Cell recovery method and apparatus

Abstract

The present disclosure provides a cell collection method and apparatus for the efficient sedimentation and retention of cells from a liquid sample onto a solid support, with low cell loss and low impact on cell morphology. The apparatus has two configurations, one for use in a centrifuge to centrifuge the sample and the other for the controlled release of the liquid after centrifugation. The apparatus includes a base that holds a solid support for receiving cells on its upper surface, which releasably holds a fluid chamber having a first opening that surrounds an aperture, with a gasket that abuts the upper surface of the support. The aperture receives the liquid sample, and the gasket defines a region where the target cells deposit. A first cap closes a second opening during centrifugation. After centrifugation, the first cap is removed, and a fluid absorption element and a cap designed to prevent positioning of the tip of the absorption element at a distance shorter than a predetermined distance from the solid support are inserted into the fluid chamber to provide for the controlled removal of the liquid.

Description

Technical Field

[0001] The present disclosure relates to a cell recovery method and apparatus for efficient sedimentation and retention of cells from a liquid sample onto a solid support, with low cell loss and low impact on cell morphology.

Background Art

[0002] There are many methods / devices used to separate cells from liquid samples. Many of them involve the use of cell centrifugation methods, which accelerate cell sedimentation towards a desired collection point. Disadvantages for many of these methods / devices include significant cell loss and altered cell morphology, which can be detrimental to subsequent analysis.

[0003] In clinical applications, various different fluid samples are routinely analyzed for the presence and characteristics of cells. These include blood, urine, cerebrospinal fluid, synovial fluid, ascites. In blood, a population of so-called "circulating rare cells" has been identified in some patients, which represent cells that have spread from a developing tissue. The most sought-after type of circulating rare cells is circulating tumor cells (CTCs), which, if present, can potentially provide important information regarding the state and prognosis of the disease.

[0004] Efficient recovery of cells in samples containing rare cells is important. Even moderate cell loss during sample processing can result in inaccurate counting or false-negative results, and samples containing rare cells may be erroneously determined to be absent. Recently, many techniques for concentrating circulating rare cells from blood have emerged.

[0005] Various methods are used to extract rare cells from patient samples (cell recovery), and as a result, typically, a fluid sample containing the cells of interest in combination with other cells (e.g., white blood cells, etc.) is obtained. The cells collected are important to the researcher, and often, microscopic studies and sequential molecular analyses of these cells are performed. To enable this analysis, it is necessary to sediment (deposit) the cells present in the sample onto the surface of a solid support that enables microscopic examination of these cells. Typically, the cells collected are sedimented onto a microscope slide (which may be a glass or plastic slide). One of the basic requirements of this process is to prevent cell loss. This is particularly important when the user is handling rare cells. In this case, the sample may contain few of these cells, and even the loss of a small number of cells can make the sequential analysis unreliable or impossible. The second major requirement is to minimize morphological changes in the cells by the cell retention process and to assist in cytological analysis.

[0006] For example, U.S. Patent No. 7,575,719 discloses a fluid sample chamber that can be sealed to the surface of a microscope slide. This sealing means can be used in combination with a filter card disposed or ultrasonically welded between the microscope surface and the sample chamber. This liquid removal method has a risk of unintended loss of the sample to the surrounding absorbent filter card medium.

[0007] U.S. Patent No. 7,628,955 discloses a chamber for a fluid sample that can be sealed to the surface of a microscope slide using an integrally molded elastomeric gasket and an oil film component. This device does not provide a means for removing liquid in a controlled manner to prevent removal of sedimented cells from the slide.

[0008] U.S. Patent No. 4,696,743 discloses a filter card disposed between a sample chamber and a deposition receiving surface such as a microscope slide. Solids present in a fluid suspension are centrifuged onto the slide surface, and the surrounding fluid is drawn into an adjacent absorbent medium. This device does not address the problem of cell loss by separating the fluid removal step from the centrifugation / sedimentation step.

[0009] U.S. Patent Publication No. 2003 / 0175850 discloses a method of selectively labeling cells using a biotin-binding antibody and binding them to a slide surface coated with an avidin / streptavidin substrate to concentrate cells of interest from a suspension. An absorbent medium absorbs unbound material or fluid, leaving a dried slide containing the sample of interest. This device does not account for the turbulent flow and shear stress resulting from the lateral flow of liquid into the medium at an uncontrolled rate. This provides the ability to remove unbound cells in the suspension, but there remains a risk of accidentally removing slide-bound target cells.

[0010] U.S. Patent No. 5,419,279 discloses a flanged hollow tube for sample deposition, which engages with a corresponding base for staining purposes to securely seal the tube to a microscope slide. There is a groove at the bottom of the tube, so an O-ring can be attached between the slide interface and the tube to prevent leakage. This device relies on the binding force between the functionalized slide surface and the cells rather than centrifugal force, in addition to promoting the capture of target cells, and furthermore does not address the removal of supernatant throughout the cell sedimentation / binding step, which can cause cell loss due to turbulence in the surrounding liquid medium.

[0011] U.S. Patent No. 5,480,484 discloses a fluid chamber sealed centrally to a support backing plate via an elastomeric gasket and metal clip. The number of fluid chambers can be varied to accommodate one or more samples, but with a change in the maximum allowable sample volume. The assembled centrifuge device floats freely within a given rotor and deposits cells from a suspension onto a slide surface when centrifugal force is applied. This device does not contemplate a simple and controlled supernatant removal method. Rather, the chamber has a relatively narrow opening and by itself is not useful for liquid removal.

