ES2910923T3 - Device and method for fluid separation by density gradient centrifugation - Google Patents

Abstract

A centrifugation container (1) comprising an insert (6), especially a test tube, for the separation of liquids into fractions of the desired density range by density gradient centrifugation, especially liquids that constitute suspensions and/or biological fluids , the insert is equipped with a partition (7) suitable for dividing the interior of the container (1) into at least two vertically arranged chambers - the upper chamber (2) and the lower chamber (3), characterized in that the partition (7 ) has an opening (4) to which is attached a guide (12) through which liquids flow, especially biological fluid, towards the lower chamber (3) of the centrifugation container (1), and said partition (7) is formed by two adjoining surfaces with openings, especially in the form of flattened disks adjusted to the cross section of the container (1) with a cross section similar to the circle, the surfaces are movably connected to each other, it can n be freely positioned relative to each other allowing closure of the opening lumen (4).

Description

DESCRIPTION

Device and method for fluid separation by density gradient centrifugation

The invention relates to a centrifuge vessel with an insert and to a fluid separation method using density gradient centrifugation. In particular, the invention is used in the separation of body fluids, eg, blood from animals, including humans, for diagnostic purposes. The solution object of the invention belongs to the field of laboratory containers, and especially tubes specifically adapted for centrifugation. Another aspect of the invention relates to the field of testing or analyzing materials by determining their chemical, physical, or biological properties, and particularly includes the analysis of liquid biological material, eg, blood.

State of the art

The collection, purification, fractionation, and/or fixation of bodily fluid samples, including blood, serve an important function, for example, in medical diagnostics and clinical trials. For conventional systems and methods for collecting blood samples on a large scale, the blood samples collected from the patient may be separated into different fractions by centrifugation, filtration, or elutriation, and then stored for later use or further testing. Separated blood components typically contain fractions of red blood cells, white blood cells, platelets, and plasma. The separation of blood into its fractions can be carried out continuously, during blood collection or in the stages after blood collection. The separation of blood into different components under highly sterile conditions is essential for many therapeutic applications and for clinical research purposes.

There are many methods to separate blood into its fractions. The methods known in the state of the art require the use of state-of-the-art specialized medical/research devices and highly qualified personnel for their correct operation.

From International Patent Application No. WO8805331, there is a known technique for the separation of white blood cells (leukocytes) from red blood cells (erythrocytes), which consists of mixing a blood sample with a working solution that adds red blood cells and thus increases its sedimentation rate. The density of the working separation fluids is selected so that the sedimentation of the white blood cells changes minimally and that the white blood cells do not settle to the bottom and, as a result, can be taken from the top of the separated liquid after the red blood cells have sediment at the bottom.

In another technique, in which the red blood cell-aggregating working solution is not mixed with blood, the blood is layered precisely on the surfaces of the separating fluids, after which the red blood cells clump or aggregate under the effect of surface contact with working separation fluids, as a result of which they settle to the bottom of that tube. There are several well-known multipolymer compounds that agglutinate red blood cells, for example, FICOLL 400 (Pharmacia Fine Chemicals, Sweden). Blood separation can occur under the influence of gravity or under the influence of centrifugation. Most white blood cells remain in the borderline phase, but these previously developed systems are not effective in separating white blood cells into subpopulations, that is, peripheral blood mononuclear cell (PBMC) and polymorphonuclear leukocyte (PMN) populations. In particular, there is an ongoing search for a single-step method using a density separation medium that would allow the separation of whole blood cells into subpopulations.

To carry out the aforementioned separation of white blood cells into subpopulations, a known method is to isolate peripheral blood mononuclear cells (PBMC) based on a centrifugation process, in which the first stage uses the Isopaque-Ficoll mixture (Nyegaard & Co., Norway) that has the component sodium metrizoate, in the next step, polymorphonuclear neutrophils are isolated using dextran or gelatin, which cause sedimentation of red blood cells. Another method uses discontinuous density gradients where two or more working separation fluids are poured in layers on top of each other. The densities are selected so that the (discontinuous) gradient is in the appropriate/required range, adjusted to the density of the separated substances.

