Cell washing apparatus and method

Abstract

This invention discloses a cell washing machine and a method for washing cells from a sample comprising cells suspended in a liquid using the cell washing machine. The cell washing machine includes a container configured to contain cells. The container includes an elongated body comprising an opening, an inner surface, a recess, and a cavity. The recess is defined by a first inner surface portion of the inner surface, the first inner surface portion being disposed between and radially outward relative to the second and third inner surface portions. The container also includes an actuation device capable of causing the container to rotate about an axis and a conduit configured to pass through the opening and to deliver fluid to and from the cavity during rotation of the container. The cavity is defined by a lower portion of the body and located below the recess, wherein the recess has an aspect ratio of at least about 2:1.

Description

[0001] This application is a divisional application of the application filed on December 14, 2017, with Chinese national application number 201780083346.4 (international application number PCT / US2017 / 066312) and entitled "Cell Washing Apparatus and Method".

[0002] Cross-references to related applications

[0003] This application is a non-provisional application of U.S. Provisional Application 62 / 434,748, filed on December 15, 2016, and claims priority to that provisional application, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0004] Embodiments of the present invention relate to the field of particle preparation, and more specifically to apparatus and methods for washing cells. Background Technology

[0005] Particle analysis, especially cellular analysis, often requires the removal or replacement of suspended particles or cells with liquid. This is used to remove interfering substances, excess contaminants, unbound labeled antibodies, detergents, permeabilizers, lysing agents, fixatives, neutralizing agents, and other substances. In some cases, cell washing can also be used to reduce unwanted interactions between cell types mediated by dissolved substances.

[0006] Traditional methods for washing particles or cells involve a variety of processes, such as precipitation and decanting, acoustic separation, centrifugation, filtration, flow through structured channels, and magnetic separation. These methods are difficult to automate, cumbersome, time-consuming, and often require disposable consumables. Therefore, there is a need for a process and apparatus for cell washing that is easily automated.

[0007] The purpose of cell washing is to remove unwanted substances that may affect further processing or subsequent analysis. Cell washing typically involves removing the suspension and resuspending the cells in a replacement liquid (generally referred to as the wash liquid). Different wash liquids may be used in different parts of the washing process, and may also include prolonged exposure to a selected wash liquid or agitation to facilitate the transfer of unwanted substances.

[0008] Cell washing can lose, damage, activate, alter, or destroy cells, leading to undesirable cell-cell interactions or differentially depleting samples of certain cell types. These effects can alter the results of subsequent analyses. For example, exposing cells to high accelerations (such as accelerations generated by the inertial effects of high-speed rotation or "centrifugal force") or high fluid shear rates can cause some cell types to rupture more than others. Therefore, measurements of washed cell mixtures may not accurately reflect the proportions of cells present in the original sample. Thus, there is a need for a process and apparatus for cell washing that preserves the characteristics and proportions of various cell types in a sample.

[0009] Washing efficiency is a measure of the quality of a washing process. A wash is efficient if it removes more unwanted substances. A process with lower washing efficiency leaves more unwanted substances behind. In some cases, multiple instances of a process can be linked to improve the washing efficiency of a combined process. While a simple dilution model is impractical for all washing processes, it serves to illustrate the concept of washing efficiency. If a washing process leaves a portion d of the fluid present at the start of the wash at the end of the wash, then after n repetitions, the remaining portion of the original fluid (including the original unwanted substances) is (d). n The washing efficiency can be expressed as the reciprocal of the remainder: in this case, it is 1 / d for a single-cycle process and 1 / d for a multiple-cycle process. n For example, if a washing process leaves 10% of the original liquid after one cycle, its washing efficiency can be expressed as 1 / 0.1 = 10. Four cycles of this type of washing process provide 1 / 0.1. 4 = 10,000 Ideal combination washing effect.

[0010] However, washing steps may not be linked together without any consequences: additional repeated washing requires more time and can increase the likelihood of cell alteration or loss. Furthermore, resuspension processes can be particularly destructive to cells because they typically rely on high fluid shear. Therefore, there is a need for a process and apparatus for cell washing that efficiently removes cells with fewer washes and resuspensions.

[0011] Cells are sensitive to the magnitude and duration of the applied force during washing; prolonged exposure to high forces can cause significant cell damage. However, in centrifugal cell washers, the time cells spend settling under applied force decreases as the force increases. Therefore, there is a need for a cell washing apparatus and process that reduces the duration of cell exposure to high forces.

[0012] Live cells are generally slightly denser than the aqueous wash liquid. In centrifugal cell washeres, the denser cells settle in a region of relatively high force through the wash liquid. The washing protocol in a centrifugal cell washer typically involves repeated cycles of settling the cells, removing as much wash liquid as possible, and resuspending the cells in fresh wash liquid. The time required for cell settling depends on the distance the cells travel through the wash liquid, and therefore takes longer when a large volume of wash liquid is present before settling. Therefore, it is desirable to settling from an initial, smaller volume of wash liquid. The washing efficiency of such processes is limited by the amount of wash liquid remaining after each removal step (often limited by mechanical constraints), compared to the total volume of wash liquid exposed to the cells. Therefore, it would be beneficial to settling the cells from an initial, smaller volume of wash liquid and then exposing them to additional wash liquid after settling. However, once the cells settle into a region of relatively high force, the additional wash liquid cannot interact significantly with the cells because the force causes the less dense liquid to float "above" the cells. Therefore, a cell washer apparatus and process are needed that allows the additional wash liquid to interact with the cells that have undergone washing during a single wash cycle.

[0013] In centrifugal cell washing, using closed containers requires complex rotating connections to add or remove liquid. However, open containers allow liquid to escape unless the container volume is larger than the volume it contains. Therefore, there is a need for an open-container cell washing machine that can hold a relatively large volume of liquid.

[0014] The embodiments of the present invention can solve these and other problems individually and collectively. Summary of the Invention

[0015] In some embodiments, the invention includes a cell washing machine having a container for containing cells. The container includes an elongated body defining an opening, a cavity, and a recess. The opening communicates with the cavity, and the cavity communicates with the recess. The recess may extend radially outward relative to a portion of the elongated body defining the cavity. The recess may be symmetrical about a vertically oriented axis. The elongated recess has a radial depth extending beyond the sidewalls forming the cavity. The recess may have an aspect ratio of about 2:1, 4:1, 10:1, or 15:1 or greater (e.g., the length-to-depth ratio when viewed in an axial cross-section). An actuation device, such as a rotor, may cause the container to spin about the axis.

[0016] The cavity may be at least partially defined by a cylindrical segment of the body and may be positioned below the recess. The inner surface defining the cavity may transition into the recess in an S-shape.

[0017] The cell washing machine may also include a conduit that passes through an opening and enters a cavity. The conduit is configured to deliver fluid into and out of the cavity during rotation of the container. The conduit terminates at an end that may be positioned adjacent to the inner wall of the container below a recess. The conduit may be offset from and parallel to the axis. The conduit may be fluidly coupled to a fluid pump.

