EP4048444B1 - Dispositif pour la stratification commandée de liquides - Google Patents

Dispositif pour la stratification commandée de liquides Download PDF

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Publication number
EP4048444B1
EP4048444B1 EP21755843.6A EP21755843A EP4048444B1 EP 4048444 B1 EP4048444 B1 EP 4048444B1 EP 21755843 A EP21755843 A EP 21755843A EP 4048444 B1 EP4048444 B1 EP 4048444B1
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EP
European Patent Office
Prior art keywords
flow
piston
chamber
wall
flow segment
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EP21755843.6A
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German (de)
English (en)
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EP4048444A1 (fr
Inventor
Mateusz ADAMSKI
Jan ZAROW
Tomasz FUJAWA
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Spark Tech Sp zoo
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Spark Tech Sp zoo
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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
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0621—Control of the sequence of chambers filled or emptied
    • 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
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0694—Creating chemical gradients in a fluid
    • 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
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • 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
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • 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
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • 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
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
    • 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
    • B01L2400/0638—Valves, specific forms thereof with moving parts membrane valves, flap valves

Definitions

  • the invention relates to a device for the controlled layering of liquids, in particular for layering of biological liquids prior separation by a density gradient centrifugation.
  • European patent application EP3349897 A1 discloses an insert for a tube for separating blood into fractions of a desired density range by density gradient centrifugation, which insert is equipped with a partition dividing the interior of the container into at least two chambers in a vertical arrangement-the upper chamber and the lower chamber. Additionally, the partition has an opening, against which the guide adjoins, on which liquids, especially biological fluid, flow into a lower chamber of the centrifugation container.
  • This application also discloses a method utilizing this insert for separating fluids using a density gradient centrifugation.
  • a significant limitation of the above invention is the necessity to ensure tightness between the upper and lower chambers.
  • patent US2014/087360 discloses an insert and a method for separating blood into blood cells populations using density gradient centrifugation in a centrifuge container.
  • the insert consists of a partition that matches the shape of the inside of the centrifuge container.
  • the partition divides the centrifuge container into an upper and a lower compartment.
  • the partition is provided with a support element below or above the partition for positioning the insert in the centrifuge container.
  • the insert is provided with at least two openings, one of which is located closer to the bottom of the centrifuge container.
  • the patent also describes a method of isolating a cell populations from a sample by density gradient centrifugation.
  • Patent US5132232A discloses apparatus and methods for collecting liquid samples, particularly blood and urine, for testing and for separating samples into fractions.
  • the apparatus consists of a centrifuge tube and an insert.
  • the insert is divided into an upper part that allows the insert to be attached to the centrifuge container and a lower part, which is an incomplete partition with an opening.
  • the full part of the partition wedges to the inside of the centrifuge container, and the opening in the partition allows for fluid collection from under the partition.
  • Invention in the patent US5314074A discloses the method for separating liquids with density gradient centrifugation with the use of an insert.
