EP3349897B1 - Dispositif et procede de separation de liquides par centrifugation en dependance de leur densité - Google Patents

Dispositif et procede de separation de liquides par centrifugation en dependance de leur densité Download PDF

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Publication number
EP3349897B1
EP3349897B1 EP16791676.6A EP16791676A EP3349897B1 EP 3349897 B1 EP3349897 B1 EP 3349897B1 EP 16791676 A EP16791676 A EP 16791676A EP 3349897 B1 EP3349897 B1 EP 3349897B1
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Prior art keywords
container
partition
separation
centrifugation
density
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German (de)
English (en)
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EP3349897B8 (fr
EP3349897A1 (fr
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Mateusz Grzegorz ADAMSKI
Patryk GUMANN
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Spark Tech Sp zoo
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Spark Tech Sp zoo
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Priority claimed from PCT/IB2016/055503 external-priority patent/WO2017046736A1/fr
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5021—Test tubes specially adapted for centrifugation purposes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0609—Holders integrated in container to position an object
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0644—Valves, specific forms thereof with moving parts rotary valves

Definitions

  • the invention relates to a centrifugation container with an insert, and a method of fluid separation using a density gradient centrifugation.
  • the invention is used in the separation of body fluids, e.g. blood of animals, including humans, for diagnostic purposes.
  • the solution which is the subject of the invention belongs to the field of containers for laboratory purposes, and especially to tubes specifically adapted for centrifugation purposes.
  • 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 analyzing liquid biological material, e.g., blood.
  • Collection, purification, fractionation and/or fixation of body fluid samples, including blood play an important role, e.g. in medical diagnostics and clinical trials.
  • blood samples collected from the patient can be separated into different fractions by centrifugation, filtration or elutriation, and then stored for later use or further testing.
  • Separated blood components usually contain fractions of red blood cells, white blood cells, platelets and plasma. Separation of blood into its fractions can be carried out continuously, during blood collection or in the stages following its collection. Separating blood into different components under highly sterile conditions is critical for many therapeutic applications and for clinical research purposes.
  • WO8805331 there is a technique known for separating white blood cells (leukocytes) from red blood cells (erythrocytes), which consists of mixing a blood sample with a working solution that aggregates red blood cells and thus increases their sedimentation rate.
  • the density of the working separation fluids is selected so that the sedimentation of white blood cells is minimally changed and that the white blood cells do not sediment to the bottom, and as a result could be taken from the upper part of the separated liquid after the red blood cells have sediment to the bottom.
  • the blood is layered precisely on the surfaces of separation fluids, after which the red blood cells agglutinate or aggregate under the effect of surface contact with the working separation fluids, as a result of which they sediment to the bottom of that tube.
  • separation fluids There are several well-known multi-polymer compounds that agglutinate red blood cells, e.g. FICOLL 400 (Pharmacia Fine Chemicals, Sweden). Blood separation can occur under the influence of gravity or under the influence of centrifugation.
  • PBMC peripheral blood mononuclear cell
  • PMN polymorphonuclear leukocytes
  • one known method is to isolate peripheral blood mononuclear cell (PBMC) based on a centrifugation process, wherein the first stage uses the Isopaque-Ficoll mixture (Nyegaard & Co., Norway) having the sodium metrizoate component, in the next stage, 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.
  • U.S. Patent Application No. US4824560 A discloses methods and means of centrifuging in a tubular container having at least two adjacent chambers that are connected to each other by a narrow, essentially capillary opening.
  • the working fluid is placed in the bottom chamber, while the fluid to be separated into fractions is placed in the upper chamber, and there is no need for special precautions to avoid liquid mixing before starting centrifugation.
  • This method has several advantages over the manual methods described above, it also has the disadvantage due to the narrow connection of both chambers which constitutes a partial barrier, even during centrifugation, This barrier prevents effective passage of blood cells between the chambers and precludes separation of the blood into fractions.