[0018] The container may also include an upper annular region defined by the upper portion of the body. The upper annular region may be positioned between the recess and the opening.

[0019] In some embodiments, the cell washing apparatus may also include a probe mounted on a lift, wherein the lift is configured to lower the probe through an opening. A sample pump may be in fluid communication with the probe. The pump and probe allow for the addition or removal of substances from the container. A controller may be electrically connected to an actuator (e.g., a rotor), a fluid pump, a sample pump, and the lift may control the operation of these components.

[0020] Embodiments of the invention also include a method comprising washing cells from a sample having cells suspended in a liquid. The method includes the step of dispensing the sample into a container comprising a cavity and a recess, wherein the cavity and the recess are arranged about a vertically oriented axis. The recess is positioned radially outward relative to a portion of a cavity forming a body. Other steps include rotating the container about its axis at a first velocity, displacing cells into the recess, causing cells to precipitate against the wall of the recess, and withdrawing at least a portion of the liquid.

[0021] The step of translocating cells into the pit includes adding washing liquid from the first aliquot of the sample into the container. The washing liquid is added through a conduit that extends into the container and terminates below the pit. The extraction step includes aspiration through the same conduit at different times.

[0022] Other steps may include adding washing liquid from a second or subsequent aliquot through a catheter and resuspending the cells. In some embodiments, the steps of withdrawing at least a portion of the liquid from the container and adding washing liquid from the second (or subsequent) aliquot may be repeated once or more to provide more effective washing. For example, the steps may be repeated 2 to 5 times in some embodiments of the invention.

[0023] The step of rotating the container at a first speed generates a force of at least about 100 or 250 x g in the pit. The method of washing cells may also include rotating the container at a second speed during the extraction step. This rotation at the second speed generates a force of at least about 25 x g in the pit. Therefore, in some embodiments, the subsequent container rotation speed may be reduced with each successive washing step.

[0024] The process of resuspending cells may include the sub-steps of adding a resuspension solution and stopping the rotation. In some embodiments, the rotation may be abruptly reversed to facilitate cell resuspension. Once the rotation is stopped, the washed cells can be removed.

[0025] The volume of the sample can be smaller than the volume of the chamber beneath the pit, and in some embodiments, it can be smaller than about 0.7 times the volume beneath the pit. The volume of the resuspension liquid can be smaller than the volume of the sample, such that after washing, the washed cells are suspended at a higher concentration than the cells in the sample before washing.

[0026] The method may include additional steps for cleaning the container for subsequent use, including adding flushing liquid, rotating the container at a third speed, and aspirating the flushing liquid through a conduit. The step of cleaning the container may include changing the rotation speed during the step of aspirating the flushing liquid.

[0027] The wash solution or resuspension solution may include suitable substances. For example, it may include about 5 mM EDTA and may also include about 0.1% to about 2% fetal bovine serum. In some embodiments, the wash solution consists of an isotonic buffer (e.g., PBS), an anticoagulant (e.g., EDTA), and a protein (e.g., fetal bovine serum). In some embodiments, fetal bovine serum may be used as the protein. Attached Figure Description

[0028] Figure 1A and Figure 1B A schematic side sectional view of an embodiment of the device of the present invention is shown.

[0029] Figure 2 It shows Figure 1A and Figure 1B A schematic side sectional view of the container of the implementation scheme.

[0030] Figures 3A to 3F The illustrated steps of an exemplary cell washing protocol are shown.

[0031] Figure 4 It shows Figures 3A to 3F The flowchart of the cell washing protocol.

[0032] Figures 5A to 5F The illustrated steps of an exemplary container cleaning solution are shown.

[0033] Figure 6 It shows Figures 5A to 5F The flowchart of the container cleaning procedure.

[0034] Figures 7A to 7F A scattering diagram is shown of the white blood cell recovery rate of an embodiment using the apparatus and method of the present invention compared to conventional washing and no washing.

[0035] Figures 8A to 8F The diagram shows the scattering plots of the WBC five-item classification analysis of the embodiment using the apparatus and method of the present invention compared to conventional washing and no washing.

[0036] Figures 9A to 9C Scattering diagrams of platelet-monocyte interactions using embodiments of the apparatus and method of the present invention are shown, compared to conventional washing and no washing.

[0037] Figures 10A to 10C Cell viability is shown in embodiments using the apparatus and method of the present invention compared to conventional washing and no washing.

[0038] Figure 11A and Figure 11B The κ / λ separation of embodiments using the apparatus and method of the present invention is shown compared to conventional washing.

[0039] Figures 12A to 12C The total cell recovery rate of embodiments using the apparatus and method of the present invention is shown compared to conventional washing.

[0040] Figures 13A to 13C The T lymphocyte recovery rate of embodiments using the apparatus and method of the present invention is shown compared to conventional washing and no washing.

[0041] Figures 14A to 14C The B cell and NK cell recovery rates of embodiments using the apparatus and method of the present invention are shown compared to conventional washing and no washing.

[0042] Figure 15 A partial side sectional view of a component according to an embodiment of the present invention is shown.

[0043] Figure 16 It shows Figure 15 Top cross-sectional view of the component shown. Detailed Implementation

[0044] Figure 1A and Figure 1B An embodiment of a cell washer according to an embodiment of the present invention is illustrated graphically. The cell washer 1 includes a container 10, which includes a body, a rotor 12, a conduit 18, and a controller 20. In some embodiments, the cell washer 1 may further include a pipette 16 and a housing 14.

[0045] Figure 2Container 10 is shown in more detail. Container 10 includes an elongated body capable of being configured to contain cell samples and washing liquid during the washing process. Container 10 can be oriented during operation so that its long axis 42 is vertical. Container 10 includes a closed bottom 36, an open top 38, and a wall 28 extending between the bottom 36 and the top 38. Wall 28 defines a cavity 30 and a recess 34.

[0046] In this document, the inner surface of wall 28 may refer to a portion of the defining cavity 30 or recess 34 of container 10. The outer surface of wall 28 may refer to a portion of container 10 separated from cavity 30 or recess 34 by a portion of wall 28.