  • the insert is in the form of a disk fitted to the inside of the centrifuge container with a plurality of capillary holes and cylindrical indentations at the edges of the disk.
  • the insert When placed in the centrifuge container the insert divides the centrifuge container into upper and lower chambers, holes and indentations in the insert enable continuous bidirectional flow of fluids between the chambers.
  • patent US5648223A describes a tube and methods for concentrating breast cancer cells using density gradient centrifugation.
  • the tube is adapted to density gradient centrifugation, additionally it is equipped with transverse constriction members narrowing its lumen.
  • the constriction members divide the tube into chambers connected to each other by permanent openings.
  • the separation container may include a body defining an internal chamber.
  • the body may define an opening, and the body may be configured to receive the sample within the internal chamber.
  • the separation container may further include a seal disposed across the opening, such that the seal may be configured to seal the opening of the body, and a plunger movably disposed at least partially inside the internal chamber.
  • the present invention aims to eliminate the above-mentioned limitations.
  • the purpose has been to: increase the possibility of regulating the flow rate of liquid, reduce both number and contact surface of the elements necessary to maintain the tightness of the system.
  • the purpose has also been to increase the precision of initial liquid flow velocity regulation through the device.
  • the aim of the invention is a device for controlled layering of liquids, in particular for layering liquids prior separation with density gradient centrifugation, the device comprising a loading chamber, and a draining chamber which includes a cylinder-shaped flow segment, and a liquid flow regulating element to regulate a liquid flow from the loading chamber to the draining chamber, characterised in that the fluid flow regulating element comprises a piston type mechanism and at least one flow transfer channel in a wall of the flow segment, the piston type mechanism comprises a piston axially movable with respect to the flow segment and the piston beeing provided with a shaft and a sealing lip tightly adhering to an inner surface of the flow segment wall, and wherein the piston has a vent in the form of a channel through its entire length, and wherein the flow transfer channel is in the form of a groove in the inner surface of the wall of the flow segment or channel in the wall of the flow segment, an inlet of the channel is located in the inner wall surface of the flow segment and an outlet of the channel in the bottom wall of the flow segment
  • the cross-sectional area of the flow transfer channel increases from the inlet to the outlet, especially in the range from approx. 1 mm 2 .
  • the draining chamber of the device for controlled layering of liquids is provided with an adapter cap for the container, in particular a container for density gradient centrifugation. It is particularly preferred if the adapter cap is provided with a thread, especially a thread that is aligned with cap thread of the container for density gradient centrifugation.
  • the sealing lip of device for controlled layering of liquids has an axial cross-section similar to letter C, ensuring self-tightness between the sealing lip and the inner wall of the flow segment preventing uncontrolled flow of liquid from the feeding chamber to the drainage chamber.
  • the sealing lip is provided with a sealing ring, in particular a rubber or silicone sealing ring placed in a groove on the sealing lip, ensuring tightness by itself by preventing uncontrolled flow of liquid from the loading chamber into the draining chamber.
  • a sealing ring prevents leaks that may occur when the flow segment and the sealing lip on the piston are made with less precision, e.g. in the process of plastic injection moulding.
  • the device for controlled layering of liquids is further provided with a piston guide fixedly connected to the loading or draining chamber, the piston guide enables only axial movement of the piston in the lumen of the flow segment. It is particularly preferred if the connection of the piston guide to the inner wall of the loading chamber or the draining chamber is made of at least one structural rib. It is also particularly preferred if both the piston and the guide are equipped with threads aligned with each other in the form of the piston thread and the guide thread.