  • Patent application US2014087360 A1 discloses an insert for a centrifuge tube suitable for use in density gradient separation.
  • the insert includes a member sized to fit within the tube for dividing the tube into a top portion and a bottom portion.
  • the insert has a support extending or depending from the member for positioning the member within the tube. At least two openings are located on the member so that a first opening is closer to a bottom end of the tube relative to a second opening when the insert is positioned in the centrifuge tube.
  • Patent US5314074 A discloses layering insert within the vessel that prevents back mixing after density gradient centrifugation.
  • the insert is capable of supporting the body of liquid dispersion to be centrifuged under regular gravity condition and enables bi-directional crossflow during centrifugation.
  • Patent US5132232 A discloses apparatus for collecting liquid analytical samples for examination, and reproducibly separating them into two or more fractions.
  • An apparatus is utilizing a container and a petter, said petter provided with an upper portion which enables attachment to the container, a lower retainer portion including a peripheral aperture, the non-apertured portion of which enables wedge-fit thereof into the container, and a body portion connecting the upper petter portion and lower retainer portion.
  • Patent US5648223 A discloses a cell-trap centrifugation tube containing a specific density gradient solution adjusted to a specific density to enrich for breast tumor cells from a cell mixture.
  • the tube allows the desired cell population to be collected by decantation after centrifugation to minimize cell loss and maximize efficiency.
  • the aim of the solution according to the invention was to obtain a tool for fast and partially automated separation of fluids into fractions of different density, e.g. biological fluids, including blood, which additionally enable the purification, isolation and fixation of biological samples.
  • fluids e.g. biological fluids, including blood
  • the essence of the invention is a centrifugation container comprising an insert, especially a test tube, for separation of 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 suitable for dividing the container's interior into at least two chambers in a vertical arrangement - the upper chamber and the bottom chamber, characterized in that the partition having an opening into which a guide adjoins, on which liquids, especially biological fluid, flow into the bottom chamber of the centrifugation container, and said partition is made of two adjacent surfaces with openings, especially in the shape of flattened discs fitted to the cross-section of the container with a cross-section similar to the circle, the surfaces being movably connected with each other, they can be freely positioned with each other enabling closing of the opening lumen.
  • the guide is spiral, funnel or vertical elements in the shape of an elongated cylinder.
  • the upper chamber of centrifugation container has additionally a vertical partition or partitions separating it into sub-chambers, each of the sub-chambers having an opening.
  • the invention also includes a method of separating a fraction with a desired density range from a sample containing fractions of different density, especially from a biological sample, comprising:
  • the step (b) is followed by an additional step or steps b) consisting of adding an additional density gradient separation medium, wherein the addition of subsequent media is from the highest to the lowest density.
  • step (d) selected fractions of different density from the liquid being separated are subjected to tests and analysis, including the possibility of being fixed, especially by the freezing method.
  • 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.
  • 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:
  • the tube insert 6 for the centrifugation container consists of a partition 7 in the form of a flat disc tightly adhering to the inner walls of the container 1, and a disc 8 equipped with a full vertical partition 11.
  • the insert in this example is placed inside a container 1 constituting a 0.23" diameter centrifugation tube.
  • Insert 6 in this example is made of plastic, but could also be made of other materials.
  • the insert 6 can also be placed in an additional container that can be attached to the centrifugation container 1, then the insert 6 is outside the container 1.
  • the wall of the tube-shaped container 1 forms a guide 12 and widens gradually into the container 1 ( Fig. 4 and 5 ), at the same time the lumen of the tube gradually decreases towards its bottom.
  • the inner wall of the container 1 is a guide 12 that allows liquids to flow from the upper chamber 2 to the bottom chamber 3 through the opening 4.
  • Liquids - especially liquids that are biological fluids for separation - flow down to the bottom of the container 1 along the guide 12, constituted by container wall, and become arranged in layers on the bottom of the container 1.
