Device and method for separating particles from a liquid

Abstract

A surface is disclosed that can be contacted with a bulk liquid to perform particle separation. The surface includes a plurality of spaced-apart ribs to retain a portion of the bulk liquid and target particles therebetween by capillary action. In general, the portion of the bulk liquid retained by the plurality of ribs on the surface forms a liquid film. One or more particles contained in the space and surrounded by the liquid film can be protected from one or more forces exerted by a liquid drainage meniscus on the surface.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 511,503, filed May 26, 2017, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the interaction between liquids and surfaces, and more particularly to the interaction between one or more particles in a liquid and a surface. The invention also relates to the separation of particles or a subset thereof present in a liquid and in contact with said surface. Background Technology

[0004] In many applications, particles can be brought into contact with a surface to isolate them from the bulk liquid or other particles within it. Subsequent removal of the bulk liquid along with any contaminating particles can be an effective method for separating and enriching the target particles. However, removing the bulk liquid from the surface may exert a force on the particles in contact with that surface. This force may result in a loss of recovery rate of the particles in contact with the surface or of the separated particles recovered from the bulk liquid.

[0005] Liquid-surface interactions occur at the gas-liquid-solid interface, where a meniscus may form where the bulk liquid contacts the surface. During drainage or filling, the meniscus passing over the surface may exert forces on the particles at the gas-liquid-solid interface, including frictional, static frictional, and shear forces. Therefore, the drainage meniscus can exert a net force on the particles in contact with the surface, thereby removing the particles from the surface.

[0006] In cell biology, the efficiency of isolating a cell population from different cell populations or from a bulk liquid can be affected during the removal of the bulk liquid by pouring or aspirating, while the desired cell population is selectively concentrated and retained on a surface, such as at the container boundary. For example, magnetically labeled cells might be enriched in a liquid film on the container surface by a magnet placed near the container. For effective separation, magnetically labeled cells should be retained on the surface when removing bulk liquid containing unlabeled cells and other contaminants.

[0007] Therefore, there is a need for an apparatus and method that can provide better protection for particles in contact with the surface when the drain meniscus passes over the particles. Summary of the Invention

[0008] The methods and apparatus disclosed herein provide a surface and method employing ribs that can help reduce particle shear on the surface under the influence of the meniscus of a bulk liquid discharge. This particle shear can be reduced by retaining a portion of the bulk liquid in the space between the first and second ribs of the surface through capillary action to form a liquid film.

[0009] As a non-limiting example, the methods and apparatus disclosed in this invention can improve the overall performance of manual dumping and automated transfer methods for separating particles from bulk liquids containing a variety of particles in terms of (1) total recovery value, (2) increasing the range of initial particle numbers to make separation of low particle numbers more effective, (3) reducing the difference in separation performance, (4) and faster separation time.

[0010] In a broader sense, an apparatus is provided for separating particles from a bulk liquid. The apparatus includes a surface to contact the bulk liquid containing particles; a plurality of ribs on the surface, including at least a first rib and a second rib spaced apart from the first rib; and a space between the first and second ribs, sized such that when the liquid in contact with the surface is removed from the surface, a portion of the bulk liquid and at least a portion of the particles therein are retained in the space by capillary action.

[0011] In some embodiments, the first rib extends along a first longitudinal axis, and the second rib extends along a second longitudinal axis.

[0012] In some embodiments, the second longitudinal axis is substantially parallel to the first longitudinal axis.

[0013] In some embodiments, the first longitudinal axis and the second longitudinal axis are typically linear.

[0014] In some embodiments, the first rib includes: a first sidewall extending away from the surface and having a first base edge and a first protruding edge; and a second sidewall extending away from the surface and having a second base edge and a second protruding edge; the first base edge and the second base edge being spaced apart by a first rib width, and the first protruding edge being connected to the second protruding edge at a first apex height; and the second rib includes a third sidewall extending away from the surface and having a third base edge and a third protruding edge; and a fourth sidewall extending away from the surface and having a fourth base edge and a fourth protruding edge, the third base edge and the fourth base edge being spaced apart by a second rib width, and the third protruding edge being connected to the fourth protruding edge at a second apex height.

[0015] In some embodiments, the height of the first vertex and the height of the second vertex are respectively between approximately 20 μm and approximately 1 mm.

[0016] In some embodiments, the device further includes a third rib spaced apart from the second rib by the spacing distance, the third rib including a fifth sidewall extending remotely from the surface and having a fifth base edge and a fifth projecting edge, and a sixth sidewall extending remotely from the surface and having a sixth base edge and a sixth projecting edge, the fifth base edge being spaced apart from the sixth base edge by the width of the third rib, and the fifth projecting edge connecting to the sixth projecting edge at a third apex height.

[0017] In some embodiments, the height of the third vertex is between approximately 20 μm and approximately 1 mm, and differs from the heights of the first and second vertices.

[0018] In some embodiments, the first protruding edge is connected to the second protruding edge via the first top wall, and the third protruding edge is connected to the fourth protruding edge via the second top wall.

[0019] In some embodiments, the fifth protruding edge is connected to the sixth protruding edge via the third top wall.

[0020] In some embodiments, the widths of the first top wall, the second top wall, and the third top wall are between approximately 1 μm and approximately 1 mm.

[0021] In some embodiments, the spacing is at least 1 μm.

[0022] In some embodiments, the spacing is less than approximately 1 mm.

[0023] In some embodiments, the spacing between adjacent ribs in the plurality of ribs is uniform.

[0024] In some embodiments, the first rib has a first cross-sectional shape in a plane orthogonal to the first longitudinal axis, and the second rib has a second cross-sectional shape in the same plane.

[0025] In some embodiments, the first cross-sectional shape is the same as the second cross-sectional shape.

[0026] In some embodiments, the first cross-sectional shape is a quadrilateral.

[0027] In some embodiments, the first cross-sectional shape is triangular.

[0028] In some embodiments, the first longitudinal axis and the second longitudinal axis are oriented relative to the flow direction of the bulk liquid thereon, such that the first longitudinal axis and the second longitudinal axis are not parallel to the flow direction.

[0029] In some embodiments, the surface includes the inner surface of the container.

[0030] In some embodiments, the first top wall and the second top wall are coplanar with the inner surface of the container.

[0031] In some embodiments, the container is a test tube.

[0032] In some embodiments, the first rib and the second rib extend along the longitudinal axis of the surface by a rib length, wherein the rib length is 5% to 95% of the longitudinal axis of the surface.

[0033] In a broad sense, a container is provided for containing a bulk liquid containing particles. The container includes: a closed bottom end having a bottom wall; an open upper end; one or more side walls extending from the bottom end to the upper end; an inner surface defining the interior of the container and an opposing outer surface; a plurality of ribs on the inner surface and extending away from the inner surface into the interior of the container, the plurality of ribs including at least a first rib and a second rib spaced apart from the first rib; and a space between the first rib and the second rib; such that when the bulk liquid is contained within the interior of the container, the bulk liquid contacts the inner surface, the first rib and the second rib, and the space between the first rib and the second rib, and the dimensions of the first rib and the second rib are determined such that when the bulk liquid in contact with the surface is removed from the surface, a portion of the bulk liquid and at least a portion of the particles therein are capillarily retained between the first rib and the second rib.

[0034] In some embodiments, the sidewall extends from the bottom end to the top end along the container axis, and wherein the first rib extends along a first rib axis parallel to the container axis.

[0035] In some embodiments, the plurality of ribs cover 5% to 95% of the area of ​​the inner surface of the sidewall.

[0036] In some embodiments, multiple ribs are located at the bottom, middle, or top of the container.

[0037] In some embodiments, the sidewall includes a plurality of ribs.

[0038] In some embodiments, multiple ribs are integrally formed with the container sidewall.

[0039] In a broader sense, a method is provided for separating particles from a bulk liquid using a device comprising a surface and a plurality of ribs on that surface, the ribs comprising: at least one first rib and a second rib spaced apart from the first rib by a distance; and a space between the first and second ribs. The method includes: contacting the device with the bulk liquid such that the bulk liquid contacts the surface, the first and second ribs, and the space between the first and second ribs; containing at least a first portion of particles in the bulk liquid in the space between the first and second ribs; removing the bulk liquid from the surface, causing a portion of the bulk liquid to capillarily remain between the first and second ribs to form a liquid film therebetween; protecting the particles contained between the first and second ribs and entrained in the liquid film from one or more forces of a drain meniscus while removing the bulk liquid from the surface; and resuspending the protected particles entrained in the liquid film in a buffer solution.

[0040] In some embodiments, the method further includes applying a first force to push the particles into the space between the first rib and the second rib.