On the other hand, US Patent Application No. US4824560 A discloses methods and means for centrifuging in a tubular vessel having at least two adjacent chambers that are connected to each other by a narrow essentially capillary opening. For operation, the working fluid is placed in the lower chamber, while the fluid to be separated into fractions is placed in the upper chamber, and it is not necessary to take special precautions to prevent the liquid from mixing before starting the centrifugation. This method has several advantages over the manual methods described above, it also has the disadvantage due to the close connection of both chambers that constitutes a partial barrier, even during centrifugation. This barrier prevents the effective passage of blood cells between the chambers and prevents the separation of blood into fractions.

Patent application US2014087360 A1 discloses an insert for a centrifuge tube suitable for use in density gradient separation. The insert includes an element sized to fit within the tube to divide the tube into an upper part and a lower part. Optionally, the insert has a support that extends or relies on the member to position the member within the tube. At least two openings are located in the member such that a first opening is closer to a lower end of the tube relative to a second opening when the insert is placed in the centrifuge tube.

US5314074 A discloses a layered insert within the container that prevents backmixing after density gradient centrifugation. The insert is capable of supporting the body of liquid dispersion to be centrifuged under normal gravity conditions and allows bi-directional cross flow during centrifugation.

US5132232 A discloses an apparatus for collecting liquid analytical samples for examination and reproducibly separating them into two or more fractions. An apparatus uses a container and a petter, said petter provided with an upper part that allows attachment to the container, a lower retaining part that includes a peripheral opening, the part without holes of which allows wedge fitting in the container, and a portion of body connecting the upper petter portion and the lower retaining portion.

US5648223 A discloses a cell trap centrifuge tube containing a specific density gradient solution adjusted to a specific density for enriching breast tumor cells from a mixture of cells. The tube allows the desired cell population to be collected by decantation after centrifugation to minimize cell loss and maximize efficiency.

The problem that arises in the manual separation methods described above is the preparation of the sample for this process and, in particular, in the stratification of poured liquids used for the separation of different density and the material being tested, for example blood. . It is important that the fluids do not mix with each other and that an interface is established between fluids of different densities. To achieve this state, various techniques and skills have been developed that allow proper stratification of the separation fluids and blood, which in most cases are carefully pipetted into a container for subsequent separation into fractions by gradient centrifugation. of density. Unfortunately, these procedures are cumbersome, difficult to perform, introduce the possibility of uncontrolled human error, and furthermore require highly qualified personnel, which is associated with high operating costs, reduces procedure repeatability, and prevents large-scale separation/splitting. scale.

The objective of the solution according to the invention was to obtain a tool for the rapid and partially automated separation of fluids into fractions of different densities, for example, biological fluids, including blood, which also allow purification, isolation and fixation. of biological samples.

Definitions

Within the description of the invention and the claims, the following terms will be understood according to the following definitions:

“vessel” is any liquid storage container that is adapted for centrifugation, for example, centrifuge tubes,

the “guide” is an element of the insert that directs the flow of liquid from the upper chamber to the lower chamber through the opening in the partition, the guide must be sized to allow the test liquid to accumulate in the separating medium at the bottom of the vessel. The guide, according to the above definition, can be the wall of the container or a different structure within the container, eg spiral, elongated sleeve, etc.

Detailed description of the invention

The essence of the invention is a centrifugation vessel comprising an insert, especially a test tube, for the separation of liquids into fractions of the desired density range by density gradient centrifugation, especially liquids that constitute suspensions and/or biological fluids , the insert is equipped with a suitable partition to divide the interior of the container into at least two vertically arranged chambers - the upper chamber and the lower chamber, characterized in that the partition has an opening to which a guide is attached, through which liquids, especially biological fluids, flow into the lower chamber of the centrifuge vessel, and said partition is formed by two adjacent surfaces with openings, especially in the form of flattened disks embedded in the cross section of the vessel with a cross section similar to the circle, the surfaces are movably connected to each other, can be positioned freely each other allowing closure of the opening lumen.

