DEVICE AND METHOD FOR FLUIDS SEPARATION BY DENSITY GRADIENT

Abstract

A device for a centrifugation container, such as a tube, is for separation of liquid fractions having a desired density range, in particular to biological and/or liquids forming suspensions. The device has a partition (7) that separates the interior of the container (1) into at least two chambers in a vertical arrangement—an upper chamber (2) and a lower chamber (3). The device having the partition (7) has an aperture (4) which can be lined up with the guide (12), on which liquids, in particular a fluid sample, can flow down from an upper chamber (3) to a lower chamber (4), of the container (1) for centrifugation. A method using the device separates out a fraction having the desired density range from the sample containing fractions of different density.

Claims

1-5. (canceled)

6. A method for separating out a fraction having a desired density range from a biological sample containing fractions of different density, comprising: a) providing a centrifuge container with a device for the centrifuge container for separating a liquid sample to fractions having a desired density range by density gradient centrifugation, the liquid sample being suspensions or biological fluids, b) filling a lower chamber of the container with medium for density gradient separation, or an upper chamber of the container is filled with the medium, wherein the medium flows through an aperture in a device partition down on a guide to the lower chamber; c) pouring the fluid sample designated to be separated to fractions of different densities into the lower chamber, by filling the upper chamber or at least one of upper sub-chambers or by attaching the upper chamber to the device partition so that the fluid sample flows down to the lower chamber through the aperture in the device partition and then along the guide and layer on the surface of separation liquids already present in the lower chamber, maintaining an interphase between liquids; d) centrifuging the separation container until the sample separates into fractions of different density.

7. The method according to claim 6, wherein step (b) is followed by an additional step or steps comprising adding an additional medium for density gradient separation, wherein additional media are added in order from highest to lowest density.

8. The method according to claim 6, wherein after step (d) selected fractions of different density from separated liquid samples are studied, tested and analyzed, wherein the selected fractions are preserved by freezing.

9. The method according to claim 6, wherein in case of separating blood to fractions of different density, each separated fraction with different density contains different blood elements including: leukocytes, platelets, erythrocytes, bone marrow cells, cells suspended in homogenate including endothelial cells, neurons, fungus, viruses, microparticles including exosomes, cellular fragments, cell organelles including nuclei, mitochondria, chloroplasts.

10. A kit comprising: e) a device for a container for centrifugation, for separation of a liquid sample to fractions of different density by density centrifugation, liquids of the liquid sample forming a suspension or comprising biological fluids, wherein the device has a partition dividing an interior of the container into an upper chamber and a lower compartment, wherein the partition has an aperture with a guide, along which the liquid sample flows, to the lower chamber of the container for centrifugation; f) at least one medium for density gradient separation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] For a better understanding presented figures illustrate several embodiments of this invention. Presented illustrations do not show all possible embodiments of the invention therefore this invention cannot be limited to solutions presented in illustrations. Illustrations present:

[0032] FIG. 1 illustrates a container in the shape of a centrifuge tube, intended for collecting fluids, especially biological material, it also illustrates the device which together with the container is used for density gradient liquids separation, according to the invention—the device enables layering of liquids in the container, prior centrifugation, one on top of another with maintaining clear interphase between them.

[0033] FIG. 2 and FIG. 3 illustrate respectively a longitudinal sectional view and a side view of a container in the shape of a centrifuge tube, wherein, for a better understanding of the invention—the discs that the partition is built of are spaced apart;

[0034] FIG. 4 and FIG. 5 illustrate respectively a side view and a longitudinal section of the tube-shaped container with visible narrowing of the inner diameter of the tube and with increasing wall thickness.

[0035] FIG. 6 illustrates a cross-section through the container-shaped tubes in the embodiment without vertical partition, and illustrates the air duct in the device partition,

[0036] FIG. 7a and FIG. 7b illustrate respectively a side view and cross-section of the upper part of the device in the form of a disk with incomplete vertical partition

[0037] FIG. 8a and FIG. 8b illustrate respectively a side view and cross-section of the upper part of the device in the form of a disk with vertical partition of rectangular shape,

[0038] FIG. 9a and FIG. 9b illustrate respectively a side view and cross-section of the upper part of the device with vertical partition build of three rectangles,

[0039] FIG. 10a and FIG. 10b illustrate respectively a side view and cross-section of the upper part of the device with vertical partition build of two intersecting rectangles forming a cross shape.

[0040] FIG. 11a and FIG. 11b illustrate a sectional and a side view of the partition disc with a cutout.

[0041] FIG. 12 illustrates one embodiment of the invention, wherein the device is fitted onto the container for centrifugation.