[0047] In this document, the recess 34 and the cavity 30 are represented separately, even though both features are defined by the inner surface of the body of the container 10. The inner surface of the body may include a first inner surface portion 37A disposed between and radially outwards a second inner surface portion 37B and a third inner surface portion 37C to form the recess 34. The cavity 30, which may taper toward axis 42, is separated from the recess 34 by the respective radii of its defining surface portions 37A and 37B. For example, the first inner surface portion 37A may define at least a portion of the recess 34, while the second inner surface portion 37B may define at least a portion of the cavity. Figure 2 As shown, the first inner surface portion 37A is farther from the major axis 42 (in a radial sense) than the second inner surface portion 37B. Figure 2 In one embodiment, the radius of the recess 34 at its bottom (corresponding to the middle inner wall of the body of the container 10) is substantially constant along the axial range of the recess 34 (except for the transition region at one or both ends of the recess 34, which lies between the first inner surface portion 34A and the second inner surface portion 34B, and between the first inner surface portion 34A and the third inner surface portion 34C). The radius of the recess 34 at its bottom may also be constant along most or all of its axial range, in which case the space formed by the recess 34 can form a cylindrical shell with a defined thickness (depth of the recess). The hollow portion or void inside the container 10 may include a cavity 30 and an upper annular region 39, which may be defined by the upper portion 40 of the body of the container 10. The extent of the recess 34 includes any transition region that is small relative to the axial range of the recess (less than about one-tenth of the axial range of the recess). The aspect ratio of the pit can be greater than approximately 2:1, 3:1, 5:1, 10:1, or 15:1 (for example, where the aspect ratio is length:depth). The radius can vary between the radius of the region surrounding the cavity 30 and the increased radius of the pit 34.

[0048] Wall 28 and the body comprising Wall 28 may be formed of a stable solid material such as glass, polycarbonate, or acrylic plastic. In some embodiments, high-density polyethylene is used to improve washing efficiency, which is attributed to reduced adhesion. Transparency is beneficial during process development because fluid behavior and cleanliness can be observed through Wall 28 during operation.

[0049] In some embodiments, container 10 and cavity 30 are symmetrical about axis 42. The outer surface of wall 28 may be shaped similarly to the inner surface of wall 28, such that wall 28 has a relatively constant thickness. This has the beneficial effect of reducing the mass of container 10 and thus reducing the torque required to make container 10 spin. The outer surface of wall 28 (and therefore the outer surface of container 10) may include a cylindrical profile and may terminate at a hemispherical bottom end, such that the outer surface of container 10 is shaped similarly to a conventional test tube.

[0050] In some embodiments, the cell washer 1 may be designed to process samples with a volume of approximately 1 mL. In such embodiments, the chamber 30 may be fitted within a cylindrical enclosure with a diameter of approximately 0.35 inches and a height of approximately 2 inches. However, in other embodiments, for other sample sizes, the cell washer 1 may be made larger or smaller by scaling to maintain force and relative volume.

[0051] The inner surface of wall 28 (which may correspond to the second inner surface portion 37B) may define at least a portion of the lower portion 32 of the container and the cavity 30. In some embodiments, an upper annular region 39 may be present in the upper portion 40 of the container 10. The lower portion 32, the intermediate portion 55, and the upper portion 40 are arranged along the axial length of the container 10. The lower portion 32, including the cavity 30, extends upward from the inner surface of the bottom 36. The intermediate portion includes a central annular region 56 with a recess 34 and begins above the lower portion 32 and extends further upward. The upper portion 40 includes the annular region 39 and, when present, begins above the central annular region 56 and extends further upward to the inner surface of the opening top 38. The wall 28 defining the cavity 30 may be formed with a smooth inner surface without sharp inner corners to avoid trapping any cells or liquids during use. The minimum inner corner radius may depend on the surface properties of the wall material and the ability of the liquid within the container 10 to wet the wall material. In some implementations, when using an aqueous fluid in a polycarbonate container, the minimum inner corner radius can be approximately 0.03 inches. The beneficial effect of separating the recess 34 from the lower portion 32 is that the recess 34 provides a location where centrifugal force confines the particles to a position separate from where the liquid is added or removed. This prevents accidental removal of particles during the washing process, thereby improving recovery rates.

[0052] Bottom 36 is the closed end forming the elongated body of container 10. Bottom 36 is positioned downwards during operation of cell washing machine 1. The orientation referred to in this specification is this orientation. The outer surface of wall 28 at bottom 36 is the physical bottom of container 10. The inner surface of wall 28 at bottom 36 is the physical bottom of cavity 30.

[0053] The lower portion 32 begins at the intersection of axis 42 and the inner surface of wall 28 at bottom 36. The purpose of the lower portion 32 is to allow selective addition or removal of liquid without affecting the cells. Cells can be retained in the recess 34 under the influence of centrifugal force while fluid is added to and removed from the lower portion 32. The inner surface of wall 28 tapers smoothly and monotonously outward from axis 42 and upward from bottom 36 until it reaches lower radius 33. Lower radius 33 is the maximum inner radius of lower portion 32. Lower portion 32 may continue further at lower radius 33 to form a cylindrical segment 31 of lower portion 32, or it may terminate at the transition of recess 34. In some embodiments, lower portion 32 is similar in shape to a test tube, having a curved lower portion and a cylindrical upper portion. In such embodiments, the radius of the cylindrical upper portion corresponds to the lower radius 33. The curved lower portion can be substantially hemispherical, parabolic, or any other contour that transitions from the intersection of the centerline and the bottom 36 to the pit radius 35. The volume of the lower portion 32 can be large enough to contain the entire sample containing the cells to be washed. The beneficial effect of the tapered lower portion 32 is that the outward-directed centrifugal force pushes relatively denser cells or particles upward along the cone towards the pit 34. A second beneficial effect is that when rotation stops, the liquid flows to the bottom center, thereby improving recovery through the probe 54.

[0054] The recess 34 may form part of a central annular region 56 within the interior region of container 10, having a maximum inner radius. The purpose of the recess 34 is to contain cells within a limited volume during the washing process, allowing the suspension to be exchanged without cell loss. The recess 34 may have a substantially constant inner radius (recess radius 35), differing in that it engages with the lower portion 32 (and with the upper portion 40, if present). In other embodiments, the recess 34 may have other shapes. For example, the recess 34 may be slightly ellipsoidal, causing cells to preferentially settle into the "deeper" portion of the ellipsoid. This can be useful when the sample has a very low cell concentration. Other shapes may also be used in other applications.

[0055] In the implementation, the inner surface of the wall 28 at the boundary of the lower portion 32 and the recess 34 may form an S-shaped transition 41 (as from...). Figure 2(See axial cross-section view shown). The beneficial effect of the S-shaped transition 41 is that it reduces particle trapping or suspension as particles move between the pit 34 and the lower portion 32. This will increase recovery and reduce residue between samples.

[0056] In an embodiment where the recess 34 has a cylindrical section, the effective volume of the recess 34 is the difference between the volume of the cavity 30 between the ends of the recess 34 and the volume of a cylinder having a radius equal to the lower radius 33 between the ends of the recess 34. Therefore, the effective volume is a cylindrical shell with one or two S-shaped tapering ends. A beneficial effect of the cylindrical section is that cells can be packed in a thin layer, making cleaning easier and subjecting the cells to similar g-forces at a given rotational rate. Another beneficial effect of the cylindrical section is the elimination of trapped fluid volume, thereby improving washing efficiency.