Preferably, the guide is spiral, funnel or vertical elements in the shape of an elongated cylinder.

Preferably, the upper chamber of the centrifuge vessel also has a vertical partition or partitions separating it into sub-chambers, each of the sub-chambers having an opening.

The invention also includes a method for separating a fraction with a desired density range from a sample containing fractions of different densities, especially from a biological sample, comprising: a) to provide a centrifuge vessel with an insert for a centrifuge vessel, especially a test tube, for the separation of liquids into fractions of the desired density range by means of density gradient centrifugation, especially liquids constituting biological fluids or suspensions,

b) fill the lower chamber of the vessel with a medium for separation on a density gradient or fill the upper chamber of the vessel with this medium, which then flows through the opening in the partition along the guide into the chamber lower;

c) pouring liquid for separation containing fractions of different density into the lower chamber filling the upper chamber or at least one sub-chamber or joining the upper chamber to the partition, so that the liquid can flow through the opening in the partition in the guide and the layer on the surface of the separating means in the lower chamber;

d) centrifugation of the container until the test sample separates into fractions of different densities. Preferably, step (b) is followed by a further step(s) b) of adding additional density gradient separation media, wherein the subsequent media addition is from higher to lower density. It is equally advantageous that, after step (d), selected fractions of different densities of the liquid being separated are subjected to testing and analysis, including the possibility of being fixed, especially by the freezing method.

Preferably, when the fraction is separated from the blood fluid, the individual fractions of the desired density range contain various blood components, including: leukocytes (lymphocytes and granulocytes), platelets, erythrocytes, bone marrow cells (megakaryocytes, erythroblasts), cells suspended in homogenate including endothelial cells, neurons, fungi, viruses, microparticles including exosomes, cell fragments, cell organelles including nuclei, mitochondria, chloroplasts.

The invention also relates to a kit comprising:

a) insert (6) for the container (1) for centrifugation, in particular the tube, for the separation of liquids into fractions of the desired density range by density gradient centrifugation, especially liquids that constitute suspensions or biological fluids, this device is equipped with a partition (7) that divides the interior of the container (1) into the upper chamber (2) and the lower chamber (3), the partition (7) has an opening (4), and in the opening there is a guide (12) on which fluids, especially separated fluid, flow into the lower chamber (3) of the container (1) for centrifugation,

b) at least one density gradient separation medium.

For a better understanding, the invention is illustrated in the embodiment illustrated in the drawing which is not a limitation of the requested protection, in which:

Fig. 1 illustrates a container in the form of a centrifuge tube with an insert, intended to collect liquids, in particular biological material, and then for their separation, which - according to the invention - allows the layering of liquids placed in the container before centrifugation;

Figures 2 and 3 illustrate a longitudinal section and a side view of a centrifuge tube container, respectively, in which, for a better understanding of the essence of the invention, the disc and the insert partition are separated;

Figs. 4 and 5 illustrate the side view and longitudinal section of the tube-shaped container, respectively, with a visible tube lumen that narrows with increasing wall thickness.

Fig. 6 illustrates a cross section through a tube-shaped container in an example without a vertical partition, but with a visible air channel,

Figs. 7a and 7b illustrate a side view and a cross section of the top of the disc-shaped insert with an incomplete partition, respectively.

Figs. 8a and 8b illustrate a side view and a cross section of the upper part of the disc-shaped insert with rectangular septum, respectively.

Figs. 9a and 9b illustrate a side view and a cross section of the upper part of the disk-shaped insert with a partition in the form of three rectangles, respectively.

Figs. 10a and 10b illustrate a side view and a cross section of the top of the disc-shaped insert with a partition in the form of two intersecting rectangles, respectively.

Figs. 11a and 11b illustrate a cross section and a side view of a notched disc-shaped partition, respectively.

Fig. 12 illustrates an embodiment of the solution of the invention in which the insert is placed in a spin container.