  • the invention also relates to a density gradient centrifugation assembly characterised in that it comprises a device for controlled layering of liquids according to the invention and a centrifuge container.
  • a density gradient centrifugation assembly characterised in that it comprises a device for controlled layering of liquids according to the invention and a centrifuge container.
  • the difference between the sum of the aperture radius of the flow segment and the depth of the flow transfer channel compared to the aperture radius of the centrifuge container is not more than 0.5 mm.
  • the advantages of the solution according to the invention include: the possibility of precise regulation of the flow rate of liquid through the system, reduction of the contact area for elements requiring tightness and reduction in their number, this reduces the production costs of the device, extends its durability and increases repeatability of layering liquids.
  • An advantage of the solution according to the invention is also the possibility to precisely determine the lumen of the opening, either as the groove in the inner surface of the wall of the flow segment or channel in the wall of the flow segment, and thus to control the initial velocity of the liquid flow through the device.
  • the device for controlled layering of liquids in an exemplary embodiment includes a loading chamber 11 communicating with a draining chamber 40, which includes a cylinder-shaped flow segment 31.
  • the device is equipped with an element for regulating the flow of liquid from the loading chamber 11 to the draining chamber 40.
  • the liquid flow regulating element consists of a piston type mechanism 19 and a flow transfer channel 32 located in the wall of the flow segment 31.
  • the piston type mechanism 19 includes a flow segment 31 and a movable piston 20 equipped with a shaft 23 and a sealing lip 22.
  • the sealing lip 22 tightly adheres to the inner wall surface of the flow segment 31 and prevents uncontrolled flow of liquid from the loading chamber 11 into the drain cha draining chamber 40.
  • the flow transfer channel 32 in the embodiment shown in Fig. 1 , 2 , and 3 are in the form of a groove in the inner wall surface of the flow segment 31. It is be appreciated that the flow transfer channel 32 may have other form, for example channel 33, where channel inlet 34 is located in the inner surface of the wall of the flow segment 31 and channel outlet 35 is located in the bottom wall of the flow segment 31, as illustrated in Fig. 6 . The inlet of the flow channel 32 is below the junction of the loading chamber 11 and the flow segment 31.
  • the cross-sectional area of the flow transfer channel 32 is variable, at channel inlet 34 it is 1 mm 2 , increases towards its outlet and terminates at the bottom edge.
  • the draining chamber 40 is provided with an adapter cap 41 with threads 42, aligned with density gradient centrifuge container cap thread, what enables tight attachment.
  • Outer wall of the flow transfer channel 32 is aligned with the inner wall of the centrifuge container 61.
  • the shape of the cross-section of the channel is selected to maintain the stiffness of the piston 20.
  • the sealing lip 22 of the piston 20 in the exemplary embodiment has an axial cross-section of a shape similar to an overturned letter C, what reduces potential leaks between the sealing lip 22 and the inner surface of the flow segment 31.
  • the sealing lip 22 is made of medical polypropylene.
  • the piston 20 has an axial vent 25 in the form of a channel through its entire length. This channel allows outflow of air from the draining chamber 40.
  • the sealing lip 22 of the piston 20 may be provided with the sealing ring 26, this further reduces potential leaks between the sealing lip 22 and the inner surface of the flow segment 31.
  • the sealing ring 26 should be made of a material with greater flexibility than the piston, e.g. rubber or silicone.
  • the device is additionally equipped with the piston guide 12 fixedly connected by four structural ribs 14 to the loading chamber 11.
  • the piston guide 12 enables more precise axial sliding movement of the piston 20 in the lumen of the flow segment 31.