[0057] In some embodiments, the pit 34 comprises an effective volume at least equal to the total volume of cells or particles in the sample. In other embodiments, the pit 34 comprises an effective volume at least equal to the total volume of the sample. This allows the entire sample to adhere within the pit 34 during cell precipitation, thereby supporting such precipitation at a nearly constant radius (and therefore a nearly constant force), regardless of the cellular portion of the sample. In other embodiments, the pit 34 comprises an effective volume at least equal to approximately 1.4 times the total volume of the sample. This larger volume supports precipitation at a nearly constant radius even when the sample is diluted during the upward shifting step, which is described in more detail below.

[0058] An upper portion 40 (when present) begins above the recess 34 and extends to a apex 38. The purpose of the upper portion 40 is to provide a location for receiving a washing liquid that traverses the recess 34, allowing cells in the recess 34 to be exposed to fresh washing liquid. The upper portion 40 tapers smoothly and monotonously inward from the recess 34 and merges with the apex 38, such that radial forces tend to force any relatively high-density material into the recess 34. The volume of the upper portion 40 may be similar to that of the lower portion 32 to accommodate the fluid initially supplied to the lower portion 32. In some embodiments, the upper portion 40 may have a larger volume than the lower portion 32 because a generally parabolic profile without fluid boundaries occupies a larger portion of the upper portion 40 than a portion of the lower portion 34. The upper portion 40 and the lower portion 32 may comprise cylindrical segments having approximately the same radius. The beneficial effect of the paired upper and lower portions surrounding the recesses is that they allow washing liquid to be added to the lower portion 32 to sweep across the cells enclosed in the recesses 34, rinsing the cells and removing dirty washing liquid from them. This increases washing efficiency while minimizing the volume of washing liquid.

[0059] As container 10 spins about axis 42, the fluid contents of container 10 are distributed radially outward and upward under the combined influence of centrifugal force and gravity. The fluid contents form a cup-shaped hollow shell, defined by wall 28 on the outside, bottom 36 on the bottom, annular lip 46 on the top, and a fluidless boundary in the middle. The fluidless boundary exhibits a generally parabolic shape, the parameters of which are determined by the rotational speed, the surface tension of the fluid, and the contact angle of the wall material relative to the fluid. VA Lubarda published a detailed analysis of the shape of the fluidless boundary in Acta Mech (2013) 224: 1365, the full text of which is incorporated herein by reference. As the rotational speed increases, the parabolic edge of the fluidless boundary descends towards the bottom 36 and comes into contact with the bottom 36, allowing the fluid to be distributed within the open shell.

[0060] The tip 38 has an opening 44 centered on axis 42 and a lip 46 surrounding the opening 44. The purpose of the tip 38 is to contain liquid in container 10 during rotation. The opening 44 may be circular and defines an opening diameter. The opening radius may be selected such that the fluid is held within container 10 at maximum operating rotational speed. In some embodiments, the maximum operating speed generates a centrifugal force of about 400 × g, and the opening radius is about 0.25 inches. The diameter of the opening 44 may be large enough to allow the introduction and removal of the sample, and small enough to prevent the fluid from escaping at maximum spin speed. In some embodiments, the diameter of the opening 44 may be between about 0.157 inches and about 0.25 inches.

[0061] The beneficial effect of the lip 46 surrounding the opening 44 is that the lip 46 retains the liquid contents when the container 10 spins and allows access to a non-spinning device such as the conduit 18 or pipette 16. In other embodiments, surface tension and gravity can retain the contents of the container 10 during spin, and the lip 46 may be absent.

[0062] The conduit 18 includes one or more tubes 64, a terminal 66, and associated fluid. The purpose of the conduit 18 is to deliver and transfer liquid to the container 10. The tube 64 has a free end located at the terminal 66 and a connecting end that connects the exterior of the container 10 to associated fluid components such as a bidirectional pump 68, one or more valves 74, one or more wash liquid reservoirs 70, and a waste liquid reservoir 72. The external fluid components are configured to deliver one or more wash liquids and remove waste liquid through the tube 64 under programmable control. This bidirectional liquid delivery may be driven by the bidirectional pump 68, which may be a single pump (such as a syringe pump or peristaltic pump) or two or more pumps, each operable in a single direction. In some embodiments, the conduit 18 may extend through the housing 14.

[0063] Tube 64 enters container 10 through opening 44 and extends downward into lower portion 32. Tube 64 may be a round tube, such as a section of a hypodermic needle shaft. Tube 64 may be straight for most of its length, but may include a bend near its free end, such that end 66 can be positioned near the tapered portion of wall 28. The straight portion of tube 64 may be positioned generally parallel to axis 42, but offset from the axis on the side toward the annular lip 46 (e.g., not collinear but parallel). In some embodiments, conduit 18 (including tube 64) does not rotate with container 10, so tube 64 is positioned not to contact any part of container 10. The off-axis position of tube 64 allows pipette 16 (of generally larger diameter) to enter without contacting tube 64 or container 10. The advantage of tube 64 extending above container 10 is that fluid can be added or removed from outside container 10. The advantage of tube 64 extending through opening 44 is that liquid can be added or removed without rotating the seal, thereby reducing system complexity. The beneficial effect of terminating tube 64 near the inner wall is that it maximizes the portion of liquid that can be removed during washing, thereby enhancing washing efficiency. It also removes the liquid with a lower g-force than that applied to particles in the pit, thus simplifying the associated fluid dynamics. The beneficial effect of tube 64 terminating below pit 34 includes the following: the added liquid washes the cells in pit 34, and the contained cells are not removed when the liquid is withdrawn. This increases washing efficiency and improves recovery rate.

[0064] The shape and position of the tube 64 as it extends through the upper portion of the container 10 have unexpected effects. For example, the tube 64 stabilizes the liquid within the container 10 during rotation and influences the shape of the wash liquid pulses. Using the tube 64 constructed as described above, the container 10 can contain a volume of fluid that would otherwise be expected to overflow through the opening 44 at the rotational rate of the container 10. As the pulses of the added wash liquid are conducted through the container 10, the tube 64 shapes the pulses of the added wash liquid, resulting in more efficient washing. Without being bound by theory, the first effect is believed to be due to the surface interaction between the tube 64 and the parabolic edge without a fluid boundary during rotation, and the second effect is due to a combination of surface tension and viscous damping effects. The cell washing machine 1 utilizes these effects to manipulate the filling level and rotational rate of the container 10 to deliver pulses of wash fluid upwards and downwards through cells deposited in the pit 34.