[0041] In some embodiments, at least a second portion of the particle entering the space responds to the first force.

[0042] In some embodiments, the first force is a magnetic attraction, and the responding particles have a first magnetic charge that is attracted to the magnet, such that the device is between the magnet and the bulk liquid, whereby the first force pushes a first portion of the particles toward the space.

[0043] In some embodiments, without a first force, the particles contained in the space empty the space.

[0044] In some embodiments, the method further includes adding an antimagnetic additive to the bulk liquid.

[0045] In some implementations, the antimagnetic additive is gadolinium.

[0046] Other features and advantages of the invention will become apparent from the following accompanying drawings and detailed description. However, it should be understood that while the detailed description and specific examples illustrate preferred embodiments of the invention, they are given in an illustrative manner only, as various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art based on this detailed description. Attached Figure Description

[0047] The invention will now be described in conjunction with the accompanying drawings, in which:

[0048] Figure 1 According to one embodiment, a perspective view of an apparatus for separating particles from a bulk liquid is shown, the apparatus having a substantially flat surface including a plurality of ribs.

[0049] Figure 1 According to another embodiment, a perspective view of an apparatus for separating particles from a bulk liquid is shown, the apparatus being shaped like a test tube and including a plurality of ribs on one of its surfaces.

[0050] Figure 1 According to another embodiment, an exploded view of two devices for separating particles from a bulk liquid is shown, the devices being shaped as circular culture containers and including a plurality of ribs on one of their surfaces.

[0051] Figure 1 D shows Figure 1 A bottom view of the petri dish C.

[0052] Figure 1 According to another embodiment, a perspective view of an apparatus for separating particles from a bulk liquid is shown, the apparatus being shaped as a rectangular culture container having a plurality of ribs on one of its surfaces.

[0053] Figure 2 It shows a structure including multiple ribs. Figure 1 A cross-sectional view of an exemplary surface of the device.

[0054] Figure 3 A shows various rib patterns. Figure 1 A top view of an exemplary surface of the device. The surface shown, including multiple ribs, may be a substantially flat surface or part of a non-flat surface.

[0055] Figure 3 B shows a front view of an experimental tube comprising multiple ribs on its inner wall. The ribs on its inner surface can be, for example... Figure 3 The pattern shown in Figure A.

[0056] Figure 4 A shows a top / front view of a surface that includes multiple ribs that have come into contact with a bulk liquid containing particles.

[0057] Figure 4 B illustrates the situation after at least a portion of the particles in the bulk liquid have been subjected to the first force and have begun to remove the bulk liquid from the surface. Figure 4 Top / front view of the surface shown in Figure A (projected onto the surface).

[0058] Figure 4 C shows that once all the bulk liquid has been substantially removed from the surface, Figure 4 Top / front view of the surface shown in B (projected from the top).

[0059] Figure 4 D shows Figure 4 The side view of surface C along line A.

[0060] Figure 4 E shows Figure 4 Front view / top view of the orthographic projection along line B on surface C.

[0061] Figure 5 A shows a cross-sectional view of the drainage meniscus that shears particles away from the surface.

[0062] Figure 5 B shows a cross-sectional view of particles protected from the effects of a draining meniscus by a liquid film extending between the first and second ribs on a surface.

[0063] Figure 6 A shows a schematic diagram of meniscus shearing in the tilting method of cell separation.

[0064] Figure 6 B shows a perspective photograph depicting the shearing effect of particles / cells on the sidewalls of the container as they pass through the drainage meniscus. Here, PMBCs were positively selected using an anti-CD45 magnetically labeled antibody. The tube was placed in a STEMCELL Silver magnet for 10 minutes, capped, the magnet tilted horizontally, and then the tube was carefully pulled out to capture the image.

[0065] Figure 7 The diagram illustrates the increased survival rate of isolated cells retained in the G tube (groove tube) and F tube (Falcon tube) after the removal of the main fluid.

[0066] Figure 8 The graph shows the purity and recovery rate of cells separated using G tubes (grooves) and F tubes (Falcon tubes) under various separation processes. "S" indicates a separation step that includes a washing step aimed at removing most of the separated cells and / or particles from the surface, and "R" indicates a rinsing step whose purpose is not to remove most of the separated cells and / or particles from the surface.

[0067] Figure 9 According to one embodiment, a perspective sectional view of a tube (G-tube) including multiple ribs on its inner sidewall is shown.

[0068] Figure 9 B shows Figure 9 Enlarged views (i) and (ii) of the boxed area shown in A.

[0069] Figure 9 C shows Figure 9 A perspective view of an exemplary tube shown in Figure A, which includes a pipette seat at its closed bottom end.

[0070] Figure 10Figure A shows a graph of the liquid film volume retained in G-tubes and F-tubes during the decanting cell separation method. This figure illustrates the results using the EasySep cell separation method with G-tubes and F-tubes containing different rib densities on their surfaces. 2-space tubes and 4-space tubes refer to tubes with rib densities of 1 / 2 and 1 / 4, respectively.

[0071] Figure 10 Figure B shows a graph of the liquid film volume retained in G-tubes and F-tubes during the aspiration cell separation method. This figure illustrates the use of G-tubes and F-tubes with different rib densities on their surfaces (e.g.,...). Figure 8 The results of the RoboSep cell isolation method (shown in Figure A).

[0072] Figure 11 The diagram shows the effect of antimagnetic additives on recovery and purity in aspiration or decanting methods using G-tubes and F-tubes for cell separation. Detailed Implementation

[0073] The ribbed surface described herein can be used in conjunction with bulk liquids containing one or more particle groups. Multiple ribs can help protect at least a portion of the particles in contact with the surface from certain forces exerted through the drainage meniscus thereon.

[0074] The particles of this invention can be any biological or non-biological particles. Biological particles can include, but are not limited to: cells, whether prokaryotic or eukaryotic, and aggregates thereof; subcellular components, such as organelles or extracellular vesicles; proteins; nucleic acids; or prions. Non-biological particles can include, but are not limited to: particles containing one or more metals and / or metalloids or any other inorganic substances; or particles containing organic substances. In some embodiments, the particle can be a combination of biological and non-biological particles. For example, the particles of this invention can contain cells complexed with one or more magnetic or magnetizable particles. The average diameter of the particles of this invention can range from angstroms to millimeters.

[0075] surface

[0076] An apparatus for separating particles from a bulk liquid 1 includes a surface 10. This surface may include or be composed of any material that can contact the particle-containing bulk liquid 13. Exemplary surfaces may include glass, one or more polymers, metals, or metalloids. In one embodiment, surface 10 may be substantially flat. Figure 1 a). In another embodiment, surface 10 may include the inner wall or inner surface of a container, including but not limited to test tubes, such as bottom-sealed test tubes, flasks, bottles, or other containers. Figure 1(be). In any case, the generally flat surface or inner wall or inner surface of the container may come into contact with the liquid, including bulk liquids containing particles 13.

[0077] rib

[0078] The device 1 includes a plurality of ribs 15 on surface 10. Figure 1 The plurality of ribs 15 includes at least the first rib 20 and the second rib 30. Figure 2 Each of the first rib 20 and the second rib 30 extends along its respective first longitudinal axis and second longitudinal axis. In one embodiment, the second longitudinal axis is substantially parallel to the first longitudinal axis. In other embodiments, the first and second longitudinal axes are generally linear. In certain embodiments, the first and second longitudinal axes may typically not be linear. In such embodiments, the first and second longitudinal axes may include zigzag, spiral, or sawtooth patterns.

[0079] like Figure 2 As shown, the first rib 20 may include a first sidewall 21a extending away from the surface 10 and having a first base edge 23a and a first projecting edge 25a. The first rib 20 may also include a second sidewall 21b extending away from the surface 10 and having a second base edge 23b and a second projecting edge 25b. Similarly, as... Figure 2 As shown, the second rib 30 may include a third sidewall 31a that extends remotely from the surface 10 and has a third base edge 33a and a third projecting edge 35a. The second rib 30 may also include a fourth sidewall 31b that extends remotely from the surface 10 and has a fourth base edge 33b and a fourth projecting edge 35b.

[0080] In some embodiments, the first base edge 23a may be spaced apart from the second base edge 23b by a first rib width w. fr Similarly, the third base edge 33a may be spaced apart from the fourth base edge 33b by the second rib width w. sr In such an embodiment, the width w of the first rib fr and / or the width of the second rib w sr It can be 1μm to about 1mm, or larger.