[0012] U.S. Patent No. 4,576,110 discloses a rotor chamber designed to deposit a cell suspension onto a microscope slide under the application of centrifugal force. At the same time, an absorption plug (shown in FIG. 9 of the patent) is disposed within a separate compartment within the rotor that projects into the same chamber, and the tip of the plug is in contact with the slide surface. When centrifugal force is applied to the rotor, particles suspended in the liquid are deposited onto the slide surface. According to this patent, since the centrifugal force is high enough to overcome capillary forces, the absorption plug does not suck up the supernatant until the rotor decelerates. On the other hand, the absorption element 3 is introduced into the system prior to centrifugation and absorption begins immediately and continues until the centrifugal force exceeds the capillary force. This can result in the loss of cells present in the solution. The concept of separating the processes of cell sedimentation and supernatant removal into mutually exclusive steps is similar to this device, but there are important differences in the approach to fluid absorption that distinguish the two designs. One of the main features of the prior art is that the absorption plug projects into the same chamber where the fluid containing the particles deposits. Even if capillary action does not function in the fluid during centrifugation, the physical entanglement of cells within the porous material alone can result in the loss of cells within the absorption plug, leading to cell loss. Furthermore, it is noted that the absorption plug, when disposed within the rotor, is in physical contact with the deposition surface. The lack of control of the flow rate of fluid to the absorption material and its approximation can result in the unintended absorption of cells weakly adhering to the deposition surface.

[0013] U.S. Patent No. 4,344,562 discloses an apparatus that uses a conventional glass slide that can be fixed within a housing using a holder. A portion of the housing is a reservoir that can receive a liquid sample, and when the glass slide is fixed within the housing, the reservoir is closed by the glass slide at its bottom. This patent aims to perform centrifugation of a small amount of liquid sample without loss of cells and enhance the accuracy for medical diagnosis and treatment purposes. A rubber O-ring is used to provide a liquid-tight seal between components. Summary of the Invention Means for Solving the Problems

[0014] The present disclosure provides a cell collection device for the sedimentation and retention of target cells from a fluid sample. The device includes a base configured to removably hold a solid support, the solid support being configured to receive cells on an upper surface thereof. The device includes a fluid chamber having a first opening with a circumferential gasket surrounding a bottom opening, the base being configured to removably hold the fluid chamber using a gasket that abuts the upper surface of the solid support so as to form a liquid-tight seal between the upper surface and the solid support, the fluid chamber having a second opening for receiving a liquid sample containing the target cells to be collected. The gasket defines a region of a selected size where the target cells deposit on the upper surface of the solid support. The device includes a first removable cap configured to close the second opening during centrifugation. The device also includes a fluid absorption element and a housing configured to receive the absorption element, the fluid chamber being configured to receive the housing after centrifugation. The housing has a bottom opening through which fluid from the fluid chamber can be absorbed by the absorption element when introduced into the fluid chamber after centrifugation. The fluid chamber includes a second removable cap configured to close the second opening and prevent the bottom tip of the absorption element from being positioned at a distance shorter than a predetermined distance from the solid support. The cell collection device is configured to be received and removably held in a centrifuge.

[0015] The fluid absorption element may have a cross-sectional area, a distribution along the axis of the absorption element, and a porosity that provides control of the fluid absorption rate and a range for positioning the tip of the fluid absorption element at a distance from the surface of the solid support. Thereby, the target cells fixed on the solid support are prevented from being detached from the surface of the solid support by the flow of the fluid absorbed by the absorption element.

[0016] The absorption element of the device may have a porosity in the range of about 1 to about 100 microns, alternatively, the porosity may be in the range of about 5 to about 50 microns, and alternatively, the porosity may be in the range of about 10 to about 20 microns. The predetermined distance from the surface of the solid substrate to the tip of the absorption element is in the range of about 0.1 mm to about 3 mm.

[0017] The device is configured to be removably held within a centrifuge, such that an axis perpendicular to the surface of the solid support, preferably an axis passing through the center of the region assigned for cell sedimentation, intersects the axis of rotation, and the long axis of the solid support is within the plane of rotation, or alternatively, the normal to the surface of the solid support intersects the axis of rotation, and the long axis of the solid support is configured to be perpendicular to the plane of rotation.

[0018] The selected size region where the target cells deposit on the upper surface of the solid support may be functionalized with a selected agent that modifies the interaction between the target cells and the surface to assist in the attachment of the target cells to the upper surface of the solid support. These agents may be selected from the group consisting of poly-L-lysine, silane coating, gelatin, fibronectin, gold or silver coating. For example, a glass slide (Merck, USA) coated with poly-L-lysine can be used.

[0019] These agents are selected to provide differential binding affinity to the target cells and other interfering cells, such that the target cells bind to the surface, while the interfering cells do not bind to the surface of the upper surface of the solid support.

[0020] The absorption element of the device may be made of porous plastic, and the porous plastic may be coated with a hydrophilic coating. The absorption element of the device may be made of a non-porous absorption material such as, for example, tissue paper, cotton, or other fibers that absorb fluid.