[0065] The bend near end 66 allows tube 64 to maintain its vertical off-axis position for most of its length, while allowing end 66 to be positioned further away from axis 42 as desired. The bend allows end 66 to be positioned within approximately 0.06 inches of wall 28 to aspirate and deliver liquid during processing. The bend can be formed at any suitable angle, including 90 degrees, 120 degrees, 130 degrees, etc. This relatively large distance reduces tolerances in the manufacture of cell washing machine 1. In an embodiment, end 66 can be within approximately 0.03 inches of wall 28 to allow conduit 18 to aspirate a larger portion of the remaining liquid after the washing phase, thereby improving washing efficiency. The section of wall 28 near end 66 can be located within the tapered portion of the lower portion 32. This position allows for the addition of liquid during rotation, which displaces sample components upward into pit 34, and allows for the removal of liquid, allowing disturbed cells to settle in pit 34.

[0066] Rotor 12 causes container 10 to spin about axis 42. Rotor 12 includes motor 48 and coupling 52. The purpose of rotor 12 is to generate a controlled centrifugal force in container 10. Motor 48 can be any of a variety of motors known in the art, such as a brushless DC motor. In some embodiments, motor 48 is capable of causing container 10 to spin at a speed of at least about 10,000 rpm to generate a radial force of about 400 × g or greater. Suitable bearings support the spinning component.

[0067] Motor 48 is electrically connected to controller 20. Controller 20 provides electrical signals to motor 48 as needed via a driver (not shown). Coupling 52 connects motor 48 to container 10. Coupling 52 may be integrally formed with container 10, such as by forming a hole in the exterior of bottom 36 for receiving a key shaft or pin of motor 48. Alternatively, coupling 52 may include a hollow portion that receives the exterior of container 10. In such embodiments, coupling 52 may include a window for viewing the process during operation.

[0068] Although rotor 12 is shown for illustrative purposes, any other suitable actuation device may be used. For example, instead of a rotor, container 10 may have a magnet therein that can be electromagnetically coupled to a coil in the surrounding container, such that the coil and the corresponding power source can move container 10 by electromotive force.

[0069] The pipette 16 includes a probe 54, a lift 56, and a sample pump 58. The purpose of the pipette 16 is to add the sample to be washed and to remove washed cells from the container 10. The pipette 16 can also remove the rinsing fluid after the container is cleaned.

[0070] Probe 54 may be an elongated tube that enters opening 44 when rotation stops. The purpose of probe 54 is to contain liquid and deliver liquid to and from container 10. Probe 54 may be a washable tube, such as those commonly used in chemical or hematological analyzers. Alternatively, probe 54 may be a disposable pipette tip coupled to a pipette mandrel. Probe 54 may also include conventional level sensing devices, such as a capacitive level sensor, to detect the level of fluid within the sample container and in container 10.

[0071] Probe 54 approaches container 10 at or near the bottom center to ensure maximum removal of material. Probe 54 enters container 10 and is positioned closer to the container axis than tube 64 to avoid collision. The advantages of using a probe separate from tube 18 for adding and removing liquid are: probe 54 can aspirate the sample, resulting in less residue compared to the more complex fluid connections of tube 18, and the probe can approach the bottom of the container without the risk of interfering with the location of end 66.

[0072] The lift 56 may consist of conventional positioning components including a motor and a sliding element. Its purpose is to raise and lower the probe 54 through the opening 44 to deliver or remove liquid. The lift 56 may also include at least one additional axis of motion for positioning the probe 54 relative to a sample container, waste container, probe washer, or liquid reservoir. A sample pump 58 is fluidly coupled to the probe 54 and provides prime mover to move the fluid within the probe 54. The sample pump 58 may be any of a variety of conventional pumps capable of delivering controlled volumes of sample, such as a syringe pump or a piston pump.

[0073] In other embodiments, catheter 18 may perform some or all of the functions of pipette 16. In such embodiments, catheter 18 may also include a catheter transport section (not shown) for positioning tube 64 relative to container 10. The catheter transport section may consist of conventional positioning components including a motor and a slider.

[0074] Some embodiments of the cell washing machine 1 may also include a housing 14. The housing 14 surrounds the container 10 and may also surround a portion of the rotor 12 and the conduit 18, such as... Figure 1A As shown. The purpose of housing 14 is to contain any leaked or atomized material resulting from the washing process. Housing 14 is fluidly coupled to conduit 18 such that conduit 18 is pressurized through a portion of housing 14 into bidirectional pump 68. Housing 14 may also include an aperture aligned with the axis of container 10 to allow probe 54 to enter. In some embodiments, housing 14 also includes a vacuum port attachable to a suction pump to remove atomized material.

[0075] Controller 20 can be a conventional controller, such as a microcomputer, microprocessor, programmable logic controller, or similar device, capable of flexibly sequencing the operations of the rotor, elevator, and fluid to execute one of a variety of cell washing protocols. During operation, the user can select a stored protocol or compile steps for a new protocol that the controller 20 will subsequently execute. Controller 20 typically controls mechanical devices such as elevator 56, rotor 12, bidirectional pump 68, sample pump 58, and other components by generating low-level signals. Drivers (not shown) convert the low-level signals into drive signals suitable for each mechanical device. In some embodiments, controller 20 may also receive signals from devices that report the status of cell washer 1. Such devices may include a tachometer or encoder for rotational feedback, a level sensor for pipette or catheter feedback, and a video signal for separation progress feedback, etc.

[0076] In some implementations, controller 20 may be embodied by a processor and a computer-readable medium coupled to the processor. The computer-readable medium may include code that can be executed by the processor to implement any of the functions described herein.

[0077] Cell washing and container cleaning

[0078] Samples may include whole blood treated with a erythrocyte lysis agent, or any other cell sample requiring washing. The wash solution may be an isotonic buffer, which in some embodiments may include additives to reduce intercellular effects. It has been found that adding approximately 5 mM EDTA to the wash solution or resuspension buffer (PBS having a concentration of approximately 1.0015 g / mL or approximately 0.1% to approximately 2% fetal bovine serum or BSA) can prevent the formation of indeterminate cell populations (potentially degranulated granulocytes) in leukocyte samples that appear near the monocyte and granulocyte populations.

[0079] Different washing liquids may be used during different parts of the washing process. A resuspension buffer different from the washing liquid may also be used in the final resuspension step. The controller can select the required added liquid according to the process by connecting the reservoir 70 containing the required liquid to the bidirectional pump 68 in a manner familiar to those skilled in the art of fluid handling via an appropriate valve in switching valve 74.

[0080] All described steps are controlled by a controller. The controller receives its instructions from a software program stored in its program memory. The controller (or a second computer operating as a user interface) accepts user instructions and (transmits the instructions to the controller, which) sequences the various mechanical components according to the selected scheme.

[0081] Exemplary cell washing processes in Figures 3A to 3F neutralization Figure 4The flowchart is illustrated in diagrammatic form. Generally, a method according to one embodiment of the present invention may include a method of washing cells from a sample comprising cells suspended in a liquid, the method comprising: dispensing the sample into a container comprising a body defining a cavity and a recess, the recess extending radially outward relative to an inner wall surface of the defining cavity; rotating the container about an axis at a first velocity; displacing cells into the recess; causing the cells within the recess to precipitate; and withdrawing at least a portion of the liquid.