[0081] In other embodiments, for example, when the first rib 20 and / or the second rib 30 may each have an inverted triangular cross-sectional shape orthogonal to the first longitudinal axis of the first rib 20 and the second longitudinal axis of the second rib 30, the first sidewall 21a and the second sidewall 21b, and / or the third sidewall 31a and the fourth sidewall 31b may extend and diverge substantially from a common point on the surface 10. In such other embodiments, the first base edge 23a and the second base edge 23b may substantially overlap each other, and / or the third base edge 33a and the fourth base edge 33b may also substantially overlap each other. Therefore, the width w of the first rib fr and / or the width of the second rib w sr It is more appropriate to cross the first sidewall 21a and the second sidewall 21b and / or the third sidewall 31a and the fourth sidewall 31b at positions spaced apart from the surface 10.

[0082] The first protruding edge 25a can be at the height h of the first rib apex. fr It connects to the second protruding edge 25b, and the third protruding edge 35a can be at the height h of the second rib apex. sr The first protruding edge 25a and the second protruding edge 25b are connected via a first top wall 27, and / or the third protruding edge 35a and the fourth protruding edge 35b are connected via a second top wall 37. In other embodiments, for example, each corresponding rib has a triangular or pointed cross-sectional shape orthogonal to the first longitudinal axis of the first rib 20 and / or the second longitudinal axis of the second rib 30, the first protruding edge 25a can be directly connected to the second protruding edge 25b, and / or the third protruding edge 35a can be directly connected to the fourth protruding edge 35b.

[0083] In embodiments where the first rib 20 and / or the second rib 30 have cross-sectional shapes in planes orthogonal to the first and second longitudinal axes respectively, the cross-sectional shapes are arcuate or circular, and the protruding edges of the corresponding sidewalls of the ribs may intersect at the height of the rib apex.

[0084] In another embodiment, the device 1 may further include a third rib 40. The third rib 40 may include a fifth sidewall 41a extending remotely from the surface 10 and having a fifth base edge 43a and a fifth projecting edge 45a. The third rib 40 may also include a sixth sidewall 41b extending remotely from the surface 10 and having a sixth base edge 43b and a sixth projecting edge 45b. In some embodiments, the fifth base edge 43a may be spaced from the sixth base edge 43b by a third rib width w. tr In such an embodiment, the width w of the third rib trThe width can be between 1 μm and approximately 1 mm, or larger. In other embodiments, for example, when the third rib 40 has an inverted triangular cross-sectional shape orthogonal to the third longitudinal axis of the third rib 40, the fifth sidewall 41a and the sixth sidewall 41b can substantially extend and diverge from a common point on the surface 10. In such other embodiments, the fifth base edge 43a and the sixth base edge 43b can substantially overlap each other. Therefore, the width w of the third rib... tr It is more appropriate to cross the fifth sidewall 41a and the sixth sidewall 41b at a position spaced apart from the surface 10.

[0085] The fifth protruding edge 45a can be at the height h of the third rib apex. tr The fifth protruding edge 45a is connected to the sixth protruding edge 45b. In some embodiments, the fifth protruding edge 45a and the sixth protruding edge 45b can be connected by the third top wall 47. In other embodiments, for example, the third rib 40 has a triangular or pointed cross-sectional shape perpendicular to the third longitudinal axis of the third rib 40, and the fifth protruding edge 45a can be directly connected to the sixth protruding edge 45b. In some embodiments, the widths of the first top wall 27, the second top wall 37, and the third top wall 47 can be between approximately 1 μm and approximately 1 mm, or greater.

[0086] In some embodiments, the height h of the first rib apex is measured in a direction orthogonal to and extending away from the surface 10. fr (For example, obtained at the first top wall 27), the height h of the second rib apex. sr (e.g., obtained at the second top wall 37) and / or the height h of the third apex rib tr (For example, obtained at the third top wall 47), this height can be between 20 μm and approximately 1 mm. In some embodiments, the height h of the first rib apex is... fr Second rib apex height h sr Each can be between approximately 20 μm and approximately 1 mm. In other embodiments, the height h of the first rib apex... fr Second rib apex height h sr The height h of the third vertex rib can be the same. In other embodiments, the height h of the third vertex rib is... tr It can be between approximately 20 μm and approximately 1 mm, and can be different from the height of the first vertex and the height of the second vertex, respectively.

[0087] The third rib 40 can be spaced apart from the second rib 30, and the second rib 30 can be spaced apart from the first rib 20 by a distance p. Figure 2The spacing distance p can be measured in a plane substantially parallel to surface 10 and between adjacent ribs, for example, between the second sidewall 21b and the third sidewall 31a. The spacing distance p can be measured at any point between adjacent ribs, for example, at any point along the adjacent sidewall of an adjacent rib. Depending on the cross-sectional geometry (obtained in a plane orthogonal to the longitudinal axes of adjacent ribs), the spacing distance p can be measured, for example, between the second base edge 23b and the third base edge 33a. Alternatively, the spacing distance p can be measured, for example, between the second protruding edge 25b and the third protruding edge 35a. Generally, the spacing distance p should be sufficient to accommodate the diameter of at least one target particle. For example, the diameter of most animal cells can be about 10 to 30 μm, and the diameter of some larger animal cells such as megakaryocytes can be about 160 μm. Furthermore, the diameter of many viruses can be about 30 nm to about 250 nm or greater. Even further, the diameter of many bacteria can be about 100 nm to about 10,000 nm or greater. In some embodiments, the spacing distance p can be at least 10 μm. In other embodiments, the spacing p can be less than 1 mm.

[0088] In some embodiments, the spacing p between adjacent ribs in the plurality of ribs 15 may be uniform. In other embodiments, the spacing p between adjacent ribs in the plurality of ribs 15 may be non-uniform. In embodiments with non-uniform spacing, the spacing may alternate between relatively large spacing and relatively small spacing.

[0089] Each sidewall of a corresponding rib in the plurality of ribs 15 can form a defined edge with surface 10. Figure 2 In the illustrated embodiment, the edge formed between each sidewall of the respective rib and the surface 10 may be influenced by the cross-sectional shape of the plurality of ribs 15 in a plane orthogonal to their longitudinal axis. The cross-sectional shape of the plurality of ribs 15 may be any polygon or a portion thereof. In some embodiments, the first rib 20 has a first cross-sectional shape in a plane orthogonal to the first longitudinal axis, and the second rib 30 has a second cross-sectional shape in that plane.

[0090] Although the first rib 20 may be referred to in the discussion below, the same discussion can be applied to the second rib 30 and / or the third rib 40. In some embodiments, the first rib 20 may include generally flat sidewalls 21a and 21b that are generally parallel to each other and intersect surface 10 at an angle between 80° and 100°. Figure 2 In some embodiments, sidewalls 21a and 21b may intersect surface 10 at an angle of approximately 90° or 90°. In some embodiments, the first cross-sectional shape is substantially square or rectangular, or quadrilateral, such as a trapezoid. In other embodiments, the first cross-sectional shape is curved inward or outward and includes a first top wall ( Figure 2 ).

[0091] In other embodiments, the first cross-sectional shape is triangular. Figure 2 In an embodiment where the first cross-sectional shape is triangular, the first and second sidewalls may intersect surface 10 at an angle of 60° to form an equilateral triangle. In other embodiments where the first cross-sectional shape is triangular, the first and second sidewalls may intersect surface 10 to form an isosceles triangle. Alternatively, the first cross-sectional shape may be arc-shaped or hemispherical. Figure 2 ).

[0092] In other embodiments, the first cross-sectional shape can be a combination of various shapes; for example, the first cross-sectional shape includes a quadrilateral with an arched or pointed top wall. Figure 2 Such a cross-sectional shape may be suitable, where particles can contact and settle onto the top walls of the plurality of ribs 15, such as a first top wall 27 or a second top wall 37, rather than in the space 50 between the first rib 20 and the second rib 30. Without being limited by the foregoing, the invention may include any shape of the top wall that can induce particles into the space 50 and away from one or more of the plurality of ribs 15. In such an embodiment, the first rib 20 may include a first vertex 29, and the second rib may include a second vertex 39, the second vertex 39 being approximately the width of the particle that can contact it. Alternatively, the widths of the first vertex 29 and the second vertex 39 may be smaller than the width of the particle that can contact it.

[0093] In some embodiments, the first cross-sectional shape is the same as the second cross-sectional shape. In other embodiments, the first cross-sectional shape is different from the second cross-sectional shape.