Figs. 13 and 13a illustrate the insert, a lateral and upper cross-section adapted to connect the upper chamber equipped with a guide in the form of an elongated cylinder, respectively.

Figs. 14 and 14a illustrate the insert, in lateral and upper cross section, equipped with a guide in the form of eight elongated cylinders, respectively.

Figs. 15 and 15a illustrate the insert, a side and top cross section equipped with a spiral guide, respectively.

Figs. 16, 16a and 16b illustrate the cross section of the insert from the side and top view, and the enlarged guide, equipped with a funnel-shaped guide, respectively.

Example 1

As shown in Fig. 1, in the first embodiment, the tube insert 6 for the centrifuge vessel consists of a flat disk-shaped partition 7 that firmly adheres to the inner walls of the vessel 1, and a disk 8 equipped with a full vertical 11 partition. The insert in this example is placed inside a container 1 which constitutes a 0.23" diameter centrifuge tube. The insert 6 in this example is made of plastic, but it could also be made of other materials. As shown in Fig. 12, the insert 6 can also be placed in an additional container which can be attached to the centrifuge container 1, then the insert 6 is outside the container 1.

The wall of the tube-shaped container 1 forms a guide 12 and gradually widens in the container 1 (figures 4 and 5), at the same time that the lumen of the tube gradually decreases towards its lower part. In this embodiment, the inner wall of container 1 is a guide 12 that allows liquids to flow from upper chamber 2 to lower chamber 3 through opening 4. Liquids, especially liquids that are biological fluids for separation , flow towards the bottom of the container 1 along the guide 12, constituted by the wall of the container, and are arranged in layers at the bottom of the container 1. The downward flow of liquid along the guide 12 prevents agitation of the separating liquids, which could cause errors in the separation of the tested liquids.

In this example, the partition 7 has the shape of a flat disk with a circular cross section (Fig. 11a, Fig. 11 b) and its shape matches the cross-sectional shape of the container 1, so the diameter on its upper face it is larger than the lower face, and the longitudinal section of partition 7 is close to a flattened inverted trapezoid. The partition 7 divides the container 1 into an upper chamber 2 and a lower chamber 3. the example partition has an opening 4 which forms a notch similar to a semicircle.

As shown in Figs. 8a and 8b, the vertical partition 11 can be in the shape of a rectangle adhering to the inner wall of the container 1, then the vertical partition 11 located on the disk 8 divides the upper chamber 2 of the container I tube-shaped into two sub-chambers 10a, 10b. In each of the halves of the disc 8 formed by the partition 11, there is a notch-shaped opening 5 which can be closed by the disc 8. In the embodiment, the notch-shaped openings 5 in the disc 8 are semicircular. In other implementations of the solution according to the invention, it is also possible to use disks 8 with other shapes of openings 5. The flow rate of liquid from upper chamber 2 to lower chamber 3 depends on the shape of openings 4, 5 and their mutual agreement with respect to others.

In this example, the notch shaped openings 4, 5 with a notch radius of 0.115” have the same shape. In other versions of the embodiment, the openings 4, 5 may have different shapes and may differ in shape, but their diameter must be greater than 0.1". With partition 7 and disk 8 arranged in such a way that openings 4, 5 do not overlap, fluid flow between upper chamber 2 and lower chamber 3 is blocked and proper fluid flow cannot take place.

In this embodiment, the container 1 is provided with a lid 9. In one version of the example, the lid 9 has a recess into which the upper part of the vertical partition 11 of the insert 6 fits and passes through. Such a position of the vertical partition II allows to change the position of the disc 8 with respect to the partition 7 by turning the protruding part of the partition, and therefore the moving part of the lid 9. The container 1 and the lid 9 have a thread and form a screw cap. Alternatively, the use of the lid 91 without a gap, where the vertical partition 11 of the insert adapts to the length of the container 1 so that, after the lid 9 is screwed on, the vertical partition 11 adheres firmly to the inner surface of the container. cap 9. Cap 9 may be made of plastic and may have a twist/turn scale calibrated. It is intended to mark the spin containers 1 and faucet 9 to facilitate the correct line/arrangement of the openings 4, 5 between them.