[0082] In step 102 and as Figure 3A As shown, probe 54 dispenses sample 140 into container 10. Container 10 may include the same or different features as described above. Probe 54 then aspirates aliquots of sample 140, descends into container 10, and deposits aliquots of sample 140 at or near the bottom of container 10. Then, elevator 56 withdraws probe 54. Figure 3A The position of sample 140 in container 10 is shown after the transfer is complete.

[0083] In step 104 and as Figure 3B As shown, the controller causes container 10 to rotate (e.g., spin) at a first speed sufficient to generate at least about 100 or 250 g at the inner wall of recess 34. Once spinning, conduit 18 dispenses wash liquid 144 at a high rate (about 1 mL / s). This volume is sufficient to displace some or all of the initial sample 140 into recess 34. In some embodiments, the high injection rate along wall 28 provides inertia to displace sample 140 rather than to completely mix it. In this and other steps involving the addition of liquid through conduit 18, controller 20 sequences the operation of bidirectional pump 68 and any suitable valve 74 to guide liquid from reservoir 70 through conduit 64 and outlet end 66.

[0084] In step 106 and as Figure 3C As shown, the spin continues at the first speed for approximately five seconds to allow cells 142 to settle onto the inner wall surface portion 37A of the pit 34. Then, the controller 20 reduces the spin speed to a lower washing speed.

[0085] In step 108 and as Figure 3DAs shown, conduit 18 injects additional wash fluid 144 at a low flow rate (approximately 200 µL / s) to fill container 10 (so that the edge of the fluidless boundary reaches the radius of the circular opening 44 located at the top 38). The shear force pulses generated during fluid injection through the interaction of the fluid-air boundary with the liquid-containing conduit 64 help to scrub the cells 142 adhering tightly to the walls of the recess 34. The added wash fluid 144 moves vertically through the cells, such that the direction of the wash fluid flow is substantially perpendicular to the direction of the centrifugal force holding the cells 142 in the recess 34. The beneficial effect of this vertical flow is that it reduces the volume of unswept cells 142. The shear force pulses can be adjusted by varying the rotation rate and the fluid injection speed. In some embodiments, controller 20 varies the rotation rate to move the fluidless boundary upwards or downwards through the recess to scrub the cells retained therein.

[0086] In step 114 and as Figure 3E As shown, conduit 18 aspirates liquid at a high rate, gradually reducing the rotational rate during aspiration to thicken the liquid layer near end 66. In this and other steps involving the removal of liquid through conduit 18, controller 20 sequences the operation of bidirectional pump 68 and any suitable valves 74 to guide liquid through conduit 64 into end 66 and into waste reservoir 72. Minimum spin rate generates a force of approximately 25 x g on cells 142 to hold cells 142 in pit 34. Washing liquid is then added to conduit 18 such that the total volume approximates the initial sample volume. Controller 20 momentarily stops or reverses rotation to resuspend cells from pit 34. Reversal may include two changes in orientation (the second change restoring the original rotational direction). These rotational changes occur within a relatively short time—less than approximately 1 second. Such rapid changes generate high tangential acceleration, which acts as an inertial force on the contents of container 10. The inertial force increases with radius, resulting in the cells deposited in pit 34 experiencing the highest inertial force. These inertial forces, combined with the viscosity and buoyancy effects in the liquid, are used to stir the cells and mix them with the adjacent parts of the liquid.

[0087] In step 112, controller 20 repeats steps 106 to 110 several times. More cycles result in better washing efficiency. In some embodiments, controller 20 sequences two or three repetitions of steps 106 to 110. During the final wash, the amount of liquid added is adjusted to suspend the particles at a desired concentration for cell counting or other subsequent treatments. The volume of liquid added may be smaller than the initial volume of the sample.

[0088] In step 110 and as Figure 3FAs shown, rotation stops, and probe 54 descends near the bottom of container 10 and aspirates the washed sample for analysis or further processing elsewhere. The system then cleans container 10 for the next use.

[0089] Exemplary container cleaning process 120 in Figures 5A to 5F neutralization Figure 6 The flowchart is illustrated in a diagrammatic way.

[0090] In step 122 and as Figure 5A As shown, the conduit 18 rapidly adds flushing liquid without causing the container 10 to spin.

[0091] At step 124, container 10 spins, and then conduit 18 aspirates flushing liquid during the spin. Controller 20 changes the rotation speed to cause the parabolic fluidless boundary to move up and down through container 10, as... Figures 5B to 5D As shown.

[0092] At step 126 and as Figure 5E As shown, when the rotation slows down, the conduit 18 aspirates all remaining accessible fluid.

[0093] At step 128, controller 20 adds flushing liquid through conduit 18 and repeats steps 124 to 126 once or more. In some embodiments, the controller sequences two or three repetitions of steps 124 to 126. More cycles improve container cleanliness and reduce residue.

[0094] At step 130 and as Figure 5F As shown, probe 54 descends to the bottom of container 10 and aspirates all remaining accessible liquid. Container 10 is then ready for reuse.

[0095] Example 1: Experimental Washing

[0096] Each of the embodiments in Examples 3-10 uses the following containers and scheme implementation methods:

[0097] The container 10 is approximately 2 inches high and has an upper and lower portion, each approximately 0.4 inches high, and a recess approximately 1 inch high (axial range or height). The upper and lower portions have cylindrical sections with an inner diameter of approximately 0.3125 inches; the recess has an inner diameter of approximately 0.4125 inches. Therefore, in this embodiment, the recess forms a cylindrical shell with a depth of approximately 0.05 inches and a maximum radius of approximately 0.206 inches. The shell depth is approximately one-eighth of the shell's outer diameter. The shell depth is approximately one-twentieth of the axial range of the recess. The diameter of the central hole is approximately 0.157 inches. The inner walls of the upper and lower portions taper away from the recess by terminating in hemispherical sections.

[0098] The example solution includes the following steps, modified as indicated by the experimental variant:

[0099] a) Using a probe, add 100µL of whole blood mixed with 400µL of erythrocyte lysis agent and label, as needed for each experiment;

[0100] b) Spin at 6460 rpm (producing approximately 100 or 250 × g at the pit wall);

[0101] c) Using a catheter, add 500µL of washing liquid at a rate of 1mL / second;

[0102] d) Allow the cells to settle against the pit walls for 5 seconds;

[0103] e) Using a catheter, add 2 mL of washing liquid at a rate of 200 µL / second;

[0104] f) Using a catheter, remove 2.4 mL of liquid at 1 mL / s while linearly reducing the spin speed to 2000 rpm (to produce approximately 25 × g at the pit walls).