[0094] The multiple ribs 15 can be oriented in any manner, as long as such orientation allows a portion of the bulk liquid to be retained by capillary action after it has been discharged from the exit surface 10. Figure 3a) In a preferred embodiment, a plurality of ribs 15 may be arranged substantially linearly along the same direction ds passing through the meniscus on surface 10, wherein the first and second longitudinal axes (each of the first rib 20 and the second rib 30) are oriented relative to the flow direction of the bulk liquid thereon such that the first and second longitudinal axes are parallel to the flow direction. In other embodiments, the plurality of ribs 15 may extend substantially linearly horizontally or substantially vertically relative to the direction through which the meniscus passes on surface 10. In a particular embodiment, the first and second longitudinal axes (each of the first rib 20 and the second rib 30) are oriented relative to the flow direction of the bulk liquid thereon such that the first and second longitudinal axes are not parallel to the flow direction. In other embodiments, these ribs may be zigzag, helical, shaded, or other patterns, and extend along the longitudinal axis in any direction along the surface relative to the flow direction of the meniscus. In fact, it is important that the orientation of the ribs effectively retains a portion of the bulk liquid by capillary action as the meniscus passes through the surface.

[0095] In embodiments of the container 100 for containing bulk liquid 113 containing particles, the container may be a test tube, flask, dish, etc. In some embodiments, the container may include: a closed bottom end having a bottom wall; an open top end; one or more side walls extending from the bottom wall to the top end; and an inner surface defining the interior of the container and an opposing outer surface. In a particular embodiment, the container 100 is a test tube (… Figure 3 b). Container 100 includes a plurality of ribs 115 on its inner surface 110 (these aspects have been generally described above). The plurality of ribs 115 extend from the inner surface 110 into the interior of container 100. As described above, the plurality of ribs 115 includes at least a first rib 120 and a second rib 130. Also as described above, the second rib 130 may be spaced apart from the first rib 120 by a distance ( Figure 3 Not shown in b; see Figure 2 The spacing defines the space 150 between the first rib 120 and the second rib 130.

[0096] In a particular embodiment, the sidewall 112 extends along the container axis from the bottom end 170 to the top end 180. The inner surface 110 of the sidewall 112 includes a first rib 120 extending along a first rib axis parallel to the container axis. A plurality of ribs 115 may cover 5% to 95% of the area of ​​the inner surface 110 of the sidewall 112. In some embodiments, the inner surface 110 and / or the sidewall 112 may include a plurality of ribs 115 extending from near the closed bottom end 170 of the container 100 to near the open top end 180 of the container 100.

[0097] In other embodiments, the ribs may not extend substantially along the entire length of the container. For example, only a portion of the inner surface 110 may include a plurality of ribs 115. In one embodiment, the plurality of ribs 115 may be located substantially at the bottom 170 of the container 100, for example, to form a clump of particles under the influence of gravity, including but not limited to centrifugal forces. In another embodiment, the plurality of ribs 115 may be located substantially at the top 180 of the container 100. In yet another embodiment, the plurality of ribs 115 may be located substantially in the middle of the container 100. Those skilled in the art will further understand that the plurality of ribs need not be positioned along the entire circumference of the sidewalls and / or inner surface of the container.

[0098] Ribs can also be provided on the surface at a density suitable for a particular downstream application. The rib density can be established by providing a desired number of ribs 15 or 115 on a surface 10 or 110 of a unit size, respectively. As an illustrative example, ten ribs with a width of 1 mm might be needed on a surface spanning 20 mm. Such a configuration would produce a density of one rib per 2 mm of surface area (in the appropriate direction). Nevertheless, the invention can cover any density of ribs provided on the surface, and thus the desired density may be limited by factors such as the feasibility of forming ribs on the surface or the ability to retain a portion of the bulk liquid passing through the surface with ribs via capillary action.

[0099] Furthermore, a surface with multiple ribs can be prepared in any manner known in the art. Injection molding can be used to manufacture the surface to form multiple ribs thereon. Alternatively, the surface including ribs can be 3D printed using any variety of substrates that can be processed by a 3D printer. Alternatively, it can be made to have multiple ribs by adhering individual ribs to a substantially smooth surface. In an alternative, the surface can be made ribbed by adhering multiple ribs arranged on a universal backing to a substantially smooth surface. Furthermore, the surface can be physically made to have multiple ribs after it has been manufactured, for example, by hand or mechanical notching or etching. Alternatively, multiple ribs can be chemically formed on the surface by applying suitable components to the surface.

[0100] space

[0101] The device 1 or container 100 also includes a space 50 located between the first rib 20 and the second rib 30, or a space 150 located between the first rib 120 and the second rib 130. Although the following discussion may focus on the device 1 and the space 50, it applies equally to the container 100 and the space 150.

[0102] The dimensions of space 50 are designed such that when the bulk liquid 13 of contact surface 10 is removed from the surface, a portion 60 of the bulk liquid 13 and at least a portion of the particles 62 are retained between the first rib 20 and the second rib 30 by capillary action (see example). Figure 4 b or Figure 4 c). The capillary retention of a portion of a bulk liquid in a space can be influenced by many factors. These factors include, but are not limited to, the contact angle between the bulk liquid and the surface, the viscosity of the bulk liquid, the volume of the space, and the distribution of hydrophobic and hydrophilic water bodies on the surface.

[0103] In applications using device 1 or container 100, the volume v of space 50 sp The tail 60 of the bulk liquid 13 retained between the first rib 20 and the second rib 30 by capillary action is a key consideration. The volume v of space 50... sp The width w of the space is 50. sp The depth d of space 50 sp And the length of the space 50 l sp The function of . For example, when the bulk liquid is characterized by low density, it may require a space with an appropriately small volume. As another example, if the bulk liquid comprises a density comparable to liquid water, the space between the ribs can be on the order of hundreds of micrometers to millimeters.

[0104] Furthermore, the volume v of space 50 can be determined such that, measured in terms of particle diameter, its size can accommodate at least one particle 62, which can be contained within the space under the influence of, for example, a first force 65 (e.g., a magnetic field provided by a magnet). In some cases, submicron particles, such as viral particles or bacteria, can be collected in the space between the first rib 20 and the second rib 30. In other cases, micron-sized cells can be collected in the space 50 between the first rib 20 and the second rib 30. In still other cases, particles of several hundred micrometers in size, such as aggregates of mammalian cells, can be collected in the space 50 between the first rib 20 and the second rib 30.

[0105] As the drainage meniscus 70 passes over the surface 10, the portion 60 of the bulk liquid 13 retained in the space 50 by capillary action, along with a portion of the particles 62 therein, is at a reduced liquid velocity. In this situation, one or more forces of the drainage meniscus 70 can be reduced relative to the portion 60 of the bulk liquid 13 retained by capillary action, thereby protecting the portion of the particles 62 contained between the first rib 20 and the second rib 30 and entrained in the portion 60 of the bulk liquid 13.

[0106] liquid film

[0107] Overall or individually, the portion 60 of the bulk liquid 13 retained by capillary action within the space 50 between the multiple ribs 15 can form a liquid film 80. Generally speaking, after the bulk liquid 13 is removed from the surface, the liquid film 80 can cover the surface 10. Figure 4 e).

[0108] Besides being affected by space v sp In addition to the influence of volume, the thickness t of the portion 60 (and liquid film 80) of the bulk liquid 13 retained by capillary action lf It may be affected by the contact angle between the liquid-gas-solid interface, the liquid viscosity, the surface adsorption of the liquid, and the velocity of the drain meniscus 70°. When the thickness t... lf When the diameter pd of the particle 62 is smaller than that of the particle 62, the particle 62 or a portion thereof can be sheared from the surface 10 from the bulk liquid 13 contained in the space 50 between the first rib 20 and the second rib 30. If the thickness t lf The diameter p of particles smaller than 62 (or aggregates of particles) d Then particle 62 may be subjected to an outward force from surface 10, thereby shearing particle 62 from surface 10 back into bulk liquid 13. Figure 5 a) For example, under typical conditions of cell separation processes, during the pouring or aspiration of bulk liquid from a container, individual cells on the container surface, such as the test tube wall, can be sheared off the surface by one or more forces of the meniscus.

[0109] If the thickness t lf The diameter p of the particle is greater than 62 d This can prevent particles 62 from being sheared off from surface 10 and / or from being sheared out from the space 50 between the first rib 20 and the second rib 30. Figure 5 (b) The above objective can be achieved by ensuring a sufficient speed of drainage at the meniscus, or by employing a surface 10 with multiple ribs 15 to slow down drainage by forming a liquid film on the surface 10 that is retained between the first rib 20 and the second rib 30 due to capillary action. These and other strategies for increasing liquid film thickness can be used individually or in combination.