[0042] FIG. 13 and FIG. 13a shows the device in a sectional side and from top view, which allows fitting separate upper chamber on top the device with guide in a form of elongated cylinder,

[0043] FIG. 14 and FIG. 14a and illustrates the device in a sectional side and top view with a guide in the form of eight elongated cylinders.

[0044] FIG. 15 and FIG. 15a shows the insert in a sectional side and top view equipped with a guide in the form of spiral,

[0045] FIG. 16, FIG. 16a and FIG. 16b show the insert in a sectional side, bottom perspective and top view, equipped with a guide in a form of a funnel.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Embodiment 1

[0046] As illustrated on FIG. 1 in the first embodiment of this invention device 6 for the centrifuge container in a form of a centrifuge tube is built of a flat circular disc 7, tightly fitted to the inner walls of the tube partition, and another circular disc 8 which both 7 and 8 constitute the device partition and of a full vertical partition 11 which is attached to disc 8. The device in this embodiment of the invention is placed inside the centrifugation container 1 which is a centrifugation tube with 0.23″ diameter. The device 6 in this embodiment is made of plastic, but could also be made of other materials. A shown in FIG. 12 the device 6 can be placed in another container that can be fitted on to the centrifuge container 1, in this case device is outside of the centrifuge container 1.

[0047] In this embodiment inner walls of the centrifuge container 1 are at the same time the guide 12 and that centrifuge container walls thickens, inner diameter of the centrifuge container decreases gradually toward its' bottom. In this embodiment of the invention inner wall of the container 1 is the guide 12, which directs the down-flow of liquids from upper chamber 2 to the lower chamber 3 via the aperture 4. Liquids—in particular biological fluids being separated to fraction—flow down to the bottom of the container 1 on and along the guide 12—being in this embodiment the internal wall of the container 1- and liquids layer one on top of the another on the bottom of the container 1. Flow-down of liquids along or on the guide 12 prevents mixing of liquids, which otherwise would impair separation of these liquids.

[0048] In this embodiment of this invention partition 7 has shape of circular disc which in transverse section has shape of a circle (FIG. 11a, FIG. 11b) and its' shape is tightly fitted to the transverse section on the container 1, therefore the diameter of the partition is longer on the top side compared to the bottom side, and its' longitudinal section closely resembles the shape of flattened inverted trapezium. Partition 7 divides container 1 to upper chamber 2 and lower chamber 3. Partition in this embodiment has an aperture 4 which is a notch in the shape resembling semicircle.

[0049] As shown on FIGS. 8a and 8b, vertical partition 11 may have a shape of rectangle, which adheres tightly to the inner walls of the container 1, whereupon vertical partition 11 attached to the disc 8 separates upper chamber 2 of the container 1 in the shape of a tube to two sub-chambers 10a, 10b. In each half of the disc 8 shaped by the vertical partition 11 is one aperture 5 in a shape of a notch, which can be closed by disc 8. In this embodiment of the invention apertures 5 in a shape of a notch in disc 8 are in a shape of semicircle. In other embodiments of the invention it is possible to use discs 8 with apertures 5 in different shapes. The shape of apertures 4, 5 and their positioning against each other determines the speed of liquids down-flow from upper chamber 2 to lower chamber 3.

[0050] In this embodiment of the invention apertures 4, 5 are in shape of a semicircular notch with 0.115″ radius and have identical shape. In different embodiments of the invention apertures 4, 5 can have various shapes, and shapes can be different from one another, however their diameter should not be bigger than 0.1″. In such arrangement of the partition 7 and disc 8 that apertures 4, 5 are not overlapping, down-flow of liquids between upper chamber 2 and lower chamber 3 is blocked and flow of liquids cannot take place.

[0051] In this embodiment of the invention container 1 is equipped with lid 9. In one embodiment of the invention lid 9 has a gap, through which protrudes upper part of the vertical partition 11 of the device 6. Such location of the vertical partition 11 enables changes of the position of the disc 8 in relation to disc 7 by turning of the protruding part of the vertical partition 11 and at the same time movable part of the lid 9. Container 1 and lid 9 has a thread and is a nut. Alternatively lid without a gap 91 can be used, wherein vertical partition 11 of the device is adjusted to the length of the container 1 in such a way that after screwing down the lid 9 vertical partition 11 tightly adheres to the inner side of the lid 9. Lid 9 may be made of polymers and can have calibrated scale for turning/screwing the lid 9. On the container 1 for centrifugation and on the lid 9 labels may be present to facilitate correct adjusting/arranging of the apertures 4, 5 positions against each other.