[0021] The porous plastic may be porous polyethylene coated with a hydrophilic coating.

[0022] The tip of the absorbent material, which is separated from the upper surface of the solid substrate, is disposed at a distance in the range of about 0.3 mm to about 3 mm from the surface of the solid substrate.

[0023] The porosity and the size of the openings of the fluid absorption element are selected to provide a selected flow rate.

[0024] The selected flow rate provides a hydrodynamic force applied to the target cells deposited on the surface, which is below the detachment threshold level of the target cells that detach from the surface of the solid support.

[0025] The solid support may be a microscope slide.

[0026] The first removable cap may include a gasket for providing a liquid-tight seal during centrifugation.

[0027] Regions on the surface of the solid support outside the region of selected size where the target cells are deposited on the upper surface of the solid support may be coated with a protective coating to prevent the adhesion of fluid accidentally spilled on the surface of the support.

[0028] The housing configured to receive the fluid absorption element includes an overflow mechanism to prevent the overflow of fluid caused by the insertion of the fluid absorption element into the fluid chamber. These overflow mechanisms may include at least one opening at the upper part of the absorbent element housing, and the opening may have any of a square, circular, or slot shape.

[0029] The present disclosure provides a method for retaining and post-treating target cells present in a fluid medium on a solid support, with an optimal retention rate for the target cells adhering to at least a part of the surface of the solid support and the preserved morphology of the target cells. The method includes subjecting the fluid medium to centrifugation to induce sedimentation of the target cells onto the surface of the solid support. After sedimentation of the fluid medium, removal is performed, characterized by a controlled rate of fluid medium removal such that the target cells sedimented on the surface are not detached from the surface by fluid flow in the vicinity of the sedimented cells. The amount of residual fluid is controlled at all stages of cell retention and post-treatment. The sedimented target cells are characterized in that their morphology does not substantially change from the morphology when the target cells are freely floating in the fluid medium.

[0030] The flow rate is controlled by the porosity of the material from which the absorption element is fabricated, the area of the opening of the housing of the absorption element, and the cross-sectional area of the absorption element.

[0031] At least a part of the surface of the solid support on which the target cells sediment is a selected size defined by the size of the opening of the fluid chamber in which the fluid containing the target cells is placed for centrifugation. The region of the selected size may be functionalized with a selected agent that modifies the interaction between the target cells and the surface to assist in the attachment of the target cells to the upper surface of the solid support.

[0032] These agents may be selected from the group consisting of poly-L-lysine, silane coating, gelatin, fibronectin, gold or silver coating. These agents are selected to provide differential binding affinity to the target cells and another cell, such that the target cells are more readily bound to the surface, while the other cell has a lower efficiency of binding to the upper surface of the solid support.

[0033] The parameters of the absorption element and the absorption element housing are selected such that the shear force associated with the fluid removal rate is low enough so that detachment of the target cells is minimized, while the same shear force is sufficient for the removal of other cells of no interest, thereby resulting in enrichment of the target cells of interest.

[0034] A further understanding of the mechanical and advantageous aspects of the present disclosure can be obtained by reference to the following detailed description and the drawings.

Brief Description of the Drawings

[0035] Embodiments of the cell collection device disclosed herein will be described by way of example only with reference to the drawings.

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Mode for Carrying Out the Invention

[0036] Various embodiments and aspects of the present disclosure will be described with reference to the details described below. The following description and drawings are illustrative of the present disclosure and should not be construed as limiting the present disclosure. Numerous detailed details are described to provide a complete understanding of the various embodiments of the present disclosure. However, in certain cases, well-known or conventional details are not described in order to provide a concise description of the embodiments of the present disclosure.

[0037] The terms "comprising" and "including" as used herein should be interpreted as being inclusive and unlimited, and not exclusive. Specifically, when used in the specification and claims, the terms "comprising" and "including" and their variants mean that the specified mechanism, step or component is included. These terms should not be construed as excluding the presence of other mechanisms, steps or components.

[0038] The term "exemplary" as used herein means "serving as an example, instance, or illustration", and should not be construed as being preferred or advantageous over other configurations disclosed herein.

[0039] The terms "about" and "approximately" as used herein are meant to cover possible variations that may exist in the upper and lower limits of the range of values, such as variations in properties, parameters and dimensions. In one non-limiting example, the terms "about" and "approximately" mean within ± 10 percent.

[0040] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art.

[0041] The phrase "target cell" as used herein means the cell of interest to be separated by the present device. In some cases, if selectivity of cell separation is not required for a particular intended use or subsequent processing method, all cells present in the sample may be of interest, and all cells present in the sample may be considered "target cells". Other cells may be present, and different mixed types of cells may be present, but only the "target cells" are separated or mainly separated.

[0042] As used herein, the term "acceleration mechanism" refers to a structural element designed to increase the fluid removal rate from the fluid chamber 44 without loss of the cells deposited on the surface of the solid support 42 after completion of centrifugation. This enables shortening of the time required for sample processing. These acceleration mechanisms are installed on the housing 48 that provides an additional fluid connection between the fluid present in the fluid chamber 44 and the absorption element 46. The acceleration mechanism may be circular in shape or may have the shape of a slot. Other shapes are possible. The acceleration mechanism is configured such that the opening of the mechanism is sufficient to increase the flow rate to the required value, and the acceleration mechanism is installed at a distance from the tip of the housing 48 such that no additional cell loss is caused by the additional shear stress applied to the cells on the solid support 42 due to the increased flow rate.