Alternatives for different shapes and arrangements of the vertical partition 8 are possible in other versions of the embodiment. As shown in Figs. 7a and 7b, it is possible that the vertical partition 11 does not adhere to the walls of the container 1, so the vertical partition 11 located on the disk 8 divides the tube into two chambers only: the upper chamber 2 and lower chamber 3, and upper chamber 2 is not divided into additional sub-chambers. In this version, the disk 8 is equipped with an opening 4 in the form of a notch, so the shape of the disk 8 in another version of the example could be limited to closing the openings 4 and the partition 7.

As shown in Figs. 9a and 9b, the vertical partition 11 can consist of three rectangular elements connected to each other by longer sides, the sides of which are very close to the inner wall of the container 1, then the vertical partition 11 located in the disc 8 divides the upper chamber 2 of the tube-shaped container 1 into three sub-chambers. In this version, the disk 8 is equipped with three notches 5, and in each of the subchambers.

As shown in Figs. 10a and 10b, the vertical partition 11 can consist of four interconnected rectangles, the sides of which are very close to the inner wall of the container 1, then the vertical partition 11 located on the disc 8 divides the part of the upper chamber 2 of the tube-shaped container 1 into four sub-chambers. In this version, the disk 8 is equipped with four notches 5, and in each of the subchambers.

The insert 6 can also be used in containers 1 with shapes other than the centrifuge tube shown in the embodiment, but these containers must be suitable for centrifugation.

Example 2

Figs. 13 and 13a shows another embodiment of the solution according to the invention, in which the insert 6 has a partition 7, which does not have an upper chamber, but allows connection, through a tube 16, to the partition of the upper chamber in the form of a container (eg test tube, bag) with a medium for separation or with a liquid for separation. Next, the partition is equipped with a guide 12 in the form of an elongated cylinder, which is attached to the partition 7 and is located at such a distance from the opening 4 that it allows the liquid to flow from the upper chamber through the tube 16 , then through the opening in the partition and along the guide to the lower chamber 3. In this embodiment, the elongated cylinder constituting the guide 12 has a length such that the test material is deposited on the surface of the gradient medium without causing significant disturbance to the separation medium.

Example 3

Figs. 14 and 14a shows another embodiment of the solution according to the invention, in which the insert 6 has a partition 7, equipped with a guide 12 in the form of 8 elongated cylinders, which are attached to the partition 7 and are at a distance of the opening 4 allows the liquid to flow from the upper chamber through the opening in the partition following the guide to the lower chamber 3. In this embodiment, the elongated cylinders constituting the guide 12 are of a length such that the test material is deposited on the surface of the gradient centrifugation medium without causing significant disturbance to the separation medium.

Example 4

On the other hand, Figs. 15 and 15a show yet another example of the solution according to the invention, in which the insert 6 has a partition 7 equipped with a guide 12 in the form of a spiral. Similar to example 2, the length of the coil should be such that the test material is deposited on the surface of the gradient centrifugation medium without causing significant disturbance to the separation medium.

Example 5

Figs. 16, 16a and 16b show yet another example of the solution according to the invention, in which the insert 6 has a partition 7 equipped with a funnel-shaped guide 12. At the same time, openings 4 in partition 7 direct the liquids from the upper chamber to flow down along the outer surface of the funnel to the bottom of chamber 3. Similarly, in example 2, the The length of the spiral should be such that the test material accumulates on the surface of the gradient centrifugation medium without causing significant disturbance to the separating medium.