  • the liquid flow down along the guide 12 prevents agitation of the separation liquids, which could cause errors in the separation of the tested liquids.
  • the partition 7 has the shape of a flat disc with a circular cross-section ( Fig. 11a, Fig. 11b ) and is closely fitted in shape to the cross-sectional shape of the container 1, so the diameter on its top side is greater than on the bottom side, and the longitudinal section of the partition 7 is close to a flattened inverted trapezoid.
  • the partition 7 divides the container 1 into an upper chamber 2 and a bottom chamber 3.
  • the partition in the example has an opening 4 constituting an indentation in the shape similar to a semi-circle.
  • the vertical partition 11 may be in the shape of a rectangle that closely adheres to the inner wall of the container 1, then the vertical partition 11 located on the disc 8 divides the upper chamber 2 of the container 1 in the shape of a tube into two sub-chambers 10a, 10b .
  • the indentation-shaped openings 5 in the disc 8 are semicircular.
  • the liquid flow rate from the upper chamber 2 to the bottom chamber 3 depends on the shape of the openings 4, 5 and their mutual arrangement with respect to each other.
  • the indentation-shaped openings 4, 5 with an indentation radius of 0.115" are of the same shape.
  • the openings 4, 5 may have different shapes and may differ in shape, but their diameter should be greater than 0.1".
  • the container 1 is provided with a lid 9.
  • the lid 9 has a gap in which the upper part of the vertical partition 11 of the insert 6 fits and passes through. Such a position of the vertical partition 11 allows changing the position of the disc 8 relative to the partition 7 by turning the protruding part of the partition, and thus the movable part of the lid 9.
  • the container 1 and the lid 9 have a thread and form a screw cap.
  • the lid 9 may be made of plastic and may have a calibrated turning/twisting scale. Markings on the centrifugation container 1 and lid 9 may be provided to facilitate correct alignment/arrangement of the openings 4, 5 in relation to each other.
  • the vertical partition 11 may not adhere to the walls of the container 1, then the vertical partition 11 located on the disc 8 divides the tube into two chambers only - the upper chamber 2 and the bottom chamber 3, and the upper chamber 2 is not divided into additional sub-chambers.
  • disc 8 is equipped with one indentation-shaped opening 4, wherein the shape of the disc 8 in another version of the example could be limited to closing the openings 4 in the partition 7.
  • the vertical partition 11 may consist of three rectangular elements connected together by longer sides, the sides of which are closely adjacent to the inner wall of the container 1, then the vertical partition 11 located on the disc 8 divides the upper chamber 2 of the container 1 in the shape of a tube into three sub-chambers.
  • disc 8 is equipped with three indentations 5, one in each of the sub-chambers.
  • the vertical partition 11 may consist of four rectangles connected together, the sides of which are closely adjacent to the inner wall of the container 1, then the vertical partition 11 located on the disc 8 divides the upper chamber 2 of the container 1 in the shape of a tube into four sub-chambers.
  • disc 8 is equipped with four indentations 5, one in each of the sub-chambers.
  • Insert 6 can also be used in containers 1 with shapes other than the centrifugation tube shown in the embodiment, but these containers must be suitable for centrifugation.
  • Fig. 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 upper chamber partition in the form of a container (e.g. test tube, pouch) with a medium for separation or with liquid for separation.
  • the partition is equipped with a guide 12 in the shape of an elongated cylinder, which is attached to the partition 7 and is located at such a distance from the opening 4 that allows liquid to flow from the upper chamber through the tube 16, then through the opening in the partition and along the guide to the bottom chamber 3.
  • the elongated cylinder constituting the guide 12 is of such a length that the test material layers onto the surface of the gradient medium without causing significant disturbances in the separation medium.
  • Fig. 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 shape of 8 elongated cylinders, which are attached to the partition 7 and are at a distance from the opening 4 allowing liquid to flow from the upper chamber through the opening in the partition following the guide to the bottom chamber 3.
  • the elongated cylinders constituting the guide 12 are of such a length that the test material layers onto the surface of the gradient centrifuging medium without causing significant disturbances of the separation medium.
  • Fig. 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 spiral-shaped guide 12.
  • the spiral length should be such that the test material layers onto the surface of the gradient centrifuging medium without causing significant disturbances in the separation medium.