[0043] As used herein, the term "overflow protection mechanism" refers to a structural element designed to prevent overflow of fluid from the fluid chamber 44 when the absorption element 46 disposed within the housing 48 is inserted into the fluid chamber 44. These overflow protection mechanisms are installed on the housing 48 and provide an additional fluid connection to the fluid present in the fluid chamber 44 when the amount of sample is excessive. These overflow protection mechanisms protect against the generation of turbulent flow within the fluid chamber 44 that would cause cell detachment from the surface of the solid support 42.

[0044] The present disclosure describes an apparatus comprising a fluid chamber assembled with a microscope slide (or other substrate) hermetically sealed using an elastomeric gasket. The slide surface is functionalized with a coating that enables capture and retention of cells and is held within a polypropylene holder that clips to the fluid chamber, thus physically securing and positioning the chamber relative to the slide. Separate from the assembly including the microscope slide, a porous absorbent element is included within a polypropylene shell, which provides means for controlling the flow rate of liquid to the porous absorbent element while improving ease of use. The absorbent element within the polypropylene shell is introduced into the assembly after completion of the centrifugation step and can be replaced with the same absorbent element and shell when the process of cell post-treatment requires multiple steps of fluid introduction and removal.

[0045] This apparatus is intended to function as a cell centrifuge device when cells of interest floating in the surrounding medium are of higher density than the surrounding medium, such that when centrifugal force is applied to the device containing the cell suspension, the more dense cells of interest preferentially sediment in the direction of the centrifugal force. Cell centrifuge devices can be broadly classified into two groups based on the method of liquid removal. In a "simultaneous liquid removal" system, an absorbent medium is placed between the fluid chamber and the slide surface, enabling removal of liquid to the adjacent absorbent medium simultaneously as centrifugal force is applied. Theoretically, prior to liquid removal, the more dense cells of interest need to have sedimented onto the slide surface; otherwise, cells remaining suspended in the medium may be drawn into and lost to the adjacent absorbent element. Another approach to cell centrifugation features a liquid-tight seal between the fluid chamber and the slide surface, which does not permit liquid removal during centrifugation. In liquid-retaining cell centrifugation, the cells of interest sediment while surrounded by a supporting liquid medium, and liquid removal is performed after centrifugation. The relatively dense cells of interest within the liquid medium still preferentially precipitate onto the slide surface.

[0046] The liquid retention cell centrifugation method exhibits numerous advantages but also has some challenges from the perspective of sample handling. When centrifuged in air, cells contrast with the complete absence of buoyancy. Instead, in a liquid medium that provides relative buoyancy (i.e., resistance to sedimentation), denser cells require a greater centrifugal force to overcome fluid resistance. The presence of cells in the liquid medium results in a reduced force pressing the sedimenting cells against the slide, which is beneficial for preserving delicate morphological features. The lack of simultaneous liquid removal offers the advantage of effectively eliminating all possible means by which cells of interest could be lost during the centrifugation process. Due to the principle of being liquid-tight, the fluid containing the cells of interest remains restricted within the chamber and in contact with the slide surface. Thus, cells floating in the liquid medium also remain within the chamber and are restricted to sediment on the slide surface.

[0047] A limitation presented by the liquid retention method of cell centrifugation is that the processes of liquid removal and cell sedimentation are separated. Therefore, a separate liquid removal method is required. Considering that cells do not receive as much centrifugal force due to the buoyancy of the surrounding medium, the contact area between the slide and the cells is not very large, which may negatively affect the adhesion of cells to the slide surface. This implies that the liquid removal process needs to be carried out as gently as possible with minimal turbulence. This apparatus and its method of use provide a method for fluid removal from the sample chamber in a controlled manner that minimizes the detachment of cells from the slide surface and maximizes the yield of sedimented cells of interest.

[0048] When removing the liquid, the cells settle within a designated area of the slide within the sample chamber. It is beneficial for the sample chamber to be removable in order to incubate or wash the cells using multiple reagents, or to assist with further downstream processing where imaging of the settled cells is required for subsequent processing. The device includes a polymer base located on top of the slide and having a protruding hook that clips to a corresponding location on the sample chamber. The clip is designed for ease of assembly but also provides a convenient means for the end user to remove the chamber without using special tools or methods.

[0049] For example, in order to facilitate reagent manipulation in downstream processes such as immunofluorescence staining, fluorescence in situ hybridization, etc., it is optimal to minimize the amount of liquid to reduce assay costs, and the device can include hydrophobic printing on surfaces other than the exposed bioadhesive area. The hydrophobicity of the printed surface functions as a convenient reagent well that can hold a small amount of liquid (the well volume is 10 - 200 μL). However, by means of holding the liquid in a controlled area, the cells of interest are also limited to the designated area, enabling precise localization of the sample and minimizing losses due to downstream processes.