[0052] Alternatively in different embodiments of the invention different shapes and positions of the vertical partition 11. As illustrated in FIGS. 7a and 7b, vertical partition 11 does not have to adhere to the inner walls of the container 1, in which case vertical partition 11 placed on disc 8 separates the tube only to two chambers—upper chamber 2 and lower chamber 3 and upper chamber 2 is not further divided to additional sub-chambers. In this embodiment of the invention, disc 8 is equipped in one aperture 5 in a shape of a notch, in the other embodiment of the invention shape of the disc 8 could be limited to the size that would enable closure of the apertures 4 in the disc 7.

[0053] As illustrated in the FIGS. 9a and 9b, vertical partition 11 can be built of three elements in the shape of a rectangle connected with each other with longer edges, which other edges adhere tightly to the inner wall of the container 1, in this embodiment vertical partition 11 placed on the disc 8 divides upper chamber 2 of the container 1 in the shape of the tube to three sub-chambers. In this embodiment, disc 8 has three notches 5, one in each of the sub-chambers.

[0054] As illustrated in FIGS. 10a and 10b, vertical partition 11 may be built of four rectangles connected with each other, which edges adhere tightly to the inner wall of the container 1, in this embodiment vertical partition 11 placed on the disc 8 divides upper chamber 2 of the container 1 in the shape of the tube to four sub-chambers. In this embodiment, disc 8 has four notches 5, one in each of the sub-chambers.

[0055] Device 6 may also be used in containers 1 shaped differently than centrifuge tube presented in this example of invention embodiment, however there has to be a method that allows to centrifuge this container.

Embodiment 2

[0056] FIGS. 13 and 13a show another embodiment of the invention, wherein the device 6 has a baffle 7, which does not have an upper chamber but allows the connection through a tube (see part 16) down to upper partition in a form of a container (for example, a test tube, pouch, bag) with separation medium or separation liquid. Subsequently, the partition is equipped with a guide 12 in a form of an elongated cylinder which is attached to the partition 7 and is situated at a distance from the aperture 4. This allows fluid flow from the upper chamber 16 through the tube followed by the aperture in the partition along the guide the lower chamber 3. In this embodiment, the elongated cylinder forms a guide 12 and its length is such that the test material spreads gently on a surface of the centrifugal medium used in the gradient separation method and it does not cause significant disturbances to the separation medium.

Embodiment 3

[0057] FIGS. 14 and 14a show another embodiment of the invention, wherein the insert 6 has a partition 7, equipped with a guide 12 in a form of eight elongated rollers which are anchored to partition 7 and are located at such distance from the aperture 4, which allows the liquid to flow from the upper chamber through the aperture, in the partition along the guide, to the lower chamber 3. In this embodiment, the length of the guide for the elongated rollers 12 is such that the test material spreads gently on a surface of the centrifugal medium used in the gradient separation method and it does not cause significant disturbances to the separation medium.

Embodiment 4

[0058] On the other hand, FIGS. 15 and 15a show yet another embodiment of the invention, wherein the device 6 has a partition 7 equipped a guide 12 in the shape of a spiral. In analogy to Example 2, the length of the coil should be such that the test material spreads gently on a surface of the centrifugal medium used in the gradient separation method and it does not cause significant disturbances to the separation medium.

Embodiment 5

[0059] FIGS. 16, 16a and 16b show yet another embodiment of the invention, wherein the insert 6 has a partition 7 provided with a guide 12 in the shape of a funnel. Wherein the four holes in the partition 7 directs the fluids from the upper chamber so as to roll down the outer surface of the funnel to the bottom of the lower chamber 3. In analogy to Example 2, the length of the coil should be such that the test material spread over a surface of the medium to the gradient centrifugation thereby causing no significant adverse to the separation medium.

Embodiment 6

[0060] Method for separation of fractions of given density from fluid sample with fractions of different density according to the invention can be achieved by, filling two sub-chambers 10a, 10b of the upper chamber 2 with two media for separation in on density gradient, first medium has density of 1.119 g/mL second medium has density of 1.077 g/mL (respectively Histopaque 1.119 and Histopaque 1.007 Sigma Aldrich), at the same time apertures 4, 5 being notches—respectively in disc 7 and disc 8—are not overlapping and remain in closed position. Next by changing the position of disc 8 by its' turning, apertures 4, 5 overlap each other in such a way that enables down-flow of mediums from the upper chamber 2 to the lower chamber 3. Down-flow occurs on and along the guide 12 which in this embodiment is the internal wall of the container 1. Media are added one by one starting from the highest density to the lowest density, and interface is established between media of different densities. Next to one of the empty sub-chambers 10, with closed down-flow between the upper chamber 2 and the lower chamber 3, fluid or mixture designated to be separated to fractions of different densities in density gradient centrifugation e.g. native or diluted blood.