[0110] However, optimizing particle separation or isolation performance involves more than simply maximizing the tail film thickness when the bulk liquid is discharged. On the one hand, an overly thick tail film may leave unwanted particles that may be close to the surface but not necessarily in contact with it. These particles may correspond to those initially randomly distributed in the bulk solution, rather than those that are brought into direct contact by, for example, magnetic force. On the other hand, the thinner the tail film, the lower the recovery rate of the desired particles, as they may be sheared off the interface during the bulk liquid discharge. Therefore, the characteristics of the multiple ribs on the surface may need to be optimized as described above to achieve an appropriate balance between maximizing the recovery rate of subsequent particles by retaining a portion of the bulk liquid through capillary action (i.e., the liquid film) and maximizing the purity of recovered subsequent particles by discharging the bulk liquid.

[0111] In practice, since different particles in a sample, such as a bulk liquid, may be present at different frequencies, the characteristics of the surface, ribs, or spaces between them can be further optimized for any given particle. For example, the optimal liquid film thickness can be varied based on the number and extent of particles on the surface to be retained within the spaces between the multiple ribs. Furthermore, the space between the first and second ribs may exclude larger particles from the liquid film; therefore, when the bulk liquid is removed from the surface, the excluded particles are easily removed even if larger particles are forcefully pushed against the surface. In this way, size selection and specific magnetic markings can be used to achieve the separation of particle mixtures.

[0112] Furthermore, for example, by applying a first force 65, at least a first portion of the particles 62 in the bulk liquid 13 can be accommodated in the space 50 between the first rib 20 and the second rib 30. This first force 65 can induce the first portion of the particles to enter the space between the first and second ribs, and at least a second portion of the particles entering the space 50 responds to the first force 65. When the bulk liquid 13 is removed from the surface 10, the first and / or second portions of the particles can be entrained in the liquid film 80 by protecting them from one or more forces of the drainage meniscus 70, and thus remain in the space 50 and the liquid film 80.

[0113] Once the bulk liquid 13 is removed from the surface 10, it may be desirable to resuspend the portion of particles protected by and within the liquid film 80 in the buffer solution. Therefore, in one embodiment, when the bulk liquid 13 is removed from the surface 10, a plurality of ribs 15 on the surface 10 can protect a portion of the particles 62 from one or more forces applied by the drainage meniscus 70, but the plurality of ribs 15 can not prevent this portion of the particles from emptying the space 50 in a downstream step of the particle separation method, for example, without the first force 65.

[0114] Furthermore, the distribution of particles on the surface can affect the thickness of the local liquid film. Therefore, in another embodiment, the size of the ribs can be determined such that the thickness of the liquid film can be influenced by the ribs, rather than by the particle distribution on or near the surface. Controlling the liquid film thickness and uniformity by using surface ribs to reduce variations in target particle recovery rates may be another advantage achieved using this design concept.

[0115] Applications in particle and / or cell separation methods

[0116] In preliminary particle and / or cell separation methods, when using EASYSep in the pouring protocol TM When (STEMCELL Technologies Inc., Canada), the meniscus shear effect was observed. Figure 6 The following will describe various aspects of exemplary schemes for pouring liquid from a test tube. In particular, in Figure 6 Figure a shows the trajectory of the gas-liquid interface (meniscus).

[0117] As in Figure 6 As can be seen from a, a method for separating particles from a bulk liquid 200 containing particles 210 can be performed using EASYSep. TM (STEMCELL Technologies, Ltd.) system or another system for separating particles from bulk liquids. Under the influence of magnet 235 (a subsequent step in the illustrated particle and / or cell separation method includes, but is not shown), particles and / or cells 215 may come into contact with the inner surface of test tube 200, which may include a first sidewall 220 and a second sidewall 230. As test tube 200 is moved away from the vertical axis, bulk liquid 210 flows down along the first sidewall 220 of test tube 200 and forms a meniscus 240, which shears off particles and / or cells 215 from its inner surface. The sheared particles 215 can be discharged downwards through the meniscus 240 to the bottom 25 of test tube 200. 0 is used for cleaning. Once the meniscus 240 has passed the first sidewall 220 and reached the bottom 250 of the tube 200, the second stage of liquid removal begins, in which the discharge of liquid 210 does not involve the meniscus. During this stage of tube drainage, only the weak fluid resistance effect generated by the discharged liquid causes particles and / or cells to move away from the surface. In the final stage of tube drainage, the meniscus moves from the bottom 250 through the second sidewall 230 to its open upper end 260, efficiently shearing particles and / or cells 215 from the second sidewall 230. This shearing effect occurs in the final stage of tube drainage and results in the desired particle and / or cell formation remaining as a “last drop” 270 at the tube opening 280. At this point, EASYSep can be stopped. TMThe (STEMCELL Technologies Ltd) protocol involves draining the test tube to ensure that the last drop containing the target particles and / or cells is retained.

[0118] For EASYSep TM The conceptual model developed by STEMCELL Technologies, Inc. identified a problem with the use of pouring or aspiration steps to remove bulk liquid from a test tube in cell separation methods. Indeed, as described above, the drainage meniscus across a generally smooth surface comprising a dense cell layer can shear cells off the surface. Such sheared cells may include the aforementioned "last drop" after the bulk liquid has been removed from the test tube. To comply with EASYSep TM (STEMCELL Technologies, Ltd.) Solution (see EASYSep) TM To achieve a satisfactory recovery (as per the manual), the "last drop" should be retained after emptying the bulk liquid from the test tube. Losing the "last drop" during tube emptying can have a significant negative impact on the recovery rate of positive selection or the purity of negative selection.

[0119] In the pouring method for removing bulk liquids, the drain meniscus only affects some sidewalls of the test tube. Figure 6 b). For example Figure 6 As shown in b, positive selection for PMBC was performed using an anti-CD45 magnetically labeled antibody. The tube was placed in a STEMCELL Silver magnet for 10 minutes, capped, and the magnet was removed from the vertical axis. The tube was then removed from the magnet before imaging. Similarly... Figure 6 As shown in b, most of the cells have detached from the sidewalls of the test tube that have undergone the drainage meniscus. Also note, for example, with Robosep... TM (STEMCELL Technologies Ltd.) When removing bulk liquids by suction, the entire inner surface of the test tube may be affected by the meniscus of the drain, which may result in a significant reduction in recovery rate.

[0120] The problem of meniscus-mediated shearing of particles and / or cells in contact with the surface during pouring or aspiration can be avoided by separating particles from the bulk fluid. This method involves providing a surface with multiple ribs, as described above, so that the liquid film is retained on the surface by capillary action. For example, the surface can be changed from a smooth surface design (hereinafter referred to as "F-tube") to a surface with multiple ribs and spaces between the ribs (referred to as "G-tube"), as described herein.

[0121] An exemplary method may include contacting a surface with a bulk liquid containing one or more particles, such that the bulk liquid contacts the surface, a first rib, a second rib, and the space between the first and second ribs. Once the bulk liquid has contacted the surface, the plurality of ribs, and the space between them, at least a first portion of the particles in the bulk liquid may be contained in the space between the first and second ribs.

[0122] Containing a first portion of a particle in the space between the first and second ribs can be passive or active. In embodiments where the containment step is active, this containment can be achieved by applying a first force to induce the first portion of the particle into the space between the first and second ribs. The first force can be attraction, magnetic attraction, pressure, or any other force that can induce the particle to move in a particular direction. In some embodiments, without the first force, the particle contained in the space can empty the space. The use of the first force can separate the first portion of the particle from other particles in the bulk liquid as needed.

[0123] In embodiments where the first force is a magnetic attraction, the responsive particles may have a first magnetic charge that is attracted to a magnet, such that a surface comprising multiple ribs lies between the magnet and the bulk liquid, thereby causing a first portion of the particle to move into space by the first force. In such embodiments, if the responsive particles are magnetic or magnetized, they will respond to the first force such that the particles are accommodated in the space between the first and second ribs. As a specific example, target cells may be linked to one or more magnetizable particles via immunoaffinity interactions (e.g., antibodies or antibody complexes, i.e., tetrameric antibody complexes), and under the influence of a magnetic force, the target cell:particle complex may be attracted to the surface in the direction of the magnetic force.

[0124] Once the first portion of particles is contained within the space between the first and second ribs, the bulk liquid can be removed from the surface. During removal of the bulk liquid from the surface, a portion of the bulk liquid remains between the first and second ribs via capillary action, forming a liquid film therebetween. The thickness of the liquid film between the first and second ribs, or multiple ribs, can be controlled or influenced as described more fully above.