[0105] g) Using a tubing, add 400µL of washing fluid and reverse the rotor twice for 0.5 seconds each time to resuspend the cells;

[0106] h) Repeat steps e), f), and g) as many times as required for each experiment. After the last repetition of step f), replace the washing liquid in step g) with resuspension buffer and stop rotating; and

[0107] i) Using a probe, aspirate washed cells and analyze them in a flow cytometer.

[0108] Example 2: Traditional Washing

[0109] a) As required for each experiment, manually load 100µL of whole blood mixed with 400µL of erythrocyte lysis agent and label into a 12×75mm polypropylene tube.

[0110] b) Add 2 mL of washing liquid; mix by inverting the container;

[0111] c) Spin at 500 × g for 5 minutes to form microparticle cells;

[0112] d) Aspirate the supernatant using a pipette;

[0113] e) Repeat steps b) through d) twice; and

[0114] f) Resuspend the microparticles in 400 µL of resuspension buffer and mix by inverting.

[0115] Example 3: Bioequivalence - Scattering

[0116] Figures 7A to 7F Scatter plots comparing leukocyte recovery rates using embodiments of the apparatus and method of the present invention with those of conventional washing and no washing are presented. The top row of plots shows the forward scattering (FS) / side scattering (SS) for unwashed cells, conventionally washed cells, and cells washed according to Example 1 (test method). The bottom row of plots shows CD45 (labeled with Krome Orange) / SS. Krome Orange is a trademark of Beckman Coulter, Inc. The light scattering and representation of WBC populations using the test method are comparable to those for conventionally washed and unwashed cells.

[0117] Example 4: Bioequivalence - Five Categories

[0118] Figures 8A to 8F Scatter plots comparing leukocyte recovery rates using the assay method with conventional washing and no washing were presented. The top row of plots shows the CD45 (labeled with FITC) / SS ratio for unwashed cells, conventionally washed cells, and cells washed according to the assay method. The bottom row of plots shows the CD14 (labeled with PE-Cy7) / SS ratio. Calculated values ​​below each set of plots show recovery rates for neutrophils, monocytes, lymphocytes, eosinophils, and basophils comparable to conventional and no washing.

[0119] Example 5: Bioequivalence - Platelets

[0120] Figures 9A to 9C The scattering patterns of CD14 (labeled with PE-Cy7) / CD41 (platelet marker labeled with APC) using the test method were compared with those of conventional washing and no washing. Figures 8A to 8F In the next row of images, the rectangular area labeled Monos is highlighted. Note that the density of monocytes showing CD41 binding is significantly reduced, especially compared to conventional cell washing methods. The assay method includes 5 mM EDTA in the washing buffer. Platelet adhesion to monocytes is significantly reduced for this assay method.

[0121] Example 6: Bioequivalence—Viability

[0122] Figures 10A to 10C Scattering patterns of leukocyte viability analysis using a testing method were compared with conventional washing and no washing. The patterns show the signal from 7-aminoactinomycin D (7AAD) / SS. 7AAD binds to dead cells but not live cells. The calculated cell viability showed a significant reduction compared to conventional cell washing and was suitable for flow cytometry analysis.

[0123] Example 7: Bioequivalence: κ / λ segregation

[0124] Figure 11A and Figure 11B Scattering patterns of B cells showing λ (labeled with PE) / κ (labeled with FITC) were compared. Diagonal lines separated κ-positive B cells from λ-positive B cells using a testing method compared to conventional washing. The calculated values ​​for signal separation were expressed as signal-to-noise ratios. These values ​​were calculated as κ = KX-median / LX-median and λ = LY-median / KY-median. The gating values ​​for κ and λ were comparable to those for conventional washing.

[0125] Example 8: Bioequivalence: Total cell recovery rate

[0126] Figures 12A to 12C Scattering patterns of ungated leukocytes used to analyze total cell recovery using a testing method were compared with conventional washing and no washing. The patterns show signals from CD45 (labeled with FITC) / SS as shown in Example 4. The calculated values ​​represent the number of cells recovered compared to the no-wash scenario.

[0127] Example 9: Bioequivalence: Cell subset recovery rate I

[0128] Figures 13A to 13C A bar graph comparing the T cell subset recovery rate using the assay method with conventional washing and no washing was presented. The bar graph shows the CD3 signal (labeled with PE-Cy5) bins. The subset cell recovery rate for the assay method (gated based on WBCs labeled as Beckman Coulter, Inc. Tetra Panel) was comparable to that with conventional washing and no washing.

[0129] Example 9: Bioequivalence: Cell subset recovery rate II

[0130] Figures 14A to 14C Scatter plots comparing B cell and NK cell recovery rates using the assay method were presented compared to conventional washing and no washing. The plots show signals from CD56 (labeled with Rhodamine) / CD19 (labeled with ECD) (gated based on WBCs labeled with Beckman Coulter, Inc. Tetra Panel). B cell and NK cell recovery rates for the assay method were comparable to those for conventional and no washing.

[0131] Figure 15 and Figure 16An embodiment including two probes, namely a dispensing probe and an aspiration probe, is shown. Having two conduits, such as two probes, has several beneficial effects. If a single probe is used, one probe must dispense clean buffer and remove waste fluid. Therefore, one beneficial effect of using a system with two probes is minimizing contamination between adjacent operations. In other words, clean buffer can be contaminated by waste fluid from previous operations. A second beneficial effect is that the two probes can be placed in different locations. The dispensing probe (which dispenses clean buffer) can be located at the top of the container, while the aspiration probe (which dispenses waste fluid) is located at the bottom. At the end of the operation, a small amount of buffer is dispensed from the upper probe to clean the inner walls of the container as the buffer falls to the bottom. This facilitates the recovery of blood cells during blood purification procedures.

[0132] Figure 15 A partial side sectional view of a component according to an embodiment of the present invention is shown. Figure 15 A container 200, which may have the configuration of the aforementioned container embodiment, is shown. A dispensing probe 202 and an aspiration probe 204, along with their distal ends, are disposed within the container 200. The aspiration probe 204 is linear and extends towards the bottom of the container 200, and may be configured to remove waste buffer. The dispensing probe 202 has a 90-degree bend at its distal end to add washing buffer to the recess 206 and any substances that may be present in the recess 206. The aspiration probe 204 may also have a 90-degree bend at its distal end, or it may be linear. Figure 16 The bend is shown more clearly. One end of the suction probe 204 can be positioned exactly below the recess 206. Although the ends of the dispensing probe 202 and the suction probe 204 are... Figure 15 and Figure 16 They are shown as orthogonal to each other, but in other embodiments they may be in the same plane or possibly aligned in a similar manner.

[0133] Figure 16 It shows Figure 15 Top cross-sectional view of the component shown. Figure 16 A top view of the distal portions of the dispensing probe 202 and the suction probe 204 is shown. Figure 16 As shown, the major axes of the dispensing probe 202 and the suction probe 204 are eccentric relative to the central axis of the container.