[0050] To maximize the ability of cells of interest to adhere to the slide surface and resist detachment, the slide surface was functionalized with a bioadhesive coating to alter the surface interaction between the sedimented cells and the slide. With a design of the device in which the slide is held on a polymer base that is clipped together with a chamber sealed to the slide via a gasket, the device is compatible with almost all microscope slides that fit within the base. Thus, a wide range of functionalized slides were tested with the device to evaluate cell adhesion performance. Since the slide is easily removable from the rest of the device components, it is compatible with almost all microscopes, including both upright and inverted microscopes. Also, slides with sedimented cells are compatible with other types of standard equipment, such as slide staining devices. Cultured SK-BR-3 cells pre-labeled with CellTracker® Green were injected into 1×PBS to create a cell suspension, which was added to the sample chamber. After centrifugation, the supernatant was removed, and the cells were fixed by incubating in ice-cold methanol for 5 minutes, after which the sample chamber was removed. The resulting slide containing the sedimented cells was washed by pipetting 1×PBS over the sample surface three times to induce cell detachment. The subsequent slide surface was imaged under FITC to evaluate cell density. As a result, poly-L-lysine showed the highest cell density. Thus, this surface modification was selected as the main slide surface of this device. Instead of poly-L-lysine, other types of coatings, such as proteins, silanes, organic polymers, inorganic metals, etc., can be used for surface functionalization. For example, other forms of polylysine, silane coatings, gelatin, fibronectin, gold or silver coatings, and other coatings that enhance cell adhesion to the solid support. For example, other methods of enhancing cell adhesion to the surface of the solid support, such as surface structure modification, etching, plasma treatment, etc., can also be used.

[0051] As the solid support, various materials can be used, such as glass, various types of plastics, or quartz.

[0052] Referring to FIGS. 1 and 2(a), FIG. 1 shows a flow diagram 100 of a cell retention method using the cell collection device disclosed herein. In step 12, a fluid sample comprising isolated cells is introduced into a fluid chamber 44 (FIG. 2(b)), which is connected to a solid support 42, both of which form part of the device described below. Then, in step 14, the fluid sample is centrifuged, thereby biasing the cells to move towards the solid support 42, inducing sedimentation of the cells, and promoting adhesion of the cells to the solid support. After centrifugation, in step 18, the fluid removal rate is carefully controlled, and in step 20, the remaining fluid is actively controlled, so that in step 16, the fluid is removed under controlled conditions. Carefully controlling the fluid is advantageous in that it prevents cell detachment from the solid support due to excessive forces that may occur when the flow rate is high and the stress level exceeds the adhesive force that holds the cells on the surface of the solid support during or after centrifugation. Also, removing the fluid after completion of the centrifugation step excludes the removal of cells that may still be present in the solution (not bound to the surface) if the centrifugation has not ended. Further, the proposed method requires control of the amount of fluid remaining in the fluid chamber after completion of the fluid removal step. This is necessary to preserve the cell morphology since the cells remain wet throughout all steps of the process. The amount of remaining fluid depends on the fluid parameters (surface tension and contact angle), and the inner diameter of the fluid chamber 44 and the distance between the solid support 42 and the tip of the absorption element 46. That amount is basically controlled by an appropriate selection of the above distance to the tip of the absorption element 46.

[0053] If multiple steps are required for post-treatment of the cells (e.g., staining, washing, etc., as shown in step 24), the addition of reagents is carried out at a controlled fluid supply rate. This can prevent detachment and removal of the cells attached to the solid support in the preceding steps of the method. The next fluid removal step is carried out using the controlled fluid removal rate and control of the remaining amount of fluid as described above, which is shown in step 26.

[0054] Following preparation step 24, the prepared cells are subjected to the necessary analysis, such as molecular analysis in step 28 or imaging in step 30.

[0055] Referring to FIGS. 2 and 3, the assembled cell holding device (generally designated 40) for cell sedimentation has reduced cell loss (increased retention rate) and improved morphology of the retained cells, and includes a substrate 42 and a fluid chamber 44, and either a wicking (suction) cap 50 shown in FIG. 2(a) or a protective cap 68 shown in FIG. 2(b), and an absorption element 46 that extends from the cap 50 into the housing 48 and is disposed within the fluid chamber 44 and is in fluid communication with the cell-containing liquid. The absorption element extends across the absorption element rod 46, the wicking cap 50, the base 52, and the fluid chamber basket 54. The absorption element 46 can be fabricated as a single-piece element, as shown further in FIG. 15, or alternatively, as shown in FIGS. 7(b), 7(c), 7(e), can be composed of a plurality of components. In the latter case, the plurality of components are arranged in contact and fluid flow is possible between these components. The absorption element 46 is assembled with the cap 50 and the housing 48, and when these are installed within the fluid chamber 44, the tip of the absorption element 46 is separated from the surface of the substrate 42 by a predetermined distance. The substrate 42 is mounted on the base 52 so as to be easily removable once the cells are collected. The fluid chamber 44 is positioned on top of the slide 42, which is inserted into the base 52 and held by the hook 30.

[0056] The fluid chamber 44 can be detachably attached to the base as will be described later. Various ways of attaching the fluid chamber 44 to a slide or a base including the slide may be based on the use of an adhesive (Figs. 16(a) - 16(b)), or using a holding mechanism 30 when the fluid chamber is pushed in, or using a holding mechanism 32 when the upper part of the element 31 is melted and formed, or mechanical holding as shown in Figs. 17(a) - 17(f) by hooks 34, 35 that are aligned by rotation for proper holding of the fluid chamber 44.