[0125] Through the interaction of the first and second ribs, or multiple ribs, with the liquid film, particles contained in the space can be entrained within the liquid film, thereby protecting them from one or more forces of the drainage meniscus when the bulk liquid is removed from the surface. By protecting the particles entrained in the liquid film from one or more forces of the drainage meniscus, the particles are retained in the liquid film once the bulk liquid is removed from the surface. Once the bulk liquid, along with any contaminants or unretained particles in the bulk liquid, has been removed from the surface, it may be necessary to separate or resuspend the particles entrained in the liquid film. Resuspension of the protected particles entrained in the liquid film can be accomplished by adding an appropriate resuspension buffer to the surface or by passively adding them to the resuspension buffer via diffusion. Resuspension may further include removing the surface from the influence of a first force or applying a second force to cause the particles to leave the surface and exit the space between the first and second ribs.

[0126] Those skilled in the art will recognize that, like many methods for separating particles from bulk liquids, this process may include a washing or rinsing step to improve the purity of the separated particles.

[0127] Containers possessing some or all of the aforementioned desired features can achieve capillary action by incorporating ribs (e.g., parallel vertical ribs) around the internal periphery of the container. Each rib is connected to the inner circumference of the test tube surface to form a sharp edge, thereby enhancing capillary ascent. Additionally, the ribs help deflect the draining meniscus and thicken the liquid film, reducing the likelihood of cells being detached from the wall by the draining meniscus. Similarly, the liquid velocity in the spaces between the ribs can be lower, resulting in less resistance to cells at the surface. Furthermore, due to capillary ascent, cells may remain hydrated while attached to the wall, thus reducing cell fixation and potential cell death due to dehydration and increased ionic strength.

[0128] In another embodiment, horizontal or helical ribs, similar to threads, can be used on the surface of the container to obtain improved performance. Cells should be retained during the liquid removal phase of cell separation, but may subsequently need to be recovered by rinsing the liquid down the container wall. Therefore, in a preferred embodiment, vertical ribs along the length of the tube can provide sufficient protection against the drain meniscus during liquid removal, but still allow cells to be washed off the wall by rinsing.

[0129] Since the entire drainage process can be performed by aspiration using a pipette located at the bottom of the container, therefore, in ROBOSep TMIn the (STEMCELL Technologies, Ltd.) scheme, the meniscus shear effect can be amplified. During such aspiration mode, particles and / or cells in contact with the tube surface can be pulled away from the surface by the discharge meniscus. As the particles and / or cells are removed from the surface, they may become entrained in the aspirated liquid and be adversely extracted from the container. This separation effect significantly reduces the recovery rate after separating the particles and / or cells from other particles and / or cells in the bulk liquid. Therefore, the method of holding cells at the container boundary of the present invention can improve the cell recovery rate in positive cell selection schemes or achieve maximum purity in negative cell selection schemes.

[0130] Therefore, the apparatus of the present invention, and the method for improving particle and / or cell separation using it, is achieved by controlling the thickness of the meniscus as the drain meniscus passes over the surface. As described herein, this improvement to the particle and / or cell separation method can be demonstrated by increasing the recovery rate and / or purity of particles and / or cells.

[0131] Furthermore, the improved particle and / or cell separation method using the apparatus of the present invention can be demonstrated by extending cell viability during the cell separation process. After contacting the particles and / or cells with the modified surface (or the inner surface of the container) and removing the bulk fluid by pouring or aspiration, the particles and / or cells are entrained in a liquid film. These particles and / or cells maintain a longer viability than those used in cell separation experiments using typical smooth-walled tubes. Figure 7 The results showed that cell viability remained consistent throughout experiments using rib-walled tubes. Conversely, cell viability consistently declined during experiments using smooth-walled tubes. This increased cell viability entrained in the liquid membrane of the rib-walled tubes allows for the use of longer protocols or "drying times" as needed.

[0132] Furthermore, the improvement of particle and / or cell separation methods using the apparatus of the present invention can be demonstrated by reducing the number of steps in the cell separation procedure (and thus reducing time and cost). In a typical cell separation procedure, particles and / or cells are brought into contact with a smooth surface (e.g., the inner surface of a container, such as a smooth tube wall) under the influence of a force (e.g., magnetic force), and the bulk fluid is removed from the surface while maintaining the influence of the force that holds the separated particles and / or cells in place. A buffer solution is then added to the test tube and mixed with the contents of the test tube, causing many cells and / or particles to detach from the surface. The buffer solution containing the particles and / or cells is then cultured (under the influence of a force) to allow the cells and / or particles to migrate back to the surface. To increase the purity of the separated cells and / or particles, the process can be repeated any number of times, which may also have the effect of reducing the overall recovery rate of the separated cells and / or particles. Figure 8The use of ribbed surfaces (or the inner surface of the container) to overcome the need for such a separation sequence is illustrated. Instead, performing one or more gentle washes yields cell and / or particle recovery rates and purity comparable to multiple separation methods. Conversely, using one or more gentle wash steps with a smooth surface (e.g., the smooth inner surface of the container) (instead of multiple separation methods) results in an undesirable reduction in the recovery rate of separated cells and / or particles compared to multiple separation methods.

[0133] exist Figure 9 Figure a shows an exemplary cross-sectional view of a G-tube employing ribs. The overall shape (diameter, length, wall thickness, edge features) of the tube 300 with ribs can be typical of standard smooth-walled plastic tubes (i.e., F-tubes) used in existing cell separation practices. On the inner surface 310 of the G-tube 300, ribs 320, as described in the present invention, extend axially along their length. In one embodiment, the ribs 320 begin approximately 10 mm from the open end 325 of the tube 300 and extend downward toward the bottom end 340. Various embodiments are evaluated by varying the cross-sectional shape of the ribs (e.g., square, triangular, circular), the height of the ribs (e.g., 250, 500, 1000 μm), and the density of the peripheral ribs (e.g., 10, 30, or 60 ribs distributed around the periphery). Rib density can be adjusted by expanding the utilization of the space therebetween and according to the expected proportion of target cells and / or particles contained in the bulk fluid. By adjusting the rib density based on the expected proportion of target cells and / or particles, the purity and recovery rate of such target cells and / or particles can be improved during the separation process. For example, with only enough space between the ribs to accommodate target cells and / or particles (in response to forces such as magnetism), the target cells and / or particles can well outnumber the non-target cells and / or particles in the space of the surface portion closest to the influence of the force.

[0134] In the pouring scheme, both the standard smooth-walled F-tube and the improved G-tube design retain a significant amount of liquid, representing EASYSep TM The "last drop" in the (STEMCELL Technologies Ltd) solution. The G tube has a volume of approximately 400 μL, while the standard F tube has a volume of approximately 200 μL, indicating that the G tube helps retain a thicker liquid film. Figure 10 Figure a shows the volume retained by the 3 G-tube design compared to the F-tube in the pouring mode. Two different drainage times (2 seconds and 10 seconds) were also compared. It can be seen that both rib density and drainage time affect the final retained volume in the liquid film. Figure 10 b shows RoboSep TMSimilar results were obtained for typical tube aspiration drainage in the (STEMCELL Technologies, Ltd.) scheme. Under these conditions, the geometry of the ribs, the density of the ribs, and the aspiration rate significantly affect the liquid film thickness and the retained liquid volume. At high rib densities, the retained liquid film may be approximately the thickness observed during tube tipping aspiration (i.e., 300 μL). For tube F, the retained film volume may be less than 10% of the retained volume of tube G. Furthermore, the retained volume can be a strong function of the rib density on the surface. At a rib density of 1 / 2, the retained volume can be 2 / 3 of that obtained with 500 μm intervals, while at a density of 1 / 4, less than 1 / 3 of the liquid volume can be retained. Therefore, rib density can be used to adjust the presented liquid film thickness.

[0135] As the aspiration rate decreases, the liquid film volume may also decrease significantly, from 2 seconds for complete drainage to 10 seconds. Therefore, the rate at which bulk liquid is aspirated from tube G can also significantly affect the liquid film volume. It should be noted that for tube F, the aspiration rate has no significant effect on the final retention volume within this range. In both cases, the tube F design retains only about 10% of the maximum retention volume of the tube G design. Therefore, tube G can provide further adjustment of the retained liquid film volume by controlling the aspiration rate.

[0136] During aspiration experiments, it was noted that the aspiration pipette sometimes stuck to the bottom of the ribbed tube 300 due to the aspiration vacuum. To address this issue, a further innovation was made to the tube 300, which includes multiple ribs 320, wherein a pair of ribs 330 can be added to the bottom 340 of the tube 300. This pair of ribs 330 acts as a pipette tip seat, preventing a vacuum from forming between the tube bottom 340 and the aspiration pipette. Figure 9 b). According to this innovation, the aspiration pipette can be placed directly on the bottom 340 of the tube to maintain consistent pipette placement without causing the pipette tip to become stuck to the bottom of the tube during aspiration.

