentation rates of less than 10^2 Svedberg units, such as biological macromolecules, resulting in an overall reduction of centrifugation time required to pellet these particles.

These devices, however, are designed to pellet very small particles by (ultra)-centrifugation and are not suitable for density gradient centrifugation. Buoyant density, which is the major criterion in density gradient centrifugation, is of minor importance for pelleting, compared to other parameters.

The objective of the present invention is to provide a device for large scale density gradient centrifugation (volume: some hundred milliliters up to some liters), which is simple, effective and which can be used in combination with large laboratory centrifuges and the appropriate standard swinging-bucket rotors, which are widely available.

The density gradient has to be mechanically stable and unwanted swirling should be effectively prevented. In particular the density gradient has to be sufficiently stable to withstand the rotational forces which occur during acceleration and deceleration of the centrifuge and which tend to cause unacceptable vortices in large volume gradient tubes.

in general, the objectives of the invention are accomplished by subdividing one large centrifugation chamber by means of stabilizing walls or lamellae into several compartments which are geometrically arranged as described below. The characteristic features of this invention are the walls which lie in radially and vertically oriented planes during centrifugation, i.e. in planes which are defined by both the direction of the centrifugal force and the axis of rotation.

This arrangement of the partition walls is schematically illustrated in Figure 1, which is an axial view (in direction of the axis of rotation), Figure 2, which is the corresponding radial view of .) compartmentalized centrifugation chamber set into the swinging-bucket rotor of a large laboratory centrifuge.

By this arrangement the walls (lamellae) most effectively counteract the rotational forces occuring during acceleration and deceleration of a centrifuge in addition to the centrifugal force, and hence most effectively prevent vortex formation without disturbing radial sedimentation or flotation of the particles. In particular, by arranging the walls exactly in radial direction the compartments are sector-shaped preventing "wall-effects", i.e. sedimentation of the particles against the walls.

Additional walls in radial-horizontal planes do not lead to further relevant stabilization.

Another characteristic feature of the invention comprises hydrostatic communication of the individual compartments at the bottom of the gradient chamber, making it feasible to layer and fractionate the density gradient simultaneously in each of the compartments.

The main advantage of this invention if that it allows very rapid and simple layering and subsequent fractionation of a large volume density gradient. For density gradient centrifugation of volumes greater than 200 ml considerable time and labour can be saved compared to distributing the same volume into a number of small gradient tubes.

Because the individual compartments are intrinsically sector-shaped ' "wall-effects" ' are avoided and hence resolving power of gradient separation is increased in comparison with common parallel-walled gradient tubes.

Furthermore it is easy to design this compartmentalized gradient chamber as a completely closed system for sterile density gradient separation.

Compared to zonal rotors, which are also suitable for large-scale density gradient centrifugation, this invention has the advantage of being potentially cheaper and more convenient. As the gradient chamber is easy to transport, the place where the gradient is layered and fractionated and the place where it is centrifuged could be separate. The advan tages over zonal rotors are most marked when relatively large biological particles such as whole cells and large subcellular particles are being separated because only weak or moderate centrifugal forces are required.

One example of the invention is presented schematically in Fig. 3 and Fig. 4, where Figure 3 is a cross-section in axial view (as Fig. 1), Figure 4 is the according perpendicular view (view in tangential direction) (both shown as the chamber is positioned during steady state centrifugation).

The essential elements required for simultaneous layering and fractionating are designed as integral parts of the centrifugation chamber. Furthermore the complete chamber is a closed system with only two connectors for inlet and outlet tubing.

The centrifugation chamber consists of these functional parts: (1) A bottom part which contains a channel with narrow openings to the base of each compartment (4 shown in Fig. 4). These inlet openings diverge as hollow oblong pyramids.

The individual compartments communicate hydrostatically by the channel. At its outmost point this common channel is connected to a pressure-fast tubing line which runs outside the chamber to its top, where it is fixed during centrifugation. All solutions enter and may also leave the centrifugation chamber by this tube. The connection between tube and common channel has to withstand the hydrostatic pressure multiplied by the centrifugal force during the centrifugation run.

(2) A middle part comprising the oblong and side by side sector shaped compartments.

(3) A top part designed similarly to the bottom part with an outlet tubing connector directly fixed to the highest point of its common channel.

Both tubing connectors can be closed by Luerstoppers if required.

The density gradient is layered, the lightest solution first, from the bottom of the chamber, and fractionated either by upward displacement, i.e. by introducing a dense solution via the bottom inlet tubing, or conversly, letting the gradient fractions out directly via the bottom tubing. In each case the corresponding layers of all different compartments meet in a common channel.

The compartmentalized centrifugation chamber can be designed as an accessory part to any large laboratory centrifuge with swinging-bucket rotor. The detailed measurements of the gradient chamber depend on the geometry of the centrifuge to be used. The centrifugation chamber may be suspended in the rotor yoke either by integral pivots or by a separate carrier.

The materials will be selected according to different needs. When the chamber is to be used for centrifugation under sterile conditions, autoclavable materials such as metals, glass or silicone are advantageous.

An exemplary model of the gradient chamber may be disassembled into two or more components for cleaning purposes. These components correspond to the functional parts described above. At the contacting surface of the bottom part facing the walls of the middle part there are complementary fitting grooves to receive and tightly fix the middle part and a gasket between both parts. When assembled the gradient chamber is tightened by means of either screws, clamps or other connecting parts. During centrifugation the components are pressed together mainly be centrifugal force.

Claims (10)

1. A compartmentalized centrifugation chamber subdivided by walls or lamellae into several hydrostatically communicating compartments to be used in connection with a centrifuge and a common swinging-bucket or fixed angle rotor, said walls or lamellae being positioned during centrifugation in planes coinciding with centrifugal force lines and further being parallel to the axis of rotation, or, at least, said walls or lamellae being positioned substantially close to said planes.

2. A compartmentalized centrifugation chamber as defined in claim 1, which is suspended into the rotor by means of a commonly available bucket or carrier.

3. A compartmentalized centrifugation chamber as defined in claim 1, which is suspended in the rotor by means of either a integral or a separate carrier accessory.

4. A compartmentalized centrifugation chamber as defined in claim 1, 2, or 3, which is a completely closed system besides defined and also closable inlet/outlet tubing connectors.

5. A compartmentalized centrifugation chamber as defined in claims 1, 2, 3, or 4, which comprises two or more components, which may be disassembled for cleaning purposes, e.g. (a) a bottom part with inlet openings for each of the compartments which communicate at one side and diverge conically or as hollow pyramids, (b) a middle part comprising the compartments, and (c) a top part, which is designed similarly as the bottom part.

6. A compartmentalized centrifugation chambers as defined in claims 1 through 5, which consists of heat-resistant (autoclavable) materials.

7. A compartmentalized centrifugation chamber as defined in claims 1 through 5, which is supplied as sterile or non-sterile disposable device.

8. A compartmentalized centrifugation chamber as defined in claims 1 through 7, which comprises valves or taps, integrated in channels or tubings.

9. A compartmentalized centrifugation chamber as defined in claims 1 through 8, which communicates during centrifugation either permanently or for a time with a nonrotating part, making exchange of fluids possible.

10. An insert, which can be used in connection with any centrifuge tube, and which thereby modifies a centrifuge tube into a compartmentalized centrifugation chamber as defined in claim 1.