Example 6

The method of separating the fraction with the desired density range from a sample containing fractions of different densities according to the invention can be carried out in such a way that in different sub-chambers 10a, 10b of the upper chamber 2, there are two media different separation media placed in a density gradient, the first medium has a density of 1.119 g/ml, the second medium has a density of 1.077 g/ml (Histopaque 1.119 and Histopaque 1.077 from Sigma Aldrich, respectively), constituting the openings 4, 5 notches - of the partition 7 and disk 8, respectively - not overlapping when it remains in the closed position. Then change the position of the disc 8 by rotating it, the notches 4, 5 overlap enough to allow the medium to flow from the upper chamber 2 to the lower chamber 3. The liquids flow along the inner wall of the container 1 which constitutes the guide 12. The position of the openings 4, 5 which constitute a notch in the partition 7 and the disc 8 of the example is adjacent to the wall of the container 1 lo constituting the guide 12. The media are added in order from higher density to lower density, and an interface is formed between media of different density. Next, a liquid or a mixture intended for separation into fractions of different densities under the influence of centrifugation, for example, native or diluted blood, is added to one of the empty subchambers 10 with a closed flow between the upper chamber 2 and lower chamber 3.

The size of the free space formed by the openings 4, 5 which are cutouts of the partition 7 and the disk 8, respectively, can be controlled by adjusting the mutual position of the partition 7 and the disk 8. By slowly rotating the upper part of the insert 11, and thus disc 8, gradually increases the fluid flow rate until the assumed liquid flow rate between chambers 2, 3 is reached. By adjusting the amount of mutual overlap between opening 4 and opening 5, the flow of liquid can be controlled to create a continuous laminar flow along the wall 12 of the centrifuge vessel 1 . The construction of the partition 7 and the disc 8 according to the invention ensures a very smooth downward flow of fluid from the upper chamber 2 towards the lower chamber 3 of the centrifuge vessel 1 in such a way that the interface between the liquids is intact and the subsequent liquid poured from the upper chamber 2 does not mix with the liquid present in the lower chamber 3.

After pouring two working density gradient separation media, these fluids are layered on top of each other due to different densities, then the test sample in the form of blood is added, it is also possible to use different types of fluids for separation, including native or diluted biological samples. The blood was first placed in the chamber 10a, and then after rotating the disc 8 of the insert 6 in such a way that the opening 4 of the partition 7 coincides at least in part with the corresponding opening 5 of the disc 8 of the insert 6, and it allows the blood to flow along the wall 12 of the container 1 from the sub-chamber 10a to the lower chamber 3 and accumulate on the surface of the separating means previously placed there. Due to the structure of the insert 6, it is not necessary to place the biological sample in the container 1 with extreme precision and care.

Next, the blood in the lower chamber 3 of the container 1 is centrifuged according to methods generally known in the art. During centrifugation, a bidirectional fluid flow occurs within different compartments formed by the separation of fluids of different density in the lower chamber 3, and a continuous density gradient is formed at the end of centrifugation, with red blood cells depositing at the bottom forming the lowest localized layer, the top layer is a liquid with a density of 1.119 g/mL, the next layer above is white blood cells with segmented nuclei, above is a layer formed by a liquid with a density of 1.077 g/ml, and a layer of white mononuclear cells above that, finally plasma is above them as the highest layer. After removing the insert, each of the cell/liquid layers can be removed by aspiration with a pipette or decantation.

Example 7

The insert and method of the invention are used, for example, to separate a desired subset of blood cells. In one embodiment, ten blood samples from healthy volunteers (20 mL venous blood) were collected into commercially available ethylenediaminetetraacetic acid (EDTA) tubes (EDTA tube, Becton Dickinson). In this experiment, the volume of the centrifugation tube 1 that constitutes the invention was 50 ml, in addition two separation media of different density (Histopaque 1.119 and Histopaque 1.077 Sigma Aldrich) were used. The separation media used were neutral in pH, isotonic to body fluids, the first separation media had a density of 1.119 g/ml, while the second had a density of 1.077 g/ml.

Next, 10 ml of separation medium with a density of 1.119 g/ml was placed in the sub-chamber 10a of the upper chamber 2 of the centrifuge vessel 1 equipped with the insert 6 according to the invention. A second medium with a density of 1.077 g/ml with a volume of 10 ml was placed in the sub-chamber 10b of the upper chamber 2 and then layered on top of the first medium using the insert 6 according to the invention described above. In the experiment, the dividing wall had a thickness of 0.08" and the notches 4, 5 of partition 7 and disk 8 had a radius of 0.115". Next, the blood collected with ethylenediaminetetraacetic acid (EDTA) was placed in the sub-chamber 10a of the upper chamber 2. Each blood sample was layered on the surfaces of the separation media with the insert 6 of the invention described above.