[0057] Fig. 4(a) is a perspective view of the absorption element 46 within the plastic housing 48, and Fig. 4(b) is a front view of the absorption element 46 within the plastic housing 48 of Fig. 4(a). The plastic housing 48 can be pushed into the cap 50 or held by an adhesive. The plastic housing 48 is equipped with an overfill protection mechanism 10 shown in Fig. 6(a). The cap 50 is equipped with a riffling (corrugation) 51 on the side for ease of use, and a hole 9 for relieving differential pressure that may be generated during the wicking process is equipped at the top of the cap 50.

[0058] Fig. 5(a) is a perspective view of the tip of the plastic housing 48 and the absorption element 46, and the tip of the housing 48 is provided with a rectangular opening 8. Fig. 5(b) shows the tip having a circular cross - sectional opening indicated by 8a. Other shapes of the opening are possible as long as the cross - sectional area in combination with other parameters of the absorption element 46 provides the required fluid flow rate during the wicking stage of the process.

[0059] Figures 6(a) to 6(e) show various implementations for controlling the wicking rate during the fluid removal process after the completion of cell sedimentation according to the proposed method. Figure 6(a) shows that the control of the fluid removal rate can be achieved by the selection of the cross-sectional area of the opening 8 of the plastic housing 48 having a rectangular shape as shown in Figure 5(a). Figure 6(b) shows how this result is achieved by the selection of the distance between the plastic housing 48 containing the absorption element 46 and the substrate 42. It should be noted that the above results can also be achieved by the combination of the cross-sectional area and the above distance. Figure 6(c) shows the control of the fluid flow rate by the selection of the geometry of the absorption element 46.

[0060] Figure 6(d) is a photograph showing the control of the fluid flow rate by using an additional capillary 14 attached to the porous material 15 and by the selection of the length and inner diameter of the capillary. Figure 6(e) shows the capillary 14 attached to the porous material 15 and having a continuously increasing cross-section.

[0061] Figures 7(a) to 7(e) stepwise show more components related to the absorption element 46 forming part of the present device. Figure 7(a) shows the tip of the absorption element 46 disposed above the functionalized surface of the solid support 42. This figure shows the porous flat component 11 near the surface used for cell retention. As will be further explained, the wicking rate is defined by the shape, the type of porous material, and the distance between the tip of the absorption element and the solid support 42, and all these parameters are optimized to reduce cell loss that may be caused by excessive flow rates. Figure 7(b) shows the absorption element 46 composed of a flat absorption chip 11 and a cylindrical porous element 82 above the absorption element 11 for an increase in the wicking capacity (the maximum amount of liquid that can be absorbed by the element 46). Other configurations of these components are also possible. For example, the element 11 can be conical with a different cross-section, and the element 82 called the cylindrical element can be fabricated with a square or rectangular cross-section.

[0062] Elements 11, 82 are arranged in contact with each other so as to ensure non-interrupted fluid flow. As a result, these components function as a single composite absorption element 46 from the perspective of wicking. Fig. 7(c) shows an additional increase in wicking ability by using a hollow cylindrical porous element 84 into which an object 82 is inserted. In a multi-component absorption element, not only the shape of the element but also the porosity can be selected, so that the wicking rate and total capacity provide the required performance. Fig. 7(d) shows a plastic housing for the absorption element 46 including a plastic cavity 48 and a cap 50, enabling good contact between the components of the absorption element 46 and allowing wicking of liquid only through a controlled opening 8 (not shown) at the bottom of the plastic cavity 48. The cap 50 is also equipped with a riffle (corrugation) on the side of the cap for ease of use.

[0063] Fig. 8(a) shows a sealing gasket 54 attached to the tip of the absorption element 46 within the housing 48 and the surface of a solid support 42 or substrate 42 that forms a cavity for reagents. Fig. 8(b) shows an embodiment of a gasket 60 with a protruding element 62 added to the gasket 54. The purpose of the protruding element 62 is to easily remove the gasket 54 from the slide at the completion of the process step prior to microscopic imaging.

[0064] Fig. 8(a) shows a sealing gasket 54 attached to the tip of the absorption element 46 within the housing 48 and the surface of a solid support 42 or substrate 42 that forms a cavity for reagents. Fig. 8(b) shows an embodiment of a gasket 60 with a protruding element 62 added to the gasket 54. The purpose of the protruding element 62 is to easily remove the gasket 54 from the slide at the completion of the process step prior to microscopic imaging.

[0065] The gasket 54 may have a circular, rectangular, or square cross-section. These can be attached to the bottom of the fluid chamber 44 by adhering them circumferentially around the area where the target cells settle, so as to extend circumferentially. Alternatively, the gasket 54 can be held in a groove that is complementary to the size and shape of the gasket incorporated in the bottom of the chamber 44. Alternatively, the gasket can be held on the surface of the solid support 42 without being attached to the chamber 44. Those skilled in the art will understand that the gasket may be optional, as other methods of creating a liquid-tight seal between the chamber 44 and the solid support 42 are known.

[0066] Figures 8(c) - 8(f) are bottom views of the fluid chamber 44 showing a groove 66 (Figure 8(c)) having a size capable of accommodating the gasket 54 (Figure 8(d)). Figure 8(e) shows the fluid chamber 44 with an additional groove 68 added to the bottom of the fluid chamber 44 to accommodate the protruding element 62 of the gasket 54 (the gasket is not present in Figure 8(e)). Figure 8(f) shows the gasket 54 with the protruding element 62 installed within the fluid chamber 44.