  • Fig. 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 the partition 7 direct liquids from the upper chamber so that they flow down along the outer surface of the funnel to the bottom of the chamber 3. Similarly, to example 2, the spiral length should be such that the test material layers onto the surface of the gradient centrifuging medium without causing significant disturbances in the separation medium.
  • the method of separating the fraction with the desired density range from a sample containing fractions of different density according to the invention can be carried out in such a way that in different sub-chambers 10a, 10b of upper chamber 2, there are two 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), with the openings 4,5 constituting indentations - of the partition 7 and disc 8, respectively - not overlapping when remaining in the closed position.
  • the indentations 4, 5 overlap enough to allow the medium to flow from the upper chamber 2 to the bottom chamber 3.
  • the liquids flow along the inner wall of the container 1 what constitutes the guide 12.
  • the position of the openings 4,5 constituting an indentation in the partition 7 and the disc 8 from the example is adjacent to the the wall of the container 1 what constitutes the guide 12.
  • the media are added in order from highest to lowest density, and a interphase is formed between the media of different density.
  • a liquid or mixture intended for separation into fractions of different density under the influence of centrifugation, e.g. native or diluted blood, is added to one of the empty sub-chambers 10 with a closed flow between the upper chamber 2 and the bottom chamber 3.
  • the size of the clearance formed by the openings 4, 5 being cutouts of the partition 7 and the disc 8, respectively, can be controlled by adjusting the mutual position of the partition 7 and the disc 8. Slowly twisting the upper part of the insert 11, and thus the disc 8, gradually increases the fluid flow rate until the assumed liquid flow velocity between the chambers 2, 3 is achieved. By adjusting the amount of mutual overlap between the opening 4 and the opening 5, the liquid flow can be controlled to create a continuous laminar flow along the wall 12 of the centrifugation container 1.
  • the construction of the partition 7 and disc 8 according to the invention ensures very smooth down flow of fluid from the upper chamber 2 into the bottom chamber 3 of the centrifugation container 1 in such a way that the interface between liquids is intact and the subsequent liquid poured from the upper chamber 2 does not mix with the liquid present in bottom chamber 3.
  • 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 turning the disc 8 of the insert 6 in such a way that the opening 4 of the partition 7 matches at least in part with the corresponding opening 5 of the disc 8 of the insert 6, and allows blood to flow along the wall 12 of the container 1 from the sub-chamber 10a to the bottom chamber 3 and to layer on the surface of separation media placed there previously. Due to the structure of the insert 6, it is not necessary for the biological specimen to be placed in container 1 with extreme precision and care.
  • the blood in the bottom chamber 3 of the container 1 is then centrifuged according to methods generally known in the art.
  • centrifugation two-way fluid flow occurs within different compartments formed by separation fluids of different density in the bottom chamber 3, and a continuous density gradient is formed at the end of centrifugation, with red blood cells settling on the bottom forming the lowest-located layer, the higher layer is a liquid with a density of 1.119 g/mL, the next layer located above is white blood cells with segmented nuclei, above is a layer formed by liquid with a density of 1.077 g/mL, and a layer of white mononuclear cells above that, finally the plasma is located above them as the highest layer.
  • each of the cell layers/or liquids can be removed by aspiration using a pipette or decantation.
  • the insert and method of the invention is used, for example, to separate a desired subset of blood cells.
  • ten blood samples were collected from healthy volunteers (20 mL of venous blood) into commercially available ethylenediaminetetraacetic acid (EDTA) tubes (EDTA tube, Becton Dickinson).
  • EDTA ethylenediaminetetraacetic acid
  • the volume of the centrifugation tube 1 constituting the invention was 50 mL
  • two separation media of different density Histopaque 1.119 and Histopaque 1.077 Sigma Aldrich
  • the separation media used had a neutral pH, were isotonic to body fluids, the first separation medium had a density of 1.119 g/mL, while the second had a density of 1.077 g/mL.
  • PBMC peripheral blood mononuclear cells
  • PMN polymorphonuclear leukocytes
  • 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, PBMC and PMN were suitable for further analysis, including but not limited to: RNA, micro-RNA, mitochondrial DNA, nuclear DNA, protein and cell phenotyping.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Centrifugal Separators (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • External Artificial Organs (AREA)