[0061] The size of the clearance created by apertures 4, 5 being the notches of respectively disc 7 and disc 8 can be controlled by regulation of positions of disc 7 and disc 8 against each other. Slow turning of the upper part of the vertical partition 11, and subsequently disc 8, causes gradual increase of the down-flow velocity up to the moment when expected velocity, of liquid down-flow from the upper chamber 2 to the lower chamber 3, is achieved. By regulation of positions of disc 7 and disc 8 against each other, liquid down-flow can be controlled in order to achieve stable laminar flow of liquid on and along the internal wall 12 of the centrifuge container 1. Construction of discs 7 and disc 8 according to the invention ensures very gentle down-flow of the liquid from the upper chamber 2 to the lower chamber 3 of the centrifuge container 1 in such a way that the surface of the liquid is intact and subsequently added liquids which down-flows from the upper chamber 2 does not mix with the liquid already present in the lower chamber 3.

[0062] After stratified down-flow of the two liquids for separation on density gradient these liquids layer one on top of the another because of different density, analyzed sample was added—blood in this case—although it is possible to use different types of separation liquids, including native or diluted biological samples. Blood was first placed in sub-chamber 10a, and next after turning the disc 8 of the device 6 in such a way that aperture 4 of the disc 7 was overlapping at least partially with respective aperture 5 in the disc 8 of the device 6 and enables down-flow of the blood on and along the inner wall 12 of the container 1 from the sub-chamber 10a to the lower chamber 3 layering it on the surface of previously placed separation media. Because of the device 6 construction it is not necessary to place the biological material in the container 1 with extraordinary precision and care.

[0063] Next blood in lower chamber 3 of the container 1 is centrifuged according to methods known in the field. During centrifugation two directional flow of liquids occurs within different compartments created by separatin liquids of different density in the lower chamber 3, at the end of centrifugation continuous density gradient establishes with red blood cells sedimenting to the bottom creating lowest placed layer, layer above is a liquid of 1.119 g/mL density, layer above is layer of polymorphonuclear cells, layer above is a liquid of 1.077 g/mL density, layer above is layer of peripheral blood mononuclear cells, layer above is the highest layer of plasma. After removing of the insert, each layer of cells/or fluid can be removed by aspiration with the use of a pipet or by decantation.

Embodiment 7

[0064] Insert and method of the invention is used, for example, for separating the desired subset of blood cells. In this embodiment of ten samples of blood were taken from healthy volunteers (20 ml of venous blood) to a commercially available tubes with versene acid (EDTA) (EDTA tube, Becton Dickinson). In this experiment, the volume of the centrifuge tube 1 of which the essence of the invention was 50 ml, was also used for the separation of two media of different densities (Histopaque 1119 and Histopaque 1077 Sigma Aldrich). For the separation of fluids used have a neutral pH, be isotonic to body fluids, the first separation medium to have a density of 1.119 g/ml, while the second had a density of 1.077 g/ml.

[0065] Then 10 ml of a medium provided for the separation of a density of 1.119 g/ml in sub-chamber 10a into the upper chamber 2 of the container 1 for centrifugation provided with the device 6 of the invention. A second fluid having a density of 1.077 g/ml with a volume of 10 ml was placed in sub-chamber 10b of the upper chamber 2, and then laminated imposed by the first medium by means of an insert 6 of the invention described above. In the experiment divider had a thickness of 0.08′ and the cutouts 4, 5, 7 and the baffle disc 8 have a radius of 0.115″. Then, the collected blood is versene acid (EDTA) provided in the upper sub-chamber 10a of the chamber 2. Each blood sample was applied to the surface layer media separation by the insert 6 of the invention described above.

[0066] In a further step, all tubes were centrifuged at 700 g (with minimal acceleration and without active braking) for 30 minutes at room temperature. In the process of density centrifugation, the blood was separated into four fractions: plasma, mononuclear white blood cells (PBMC), white blood cells with a segmented nucleus (PMN), and Czerwonki cells. Purity fraction of PBMC and PMN was confirmed by flow cytometry. Purity PBMC and PMN in the fractions was 95% and 92%. PBMC and PMN were undetectable in plasma fractions. Isolated plasma, PBMC and PMN were suitable for further analysis, including, but not limited to aPatryk, nalysis: RNA, micro-RNA, mitochondrial DNA, nuclear DNA, proteins and phenotyping of the cells.