[0067] Once the target cells have settled on the upper surface of the solid support 42, the fluid chamber 44 is filled with cell-free liquid. At this point, the absorption element 46 (installed within the housing 48 together with the wicking cap 50) is inserted into the fluid chamber 44 and secured by the leak-proof wicking cap 50. The purpose of the absorption element 46 is to draw liquid from the fluid chamber 44 so that the cells are not damaged, deformed, or detached from the surface of the support 42 during liquid removal. This is achieved primarily by the design parameters of the absorption element 46 and secondarily by the design parameters of the housing 48 and the cap 50, which, in combination with the design parameters of the housing 48, define the distance between the absorption element 46 and its tip and the surface of the support 42.

[0068] With respect to the absorption element 46, the fluid absorption element is designed such that the cross-sectional area, the distribution along the axis of the fluid absorption element 46, and the porosity are within a range that provides control over the rate of fluid absorption into the absorption element 46 along its length and the rate of fluid absorption to the tip spaced from the upper surface of the solid support 42, thereby providing controlled removal of liquid from the fluid chamber 44. The mechanical design of the housing 48 is also optimized to assist in the optimal removal of liquid. A first one is the design of the housing 48 and the cap 50, which is to space the tip of the absorption element 46 from the upper surface of the solid support 42 and to position the tip of the fluid absorption element at a distance shorter than a predetermined distance from the surface of the solid support 42. The distance at which the tip of the absorption element 46 is positioned from the upper surface of the solid substrate 42 on which the target cells are deposited is important for the following reasons.

[0069] The detachment of target cells attached to the surface of the solid support 42 is mainly caused by shear stress resulting from fluid flow. The shear stress on the target cells generated by the fluid flow is defined by the differential of the fluid velocity with respect to distance. The absolute value of the velocity is defined by the flow rate of the fluid, which depends on the design parameters of the absorption element 46 and the selected porous material in which it is fabricated. An increase in the distance between the solid support 42 and the tip of the absorption element 46 reduces the shear stress and decreases the loss of target cells. On the other hand, this also slows down the removal of the fluid and may prevent the complete removal of the fluid from the fluid chamber 44, making the process suboptimal or inefficient. Optimization of all the design parameters described above and implementation of the flow increase or acceleration mechanisms 17, 17a shown in FIGS. 10(a) and 10(b) shorten the wicking time for removing the fluid from the fluid chamber 44 and enable fluid removal with reduced cell loss at an acceptable fluid removal time. The fluid flow provided by the acceleration mechanisms 17, 17a shown in FIGS. 10(a) and 10(b) can be significantly higher compared to the flow provided by the absorption element 46 passing through the tip 8 of the housing 48. However, until the absorption element is introduced into the fluid chamber 44, most of the fluid does not contain cells, and since the distance between the mechanism 17 or 17a and the support 42 is selected to be quite large, no cell detachment occurs in relation to this fluid flow.

[0070] In addition to the distance from the upper surface of the substrate 46 on which the chip is installed, the housing 48 may be provided with an overflow prevention mechanism. FIG. 9 is a view of the absorption element housing 48 showing an overflow prevention mechanism 10 having an opening in the upper wall of the housing 48 to facilitate the inflow of fluid into the housing 48. The protection mechanism is installed at the upper part of the housing 48, and these are close to the part of the absorption element 46 having a large cross-sectional area. Also, the cross-sectional area of the overflow mechanism is large, for the direct fluid flow to the part of the absorption element 46 having a large volume of porous material. As a result, an excessive amount of fluid that may initially be present in the fluid chamber 44 is absorbed, and the fluid chamber 44 does not overflow. Further, these mechanisms are installed far away from the surface of the solid support 42 where the cells are sedimented at the centrifugation stage. Thereby, the increase in the flow rate due to the overflow mechanism does not cause the detachment of the cells from the surface and does not result in cell loss. An example of the opening of the overflow mechanism in the housing 48 is shown in FIG. 9.

[0071] FIG. 10(a) shows a first embodiment of the absorbent housing fluid chamber 48 having a flow acceleration mechanism 17 which is an elongated slot. FIG. 10(b) shows a second embodiment of the absorbent housing 48 having a mechanism 17a which is a circular hole. The presence of the mechanism 17 or 17a provides an increase in the wicking rate without adverse effects on cell retention. This is achieved by arranging the mechanism 17 or 17a further away from the tip of the housing 48 and thus at a large distance from the surface 42 where the cells sediment. An increase in the wicking rate may be required to reduce the sample processing time.

[0072] Figure 11(a) shows a perspective view of a solid support 42 in the form of a glass slide with the functionalized area on the surface of the glass slide surrounded by a gasket 54 in a state where the fluid chamber 44 is removed. Figure 11(b) shows the reinstalled fluid chamber 44. The ability to remove and reinstall the fluid chamber 44 enables visual control, including microscopy (if necessary), at various stages of the process. Also, after the completion of the microscopy stage, reinstallation of the fluid chamber 44 may be required for post-treatment of the cells.