  • the piston 20 and the guide 12 may be equipped with threads aligned with each other in the form of the piston thread 21 and the guide thread 13. The threading increases the precision of liquid flow through the flow transfer channel 32.
  • the piston shaft 23 is equipped with a key 24 which facilitates the rotary movement of the piston 20.
  • the piston shaft 23 has a cross-shaped end that fits into a socket 52 in the form of a cap 51 with a bore.
  • a density gradient centrifugation kit shown in Fig. 5 , comprises a device for controlled layering of liquids and a centrifuge container 61.
  • the kit enables attachment of centrifuge container, in this case a test tube, to the device.
  • the centrifugation kit comprises a container 61 with a thread 62 aligned to the thread 42 of the adapter cap 41 and a device for the controlled layering of liquids.
  • the sum of the aperture radius of the flow segment 31 and the depth of the flow transfer channel 32 is equal to the aperture radius of the centrifuge container 61.
  • the performed tests have shown that the best results, preventing formation of droplets in the flow transfer channel 32, are obtained when the sum of the aperture radius of the flow segment 31 compared to the aperture radius of the centrifuge container 61 is within ⁇ 0.25 mm.
  • the adapter cap is provided with a crushing rib 44 in ring shape which is crushed when the container 61 is screwed onto the adapter cap 41 ( Fig. 9 ), this causes its deformation and additional sealing of the connection between the container 61 and the adapter cap 41.
  • the adapter cap may be additionally equipped with a positioning element 43, for example of a triangular shape, which conforms to the shape of the place where the container is placed in the stand (not shown).
  • the positioning element 43 enables the repetitive positioning of the flow transfer channel 32, which may be used especially in the case of automation of controlled layering of liquids based on visual process control systems.
  • the thread 21 of the piston 20 is screwed onto the thread 13 of the guide 12.
  • the piston 20 is screwed in all the way so that the lip of the piston 22 is above the upper edge of the inlet 34 of the flow transfer channel 32, blocking the flow of liquid from the loading chamber 11 to the draining chamber 40.
  • the device is then screwed onto a 15ml falcon centrifugation container 61 by screwing the thread 42 of the cap 41 onto the thread 62 of the container 61.
  • the device with the attached container 61 is placed in a stand.
  • First density gradient separation liquid- Histopaque 1077 Sigma-Aldrich - in the volume of 4ml is poured into the loading chamber 11.
  • a socket 52 is placed and by turning the cap 51 with the key 52, the thread 21 of the piston 20 is partially screwed out from the thread 13 of the guide 12. After shifting the sealing lip 22 of the piston 20 below the inlet 34 of the channel 33 in the flow segment 31, the flow of liquid through the flow transfer channel 32 from the loading chamber 11 to the draining chamber 40 is enabled. After emptying the loading chamber 11, the liquid flow was disabled by screwing the piston 20 so that the lip of the piston 22 was above the upper edge of the flow transfer channel 32. The socket 52 was then removed from the key and 4 ml of whole blood was poured into the loading chamber 11.
  • a socket 52 is placed and by turning the cap 51 with the key 52, the thread 21 of the piston 20 is partially screwed out from the thread 13 of the guide 12.
  • the flow of liquid through the flow transfer channel 32 from the loading chamber 11 to the draining chamber 40 is enabled.
  • the blood flows creating an interface on the density gradient separation liquid.
  • the initial flow rate should on one hand prevent formation of droplets in the flow transfer channel 32 and on the other ensure continuous flow, for this embodiment the diameter of the flow transfer channel 32 should be about 3 mm 2 .
  • After pouring about 0.5 ml of blood aperture of flow transfer channel 32 was gradually increased from about 3 mm 2 to about 5.5 mm 2 .
  • the container 61 After emptying the loading chamber 11, the container 61 detached from the insert, capped, and was then centrifuged in order to separate the blood into fractions, subsequently the desired blood fractions were isolated. When handling the container 61 before and after centrifugation, great care was taken to avoid shaking.