Claims (7)

  1. Récipient de centrifugation (1) comprenant un insert (6), notamment un tube à essai, pour la séparation de liquides en fractions de la gamme de densité souhaitée par centrifugation à gradient de densité, notamment de liquides constituant des suspensions et/ou des fluides biologiques, l'insert est équipé avec une cloison (7) adaptée pour diviser l'intérieur du conteneur (1) en au moins deux chambres disposées verticalement - la chambre supérieure (2) et la chambre inférieure (3), caractérisée en ce que la cloison (7) présente une ouverture (4) auquel se raccorde un guide (12) sur lequel des liquides, notamment du fluide biologique, s'écoulent dans la chambre inférieure (3) du récipient de centrifugation (1), et ladite cloison (7) est constituée de deux surfaces adjacentes percées d'ouvertures , notamment en forme de disques aplatis adaptés à la section transversale du conteneur (1) de section similaire au cercle, les surfaces étant reliées mobiles entre elles, elles peuvent être librement positionnées l'une avec l'autre permettant la fermeture de e e ouverture (4) lumière.
  2. Récipient de centrifugation (1) comportant l'insert (6) selon la revendication 1 caractérisé en ce que le guide (12) est une spirale, un entonnoir ou des éléments verticaux en forme de cylindre allongé.
  3. Récipient de centrifugation (1) comportant l'insert (6) selon la revendication 1 caractérisé en ce que la chambre supérieure (2) comporte en outre une cloison verticale (11) ou des cloisons la séparant en sous-chambres (10), chacune des des sous-chambres (10) ayant une ouverture (4, 5).
  4. Procédé de séparation d'une fraction avec une gamme de densité souhaitée à partir d'un échantillon contenant des fractions de densité différente, notamment à partir d'un échantillon biologique, comprenant :
    a) fournir un récipient de centrifugation (1) selon l'une quelconque des revendications 1 à 3 pour la séparation de liquides en fractions de la plage de densité souhaitée par centrifugation à gradient de densité, en particulier de liquides constituant des suspensions ou des fluides biologiques,
    b) remplir la chambre inférieure (3) du conteneur (1) avec un milieu de séparation dans un gradient de densité ou remplir la chambre supérieure (2) du conteneur (1) avec ce milieu, qui s'écoule ensuite par l'ouverture dans le cloison le long du guide (12) jusqu'à la chambre inférieure ;
    c) verser du liquide de séparation contenant des fractions de densité différente dans la chambre inférieure (3) en remplissant la chambre supérieure (2) ou au moins une sous-chambre (10a, 10b) ou en attachant la chambre supérieure à la cloison, de sorte que le le liquide peut s'écouler à travers l'ouverture (4) de la cloison sur le guide (12) et se répandre à la surface de la chambre inférieure (3) du média de séparation ;
    d) centrifugation du récipient (1) jusqu'à ce que l'échantillon d'essai soit séparé en fractions de densité différente.
  5. Procédé selon la revendication 4, caractérisé en ce qu'après l'étape (b) est suivie d'une ou des étapes supplémentaires b) consistant à ajouter un milieu supplémentaire de séparation à gradient de densité, dans lequel l'ajout de milieux ultérieurs se fait du plus haut au plus densité la plus faible.
  6. Procédé selon la revendication 4, caractérisé en ce qu'après l'étape (d), des fractions sélectionnées de densité différente du liquide à séparer sont soumises à des tests et analyses, incluant la possibilité d'être fixées, notamment par la méthode de congélation.
  7. Procédé selon la revendication 6, caractérisé en ce que lorsque la fraction est séparée du liquide sanguin, les fractions individuelles de la gamme de densité souhaitée contiennent divers composants sanguins, notamment : leucocytes (lymphocytes et granulocytes), plaquettes, érythrocytes, moelle osseuse cellules (mégacaryocytes, érythroblastes) en suspension dans un homogénat cellulaire comprenant des cellules endothéliales, des neurones, des champignons, des virus, des microparticules comprenant des exosomes, des fragments cellulaires, des organites cellulaires comprenant des noyaux, des mitochondries, des chloroplastes.
EP16791676.6A 2015-09-15 2016-09-15 Dispositif et procede de separation de liquides par centrifugation en dependance de leur densité Active EP3349897B8 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PL413910A PL237582B1 (pl) 2015-09-15 2015-09-15 Insert do pojemnika do wirowania, zwłaszcza, do probówki, do wydzielenia frakcji o pożądanym zakresie gęstości metodą wirowania na gradiencie gęstości oraz pojemnik do wirowania zawierający ten insert
PCT/IB2016/055503 WO2017046736A1 (fr) 2015-09-15 2016-09-15 Dispositif et procédé pour la séparation de fluides par gradient de densité