[0134] Some exemplary washing procedures that can be performed using embodiments of the present invention can be described as follows. A first washing procedure is a "sample washing," in which a sample such as blood (e.g., 100 μL of blood) is washed. A second washing procedure may be a "lysis washing," in which non-cellular products of cell lysis are removed from the blood sample. A third washing procedure is a "batch washing," in which a larger volume (e.g., 500 μL) of blood is washed. In some embodiments, the complete workflow may include a first washing procedure (sample washing) followed by a second washing procedure (lysis washing).

[0135] Sample washing (e.g., washing a single 100µL sample).

[0136] Step 1. Dispense the sample (e.g., blood) into a container.

[0137] Step 2. Dispense fresh wash buffer into the container using the dispensing probe located in the upper region of the container.

[0138] Step 3. Rotate the container back and forth to create a vortex in the sample.

[0139] Step 4. Spin the container at maximum speed so that the sample components fall into the container pits as particles.

[0140] Step 5. As the container rotation speed decreases, use the aspiration probe located near the bottom of the container to remove the waste buffer.

[0141] Step 6. Perform steps 2 through 4 a total of four or more times.

[0142] Step 7. Spin the container back and forth at regular intervals. Each interval should be slower than the previous one. This is to separate any cells that may have adhered to the wall.

[0143] Step 8. While the tube is slowly spinning, use the upper probe to dispense a small amount of buffer solution into the container to clean the container walls.

[0144] Pyrolysis and washing

[0145] Step 1. Transfer the lysed solution to a container using an external pipette. The lysing solution may include the sample and the lysing agent. For example, the lysing solution may include 100 μL of blood and 2.0 mL of IOTest Lyse (an ammonium chloride-based erythrocyte lysing solution available from Beckman Coulter, Inc.). The total volume may be greater than 2.0 mL (e.g., 2.1 mL).

[0146] Step 2. Immediately spin the container at maximum speed so that the blood is broken into microparticles and falls into the pit.

[0147] Step 3. As the rotation speed of the container decreases, remove the lysis supernatant (using the lower suction probe).

[0148] Step 4. Stop rotating the container.

[0149] Step 5. Dispense fresh wash buffer using the above-mentioned partial dispensing probe.

[0150] Step 6. Spin the container back and forth to create vortices in the sample.

[0151] Step 7. Spin the container at maximum speed so that the sample components fall into the container pits as particles.

[0152] Step 8. As the container's rotation speed decreases, use the lower aspiration probe to remove the waste buffer solution.

[0153] Step 9. Spin the container back and forth at regular intervals. Each interval should be slower than the previous one. This can be used to separate any cells that may have adhered to the wall.

[0154] Step 10. While the container is slowly spinning, use the upper partitioning probe to dispense a small amount of buffer solution to clean the container walls.

[0155] Batch washing (500uL sample)

[0156] 1. Use the same process as for sample washing, but each step takes longer. .

[0157] While various aspects of this specification are highlighted by reference to specific embodiments, these disclosed embodiments are merely examples of the principles of the subject matter. Unless otherwise indicated or clearly contradicted by the context, the invention covers any combination of features in the described embodiments.

[0158] Unless otherwise specified, all figures used in this specification and claims to represent features, items, quantities, parameters, characteristics, periods, etc., shall be understood to be modified in all cases by the term "about". As used herein, the term "about" means that the feature, item, quantity, parameter, characteristic, or period so defined covers a range of ±20% above or below the value of said feature, item, quantity, parameter, characteristic, or period. Therefore, unless stated to the contrary, the numerical parameters listed in the specification and appended claims are variable approximations.

[0159] The terms “a,” “an,” “the,” and similar pronouns used in the context of describing the invention (especially in the context of the following claims) should be understood to cover both the singular and plural unless otherwise specified herein or the context clearly contradicts. All methods described herein can be performed in any suitable order unless otherwise specified or the context clearly contradicts. The use of example or exemplary language (e.g., “such as”) is intended only to better illustrate the invention and not to limit the scope of the claims. No language in this specification should be construed as indicating that any element not protected by the claims is necessary for carrying out the invention.

Claims

1. A cell washing machine, comprising: A container configured to contain cells, the container including an elongated body including an opening, an inner surface, a recess and a cavity, the recess being defined by a first inner surface portion of the inner surface, the first inner surface portion being disposed between and radially outward relative to the second inner surface portion and the third inner surface portion; as well as An actuating device that enables the container to spin about an axis; as well as A conduit configured to pass through the opening and to deliver fluid to and from the cavity during rotation of the container; The cavity is defined by the lower portion of the body and is located below the recess, wherein the recess has a length-to-depth aspect ratio of at least about 2:

1.

2. The cell washing machine according to claim 1, wherein the transition from the first inner surface portion to the second inner surface portion is an S-shaped transition.

3. The cell washing machine according to claim 1 or claim 2, wherein the catheter includes a tip disposed adjacent to the wall of the container and below the recess.

4. The cell washing machine according to any one of claims 1 to 3, wherein the conduit is in fluid communication with a fluid pump.

5. The cell washing machine according to any one of claims 1 to 4, further comprising: A probe is connected to an elevator, which is configured to lower the probe through the opening, and the probe is in fluid communication with a pump.

6. The cell washing machine according to any one of claims 1 to 5, further comprising: A controller configured to control one or more of the rotor, the fluid pump, the sample pump, and the elevator.

7. The cell washing machine of claim 1, further comprising a dispensing probe and an aspiration probe passing through the opening and entering the container.

8. The cell washing machine of claim 7, wherein each of the dispensing probe and the aspiration probe includes a curved end.

9. The cell washing machine of claim 7, wherein the aspiration probe terminates at a point lower than the dispensing probe within the container.

10. A method for washing cells from a sample comprising cells suspended in a liquid using a cell washing machine according to claim 1, the method comprising: a) Dispensing the sample into a container, the container comprising a body defining a cavity and a recess, the recess extending radially outward relative to an inner wall surface defining the cavity; b) Rotate the container about its axis at a first speed; c) Displace the cell into the pit; d) To cause the cells in the pit to precipitate; as well as e) Extract at least a portion of the liquid; The washed cells are formed after step e), and the translocation step includes: Washing liquid for the first aliquot sample is added through a conduit that extends into the container and terminates below the recess, wherein the extraction step includes aspiration through the conduit.

11. The method of claim 10, further comprising: f) The washing liquid of the second aliquot sample is added through the conduit.

12. The method of claim 10 or 11 further comprises resuspending the washed cells.

13. The method of claim 12, wherein the resuspension step comprises adding the resuspension liquid to the container and stopping the rotation.

14. The method of claim 12 or 13, wherein the resuspension step includes reversing the rotation direction of the container.

15. The method according to any one of claims 10 to 14, further comprising rotating the container at a second speed during the extraction step.

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