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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)

Claims (12)

  1. Un dispositif pour le stratification contrôlée de liquides, en particulier pour le stratification de liquides avant séparation par centrifugation à gradient de densité, le dispositif comprenant une chambre de chargement (11), une chambre de drainage (40) qui comprend un segment d'écoulement en forme de cylindre (31) et un élément de régulation de flux de liquide pour réguler un flux de liquide de la chambre de chargement (11) à la chambre de drainage (40), caractérisé en ce que l'élément de régulation de flux de liquide comprend un mécanisme de type piston (19) et au moins un canal de transfert de flux (32) dans la paroi du segment d'écoulement (31), le mécanisme de type piston (19) comprenant le piston (20) mobile axialement par rapport au segment d'écoulement (31), le piston (20) étant pourvu d'une tige (23) et d'une lèvre d'étanchéité (22) adhérant étroitement à la surface intérieure de la paroi du segment d'écoulement (31), et où le piston (20) a un évent (25) sous forme de canal sur toute sa longueur, et où le canal de transfert de flux (32) est sous forme de rainure dans la surface intérieure de la paroi du segment d'écoulement (31) ou canal (33) dans la paroi du segment d'écoulement (31), une entrée (34) du canal (33) se trouvant dans la surface murale intérieure du segment d'écoulement (31) et une sortie (35) du canal (33) dans la paroi de fond du segment d'écoulement (31), et l'entrée (34) du canal de transfert de flux (32) est située en dessous de la jonction de la chambre de chargement (11) et du segment d'écoulement (31).
  2. Le dispositif selon la revendication 1, caractérisé en ce que la zone de section transversale du canal de transfert de flux (32) augmente de l'entrée (34) à la sortie (35), en particulier dans la gamme d'env. 1 mm2.
  3. Le dispositif selon la revendication 1, caractérisé en ce que la chambre de drainage (40) est pourvue d'un capuchon adaptateur (41) pour un conteneur, en particulier un conteneur pour centrifugation à gradient de densité.
  4. Le dispositif selon la revendication 3, caractérisé en ce que le capuchon adaptateur (41) est pourvu d'un filetage (42), en particulier un filetage qui s'adapte dans le filetage (62) du conteneur (61).
  5. Le dispositif selon la revendication 3, caractérisé en ce que le capuchon (41) possède également une côte de broyage (44).
  6. Le dispositif selon la revendication 1, caractérisé en ce que la lèvre d'étanchéité (22) a une section transversale axiale similaire à une lettre C, assurant l'auto-étanchéité entre la lèvre d'étanchéité (22) et la paroi intérieure du segment d'écoulement (31) empêchant le flux de liquide non contrôlé de la chambre d'alimentation (11) à la chambre de drainage (40).
  7. Le dispositif selon la revendication 1, caractérisé en ce que la lèvre d'étanchéité (22) est pourvue d'un anneau d'étanchéité (26), en particulier un anneau d'étanchéité (26) en caoutchouc ou en silicone, placé dans la rainure de montage (27) sur la lèvre (22), assurant une auto-étanchéité empêchant le flux de liquide non contrôlé de la chambre (11) dans la chambre de drainage (40).
  8. Le dispositif selon la revendication 1, caractérisé en ce qu'il comprend en outre un guide de piston (12) fixé de manière fixe à la chambre de chargement (11) ou à la chambre de drainage (40), où le guide de piston (12) est configuré pour permettre un mouvement axial du piston (20) dans la lumière du segment d'écoulement (31).
  9. Le dispositif selon la revendication 8, caractérisé en ce que la connexion du guide (14) du piston (12) à la paroi intérieure de la chambre d'alimentation (11) ou de la chambre de drainage (40) est réalisée par au moins une nervure structurelle (14).
  10. Le dispositif selon la revendication 8, caractérisé en ce que le piston (20) et le guide (12) sont pourvus de filetages adaptés sous forme de filetage de piston (21) et de filetage de guide (13).
  11. L'ensemble pour une centrifugation à gradient de densité, caractérisé en ce qu'il comprend le dispositif tel que défini dans l'une quelconque des revendications précédentes 1 à 10 et le conteneur de centrifugeuse (61).
  12. L'ensemble selon la revendication 11, caractérisé en ce que la différence entre la somme du rayon d'ouverture du segment d'écoulement (31) et la profondeur du canal de transfert de flux (32) par rapport au rayon d'ouverture du conteneur de centrifugeuse (61) n'est pas plus de 0,5 mm.
EP21755843.6A 2020-06-30 2021-06-24 Dispositif pour la stratification commandée de liquides Active EP4048444B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PL434517A PL434517A1 (pl) 2020-06-30 2020-06-30 Urządzenie do kontrolowanego warstwowego nalewania cieczy
PCT/PL2021/050044 WO2022005314A1 (fr) 2020-06-30 2021-06-24 Dispositif pour la stratification commandée de liquides

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EP4048444A1 EP4048444A1 (fr) 2022-08-31
EP4048444B1 true EP4048444B1 (fr) 2024-05-22

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EP21755843.6A Active EP4048444B1 (fr) 2020-06-30 2021-06-24 Dispositif pour la stratification commandée de liquides

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PL (1) PL434517A1 (fr)
WO (1) WO2022005314A1 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648223A (en) * 1994-08-31 1997-07-15 Activated Cell Therapy, Inc. Methods for enriching breast tumor cells
US10220384B2 (en) * 2011-08-09 2019-03-05 Jae Go Kwon Devices and methods for overlaying blood or cellular suspensions
PL237582B1 (pl) * 2015-09-15 2021-05-04 Spark Tech Spolka Z Ograniczona Odpowiedzialnoscia 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
KR102638609B1 (ko) * 2017-07-27 2024-02-19 바이오메리욱스, 인코포레이티드. 격리 튜브

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PL434517A1 (pl) 2022-01-03
WO2022005314A1 (fr) 2022-01-06

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