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EP3349897A1 EP3349897A1 (fr) 2018-07-25
EP3349897B1 true EP3349897B1 (fr) 2022-01-26
EP3349897B8 EP3349897B8 (fr) 2022-02-16

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CN (1) CN108367288A (fr)
DK (1) DK3349897T3 (fr)
ES (1) ES2910923T3 (fr)
PL (1) PL237582B1 (fr)

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US20210220817A1 (en) * 2018-12-08 2021-07-22 Min Wei Apparatus For Manufacturing Cell Therapy Product
TWI683702B (zh) * 2019-01-31 2020-02-01 瑩芳有限公司 採檢容器
SE545603C2 (en) * 2019-08-22 2023-11-07 Grimaldi Dev Ab Separating particles through centrifugal sedimentation
PL434517A1 (pl) * 2020-06-30 2022-01-03 Spark-Tech Spółka Z Ograniczoną Odpowiedzialnością Urządzenie do kontrolowanego warstwowego nalewania cieczy
CN113358451A (zh) * 2021-03-30 2021-09-07 刘开 一种新型离心层析提取器

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5132232A (en) * 1985-07-30 1992-07-21 V-Tech, Inc. Method and apparatus for preparation of liquids for examination
IL100828A (en) * 1992-01-31 2002-05-23 Novamed Ltd Method and means for density gradient centrifugation
US5648223A (en) * 1994-08-31 1997-07-15 Activated Cell Therapy, Inc. Methods for enriching breast tumor cells
US6516953B1 (en) * 1998-12-05 2003-02-11 Becton, Dickinson And Company Device for separating components of a fluid sample
US7074577B2 (en) * 2002-10-03 2006-07-11 Battelle Memorial Institute Buffy coat tube and float system and method
JP4853295B2 (ja) * 2007-01-11 2012-01-11 株式会社島津製作所 遠心分離機の遠沈管
US9683983B2 (en) * 2011-05-05 2017-06-20 Stemcell Technologies Inc. Method and insert for density gradient separation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NYSTAD BENT HELGE ET AL: "Technical Condition Assessment and Remaining Useful Life Estimation of Choke Valves subject to Erosion", 1 January 2010 (2010-01-01), XP055790099, Retrieved from the Internet <URL:https://www.researchgate.net/publication/272741419_Technical_Condition_Assessment_and_Remaining_Useful_Life_Estimation_of_Choke_Valves_subject_to_Erosion#fullTextFileContent> [retrieved on 20210325], DOI: 10.13140/rg.2.1.1564.3684 *

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DK3349897T3 (da) 2022-04-04
PL237582B1 (pl) 2021-05-04
EP3349897B8 (fr) 2022-02-16
EP3349897A1 (fr) 2018-07-25
ES2910923T3 (es) 2022-05-17
PL413910A1 (pl) 2017-03-27
CN108367288A (zh) 2018-08-03

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