In the next step, all tubes were centrifuged at 700g (with minimal acceleration and no active braking) for 30 minutes at room temperature. As an effect of density gradient centrifugation, the blood was separated into four fractions: plasma, peripheral blood mononuclear cells (PBMC), polymorphonuclear leukocytes (PMN), and red blood cells. The purity of the PBMC and PMN fractions was confirmed by flow cytometry. The purity of PBMC and PMN in their fractions was 95% and 92%, respectively. PBMC and PMN cells were not detectable in the plasma fraction. Isolated plasma, PBMCs and PMNs were suitable for additional analyses, including but not limited to: RNA, microRNA, mitochondrial DNA, nuclear DNA, protein and cell phenotyping.

Claims (7)

1. A centrifuge container (1) comprising an insert (6), especially a test tube, for separating liquids into fractions of the desired density range by density gradient centrifugation, especially liquids constituting suspensions and/or biological fluids, the insert is equipped with a partition (7) suitable for dividing the interior of the container (1) into at least two vertically arranged chambers - the upper chamber (2) and the lower chamber (3), characterized in that the partition (7) has an opening (4) to which is attached a guide (12) through which liquids flow, especially biological fluid, towards the lower chamber (3) of the centrifugation container (1), and said partition (7) it is formed by two adjoining surfaces with openings, especially in the form of flattened disks adjusted to the cross section of the container (1) with a cross section similar to the circle, the surfaces are movably connected to each other, pu They can be positioned freely with respect to each other allowing the closure of the opening lumen (4). 2. The centrifugation container (1) comprising the insert (6) according to claim 1, characterized in that the guide (12) is a spiral, a funnel or vertical elements in the form of an elongated cylinder. 3. The centrifuge container (1) comprising the insert (6) according to claim 1, characterized in that the upper chamber (2) additionally has a vertical partition (11) or partitions that separate it into sub-chambers (10), each one of the sub-chambers (10) has an opening (4, 5). 4. A method for separating a fraction with a desired density range from a sample containing fractions of different densities, especially from a biological sample, comprising: a) provide a centrifuge container (1) of any claim from 1 to 3 for the separation of liquids into fractions of the desired density range by density gradient centrifugation, especially liquids that constitute suspensions or biological fluids, b) filling the lower chamber (3) of the vessel (1) with a medium for density gradient separation or filling the upper chamber (2) of the vessel (1) with this medium, which then flows through the opening in the partition along the guide (12) to the lower chamber; c) pouring liquid for separation containing fractions of different density into the lower chamber (3) by filling the upper chamber (2) or at least one sub-chamber (10a, 10b) or fixing the upper chamber to the partition, so that the liquid can flow through the opening (4) in the partition in the guide (12) and spill on the surface of the separating medium of the lower chamber (3); d) centrifuging the container (1) until the test sample separates into fractions of different densities. 5. The method according to claim 4, characterized in that after step (b) there follows an additional step or steps b) consisting of adding an additional density gradient separation medium, in which the addition of media Posterior is from highest to lowest density. 6. The method according to claim 4, characterized in that after step (d), selected fractions of different densities of the liquid being separated are subjected to tests and analysis, which includes the possibility of being fixed, especially by the freezing method. 7. The method according to claim 6, characterized in that when the fraction is separated from the blood liquid, the individual fractions of the desired density range contain various blood components, including: leukocytes (lymphocytes and granulocytes), platelets, erythrocytes, cells bone marrow cells (megakaryocytes, erythroblasts) suspended in a cellular homogenate including endothelial cells, neurons, fungi, viruses, microparticles including exosomes, cell fragments, cell organelles including nuclei, mitochondria, chloroplasts.