[0137] In further experiments, the possibility of adding a diamagnetic additive to the bulk liquid to remove unmagnetically labeled cells from the surface or container wall near the magnet (i.e., from the space between the first and second ribs) was tested. The diamagnetic liquid (e.g., gadolinium) adds an extra force to the cell separation process because the liquid may be more magnetic than the cells (and the volume they occupy), creating a net repulsive force to move unmagnetically labeled cells away from the container surface adjacent to the magnet. Thus, the G-tube and the diamagnetic additive can work synergistically. The G-tube helps retain the liquid near the wall where the target cells are clustered during cell separation; the thicker the liquid film, the higher the recovery rate of the target cells, as it better protects the cells from meniscus shear. However, with a thicker liquid film, the retention of unlabeled cells, which are randomly distributed in the bulk liquid of the tube and thus in the liquid film on the container wall, may also be higher. With the addition of the diamagnetic liquid, unlabeled cells can be separated from the liquid film volume obtained from the container wall adjacent to the magnet. Therefore, by altering the geometry of the cell wall to thicken the liquid film, the recovery rate of target cells can be improved, while the purity can be increased by using antimagnetic additives to separate non-target cells from the cell wall. Figure 11 a, 11b).

[0138] The apparatus and particle and / or cell separation method of the present invention provide a surface with suitable ribs that can help protect particles and / or cells on the contact surface by forming a liquid film on the ribs on the surface, thereby helping to reduce meniscus peeling of particles and / or cells on the drain / fill surface. The overall performance of manual pouring and automatic pipetting can be improved in the following ways: (1) overall particle recovery rate and purity value, (2) increased range of initial cell numbers, enabling more efficient separation at low cell numbers, (3) reduced differences in separation performance, and (4) faster separation time.

[0139] The following non-limiting examples provide further details that can help those skilled in the art understand the subject matter of the invention.

[0140] Exemplary embodiments

[0141] Example 1: Preliminary Prototype Manufacturing

[0142] Although any surface can be used to test the surface concept for the applicable ribs, the initial testing focused on surfaces suitable for EasySep. TM Silver magnets are available in 14mL test tube sizes and can be used with RoboSep. TM The design also included testing of other concepts, including Eppendorf-type (2 mL) and 50 mL tube sizes. Figure 9A view of the prototype G-tube is shown. The overall shape (diameter, length, wall thickness, edge features) is replicated from a standard 14 mL F-tube. Inside the G-tube, multiple ribs extend axially along its length. These ribs begin approximately 13.5 mm from the top of the tube's opening and extend towards the hemispherical, closed bottom of the tube. Many designs can be created by varying the shape of the ribs (square, triangle), their height (250, 500, 1000 μm), and their spacing (density). One possible embodiment could include square ribs with a spacing of 900 μm and a height of 500 μm. Alternative embodiments could include triangular ribs or ribs with a vertex width smaller than the cell diameter to address cell retention on the top surface of the ribs.

[0143] Example 2: Volume Preservation

[0144] Experiments were conducted to measure and compare the volume of fluid remaining in the smooth-walled F tube and the ribbed G tube after aspiration. The experiments were performed using phosphate-buffered saline containing protein (i.e., 10% newborn calf serum). First, 5 mL of liquid was added to each tube, then the liquid was removed by pouring or aspiration; the tube was kept inverted or a vacuum was applied for a specified time of 2 or 10 seconds. The volume of fluid remaining in the tube was measured using the mass balance method. Figure 10 a and 10b summarize the results.

[0145] In the pouring method, both the F-tube and G-tube designs retain a significant amount of liquid, which represents EasySep's... TM The protocol typically retains the "last drop." The volume in tube G is approximately 400 μL, and in tube F, approximately 200 μL. When the tubes are inverted for 10 seconds instead of 2 seconds, less liquid is retained because more droplets would drip from the tubes during this time. An interesting aspect of these results is the small change in volume retained during pouring. The volume poured out may be influenced by the balance between interfacial tension and the weight of the liquid near the tube opening. For a falling droplet, its weight may exceed the interfacial tension. Therefore, this relationship limits the amount of liquid that can be drained from the tubes by pouring.

[0146] For aspiration, the retained liquid volume decreases proportionally with rib density and, as expected, converges to F-tube (0 ribs). Unlike the pouring process, where the retained volume is controlled by the balance between interfacial tension and weight, in aspiration, the retained volume is controlled by the capillary action of the ribs, and resistance is generated as liquid is expelled from the ribs. Therefore, the retained volume is directly proportional to the number and size of the ribs on the surface. A larger volume retained in the tube (i.e., a liquid film) can reduce the purity of target cells because contaminating cells may remain in the retained volume. If the recovery rate of target cells remains constant, the presence of ribs increases the retained liquid volume, thereby reducing the purity of each wash. Therefore, careful rib design is necessary.

[0147] Example 3: Evaluation of injection-molded G-tubes in RoboSep suction solutions

[0148] (A) Using standard RoboSep TM Operating procedures in RoboSep TM Experiments targeting CD19-positive cells were conducted on the platform. The only difference in experimental conditions was the tubes used during separation. Three 0.5 mL donor samples were separated using F tubes, and three 0.5 mL samples were separated using G tubes. Table 1 lists the recovery rates of target cells for each tube type. The results show that purity was relatively unaffected, while the recovery rate of target cells was increased by more than 2 times.

[0149] Table 1

[0150] Pipe type %purity % recovery rate F-pipe-1 95.1 37.27 F-pipe-2 91.2 28.65 F-tube-3 87.6 41.14 G-tube-1 95.5 84.85 G-tube-2 94.7 76.78 G-tube-3 88.3 87.41

[0151] (B) Using standard RoboSep TM Operating procedures in RoboSep TM Experiments targeting CD56-positive cells were conducted on the platform. The only difference in experimental conditions was the tubes. Three 0.5 mL donor samples were separated using F tubes, and three 0.5 mL samples were separated using G tubes. Table 2 lists the recovery rates of target cells for each tube type. The results show that purity was relatively unaffected, while the recovery rate of target cells was increased by more than 3 times. Furthermore, the general applicability of this design to different cell type selections was confirmed.

[0152] Table 2

[0153] Pipe type %purity % recovery rate F-pipe-1 97.5 16.7 F-pipe-2 95.5 19.02 F-tube-3 97.2 19.94 G-tube-1 97.4 56.94 G-tube-2 96.5 61.03 G-tube-3 98 62.15

[0154] (C) Experiments targeting CD3-positive selection were performed on the RoboSep platform using standard RoboSep procedures. The only difference in experimental conditions was the tube design. Three 0.5 mL donor samples were isolated using F tubes, and three 0.5 mL samples were isolated using G tubes. Table 3 lists the average target cell recovery rates for each tube type. The results show that purity was relatively unaffected, while the target cell recovery rate was increased by more than 1.5 times. However, this example also demonstrates a slight decrease in purity with increasing volume retention in the G tube design.

[0155] Table 3

[0156] Pipe type %purity % recovery rate F-pipe-1 99.6 40.5 G-tube-1 95.5 66.0

[0157] Example 4 – Comparison of tube surface designs using releasable particles in the RoboSep process

[0158] Using standard RoboSep TMOperating procedures in RoboSep TM Experiments targeting CD19 positive selection were conducted on the platform. The only difference in experimental conditions was the tube design. Four 0.5 mL donor samples were isolated in triplicate using F tubes and G tube designs with three different rib densities. In this experiment, the rib density on the wall was compared to optimize the performance of the G tubes. In each case, three replicate samples were isolated, and purity and recovery were evaluated (Table 5). No significant effect of surface rib density on the final purity of CD19 positive selection was found. However, rib density significantly affected the recovery rate of CD19 positive cells. The results showed that the recovery rate increased with increasing rib density. The recovery rate obtained with the maximum rib density tested (60 ribs per tube) was significantly higher than that with designs of 15 or 30 ribs (P < 0.0001).

[0159] Table 4

[0160]

[0161] Example 5 – Synergistic effect of G-tubes and antimagnetic additives

[0162] Experiments were conducted to evaluate the synergistic effect of using a diamagnetic additive (gadolinium) with G-tubes in magnetically labeled cell purification. G-tubes help retain a liquid film on the tube wall, where target cells accumulate near the magnet during cell separation. Simultaneously, the diamagnetic additive, such as chelated Gd... 2+ Non-target, unlabeled cells are separated from the tube wall adjacent to the magnet. In this separation, the countercurrent flow of labeled and unlabeled cells can improve the purity of target cells in the liquid film after bulk liquid aspiration. Figure 11 Figures a and 11b show data from selection experiments for CD3+ and CD19+, respectively. Similar results were found in further replicate experiments. These experiments involved direct comparisons between F-tubes and G-tubes, as well as comparisons between pouring and aspiration liquid separation methods. Clearly, the antimagnetic additive can compensate for the loss of cell purity due to the increased retention volume in G-tubes, while maintaining increased recovery rates due to slow surface drainage caused by surface ribs. The following is a summary of some results:

[0163] • For the antimagnetic additive, the purity was significantly improved (p<0.0001, p<0.0001), but had no significant effect on the recovery rate (p=0.12, p=0.68).

[0164] The antimagnetic additive works in conjunction with the inversion process, which is unexpected, as the mixing that occurs during tube inversion might be considered detrimental to separation. Therefore, the antimagnetic additive can be used to improve purity.

[0165] • Compared to F tubes (~0.5 logit purity), G tubes with and without additives show a greater relative improvement (~1.0 logit purity).

[0166] Therefore, the antimagnetic additive improved the purity of both F and G tubes in both the pouring and suction methods, without negatively impacting the recovery rate. However, the relative improvement in purity was greater in the G tube, confirming a synergistic effect.

Claims

1. An apparatus for separating particles from a bulk liquid, the apparatus comprising: Surfaces in contact with bulk liquids containing particles; The surface has a plurality of ribs, each rib including at least a first rib and a second rib spaced apart from the first rib by a distance, the first rib extending along the axis of the first rib, and the distance between the ribs being 1 μm-1 mm; and The space between the first rib and the second rib is sized such that when liquid in contact with the surface is removed from the surface along the axis of the first rib, a portion of the bulk liquid and at least a portion of the particles in the bulk liquid are retained in the space by capillary action. The surface is the inner wall of the container, and the axis of the first rib is parallel to the axis of the container; as well as The plurality of ribs extend from the inner sidewall into the interior of the container.

2. The apparatus according to claim 1, characterized in that: The first rib extends along a first longitudinal axis, and the second rib extends along a second longitudinal axis.

3. The apparatus according to claim 2, characterized in that: The second longitudinal axis is substantially parallel to the first longitudinal axis.

4. The apparatus according to claim 3, characterized in that: The first longitudinal axis and the second longitudinal axis are linear.

5. The apparatus according to claim 1, characterized in that: The first rib includes: a first sidewall extending away from the surface and having a first base edge and a first protruding edge; and a second sidewall extending away from the surface and having a second base edge and a second protruding edge; the first base edge and the second base edge are spaced apart by a first rib width, and the first protruding edge connects to the second protruding edge at a first apex height; and The second rib includes: a third sidewall extending away from the surface and having a third base edge and a third protruding edge; and a fourth sidewall extending away from the surface and having a fourth base edge and a fourth protruding edge; the third base edge being spaced apart from the fourth base edge by the width of the second rib, and the third protruding edge being connected to the fourth protruding edge at a second apex height.

6. The apparatus according to claim 5, characterized in that: The heights of the first vertex and the second vertex are respectively between 20 μm and 1 mm.

7. The apparatus according to claim 5, characterized in that, Also includes: A third rib, spaced apart from the second rib by the spacing distance, the third rib comprising: a fifth sidewall extending away from the surface and having a fifth base edge and a fifth protruding edge; and a sixth sidewall extending away from the surface and having a sixth base edge and a sixth protruding edge; the fifth base edge being spaced apart from the sixth base edge by the width of the third rib, and the fifth protruding edge being connected to the sixth protruding edge at a third apex height.

8. The apparatus according to claim 7, characterized in that: The height of the third vertex is between 20 μm and 1 mm, and is different from the height of the first vertex and the second vertex.

9. The apparatus according to claim 7, characterized in that: The first protruding edge is connected to the second protruding edge via a first top wall, and the third protruding edge is connected to the fourth protruding edge via a second top wall.

10. The apparatus according to claim 9, characterized in that: The fifth protruding edge is connected to the sixth protruding edge via the third top wall.

11. The apparatus according to claim 10, characterized in that: The widths of the first top wall, the second top wall, and the third top wall are between 1 μm and 1 mm.

12. The apparatus according to claim 11, characterized in that: The spacing between adjacent ribs in the plurality of ribs is uniform.

13. The apparatus according to claim 2, characterized in that: The first rib has a first cross-sectional shape obtained in a plane orthogonal to the first longitudinal axis, and the second rib has a second cross-sectional shape taken in the same plane.

14. The apparatus according to claim 13, characterized in that: The first cross-sectional shape is the same as the second cross-sectional shape.

15. The apparatus according to claim 13 or 14, characterized in that: The first cross-sectional shape is quadrilateral.

16. The apparatus according to claim 13 or 14, characterized in that: The first cross-sectional shape is triangular.

17. The apparatus according to claim 2, characterized in that: The first longitudinal axis and the second longitudinal axis are oriented relative to the flow direction of the bulk liquid thereon, such that the first longitudinal axis and the second longitudinal axis are not parallel to the flow direction.

18. The apparatus according to claim 9, characterized in that: The first top wall and the second top wall are coplanar with the inner surface of the container.

19. The apparatus according to claim 18, characterized in that: The container is a test tube.

20. The apparatus according to any one of claims 1 to 10, characterized in that: The first rib and the second rib extend along the longitudinal axis of the surface by one rib length, and the rib length is 5%-95% of the longitudinal axis of the surface.

21. A container for containing bulk liquids containing particles, characterized in that, The container includes: A closed bottom end, having: a bottom wall; an open upper end; and one or more side walls extending from the bottom wall to the upper end, the one or more side walls defining the container axis; Define the inner surface of the container and its opposite outer surface; A plurality of ribs extending parallel to the container axis on the inner surface extend from the inner surface into the interior of the container. The plurality of ribs includes at least a first rib and a second rib spaced apart from the first rib by a distance of 1 μm-1 mm. The space between the first rib and the second rib; Thus, when the bulk liquid is contained inside the container, the bulk liquid contacts the inner surface, the first rib and the second rib, and the space between the first rib and the second rib; and the dimensions of the first rib and the second rib are configured such that when the bulk liquid in contact with the surface is removed from the surface along the container axis, a portion of the bulk liquid and at least a portion of the particles in the bulk liquid are retained therein by capillary action.

22. The container according to claim 21, characterized in that: The plurality of ribs cover 5% to 95% of the area of ​​the inner surface of the sidewall.

23. The container according to claim 21 or 22, characterized in that: The plurality of ribs are located at the bottom, middle or top of the container.

24. The container according to claim 21, characterized in that: The sidewall includes the plurality of ribs.

25. The container according to claim 21, characterized in that: The plurality of ribs are integrally formed with the sidewall of the container.

26. A method for separating particles from a bulk liquid using an apparatus, wherein, The device includes: a surface, which is the inner wall of a container; a plurality of ribs extending along the container axis and from the inner wall into the interior of the container; the plurality of ribs including at least a first rib and a second rib spaced apart from the first rib by a spacing of 1 μm-1 mm, wherein the first rib extends along a first rib axis parallel to the container axis; and a space between the first rib and the second rib. The method includes: The device is brought into contact with the bulk liquid, thereby bringing the bulk liquid into contact with the surface, the first rib and the second rib, and the space between the first rib and the second rib; At least a first portion of the particles in the bulk liquid is contained in the space between the first rib and the second rib; The bulk liquid is removed from the surface along the axis of the first rib, so that a portion of the bulk liquid is retained between the first rib and the second rib by capillary action to form a liquid film therebetween; When the bulk liquid is removed from the surface, the particles contained between the first and second ribs and entrained in the liquid film are protected from one or more forces acting on the drainage meniscus; and The protected particles entrained in the liquid film are resuspended in the buffer solution.

27. The method according to claim 26, characterized in that: It also includes applying a first force to cause the particles to enter the space between the first rib and the second rib.

28. The method according to claim 27, characterized in that: At least a second portion of the particle that has entered the space responds to the first force.

29. The method according to claim 28, characterized in that: The first force is a magnetic attraction, and the responding particle has a first magnetic charge that is attracted to the magnet, such that the device is positioned between the magnet and the bulk liquid, thereby causing the first force to move a first portion of the particle toward the space.

30. The method according to claim 28 or 29, characterized in that: In the absence of the first force, the particles contained in the space will empty the space.

31. The method according to claim 28 or 29, characterized in that: It also includes adding antimagnetic additives to the bulk liquid.

32. The method according to claim 31, characterized in that: The antimagnetic additive is gadolinium.

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