[0073] Figures 12(a) - 12(c) show the cell holding device during the fluid introduction stage into the fluid chamber 44, where the first screw cap 68 is removed, a fluid sample flows into the fluid chamber 44, and then the screw cap 68 is screwed onto the fluid chamber 44. The screw cap 68 is used during the centrifugation stage of the process to prevent accidental leakage of the fluid containing the cells. This is achieved by using a gasket 70 shown in Figure 12(c), which is in the form of an O-ring that provides a liquid-tight connection between the fluid chamber 44 and the screw cap 68. The gasket 70 can have other shapes (e.g., flat) as long as a liquid-tight connection is achieved. As described above, the cap 68 is used for the centrifugation stage for the purpose of preventing leakage of the fluid containing the cells from the fluid chamber 44. No liquid removal is expected during the centrifugation step, which enables the cells to be maintained in the liquid and prevents damage to the cells that exists in devices described in other patents or available on the market. After centrifugation is complete, the cap 68 is replaced with a second screw cap 50 that includes an absorption element 46. Liquid removal is performed at this stage, and the liquid is removed in a controlled manner by appropriate selection of the porosity of the material used in the manufacture of the absorption element 46, the configuration / cross-section of the absorption element, and the distance between the tip of the absorption element and the surface of the solid support 42. The distance is defined by the design of the housing 48 and the design of the cap 50.

[0074] Referring to FIGS. 2(c) and 2(d), the device 40 is configured to be removably held within a centrifuge, and an axis 41 that is perpendicular to the surface of the solid support 42 and passes through the center of the region where the target cells are deposited on the upper surface of the solid support 42 intersects the axis of rotation 49 within the centrifuge. The orientation of the device within the centrifuge may vary depending on the particular centrifuge used, such that the long axis of the solid support 42 is within the plane of rotation (as shown in FIG. 2(b)), or the long axis of the solid support 42 is arranged within the centrifuge to be perpendicular to the plane of rotation, as shown in FIG. 2(c). Due to the axial symmetry of the internal cavity of the fluid chamber 44, other orientations are also possible.

[0075] FIG. 13 shows the base 52 of the cell holding device with holes 80 for visualization of the fluid and cells without removing the fluid chamber 44. FIG. 14 shows the solid support 42 of the cell holding device with a section 86 of the surface functionalized to capture cells and a Teflon protective layer 82 around the functionalized region 86.

[0076] FIG. 15(a) shows a shaped absorbent element 46 with a conical tip 25 having a circular cross-section 28. FIG. 15(b) shows a shaped absorbent element 46 with a flat tip having a triangular shape 27 and a rectangular cross-section 29.

[0077] FIGS. 16(a) - 16(b) show other embodiments of the proposed device, showing the state where the absorbent element is removed from the assembly (FIG. 16(a)) and the assembled device where the absorbent element is in contact with the fluid (FIG. 16(b)). FIGS. 16(c) - 16(d) show other embodiments of the proposed device where the fluid chamber 44 is directly adhered to the surface of the slide 42. FIG. 16(c) shows a cap 68 attached to the fluid chamber 44 before the centrifugation step of the process. FIG. 16(d) shows a encapsulated absorbent element 46 made of absorbent tissue paper inserted into the fluid chamber 44 to remove the fluid after completion of the centrifugation step.

[0078] Figures 17(a) to 17(f) show examples of various ways to hold the components of the assembly. The perspective view of Figure 17(a) shows a base 52 provided with hooks 30 integrally formed along two opposing long edges, which are used to hold the fluid chamber assembly 44. Installed in the center of the base 52 is a hole 80. Installed along two opposing long edges of the base 52 are tabs 37, which are used for slide retention.

[0079] Figure 17(b) shows a base 52 that uses an integrally formed pin 31 instead of the hook 30 as in Figure 17(a). Since the pin 31 is compressible using heat, when the fluid chamber 44 engages with the base 52, the mechanism 32 shown in Figure 17(c), which is generated by the heat-induced compression of the pin 31, prevents the fluid chamber 44 from falling off the base 52. When the pin 31 is compressed by heat, a plastic mass 32 larger than the opening through which the pin protrudes is generated, thereby holding the fluid chamber 44 in place.

[0080] Figures 17(d) and 17(e) show other embodiments in which the retention of the fluid chamber is achieved by a retainer 33 that engages with a hook 34 attached to the fluid chamber 44 during rotation of the fluid chamber 44. A stopper 35 is added to the hook 34 to prevent the disengagement between the base 52 and the fluid chamber 44. Figure 17(d) shows the assembly before the attachment of the fluid chamber 44 to the base 52, and Figure 17(e) shows the assembled device.

[0081] Figure 17(f) shows a fluid chamber assembly 44 fixed to the base 52 using a retainer 33 and a hook 34 when the fluid chamber 44 is engaged by sliding it in the direction of the long edge of the base 52, which is in contrast to the embodiment of Figure 17(e) where the engagement is achieved by the rotation of the fluid chamber 44.

[0082] FIG. 17(g) shows a bottom view of the base 52 with the slide 42 held on the upper surface of the base 52. The slide 42 includes a region 86 functionalized with a selected agent. Surface functionalization is known to be used for the improvement of cells adhering to the surface. By the selection of a specific type of functionalization, it becomes possible to increase the retention rate of a specific type of cell, which can be used as a method for concentrating the collected cells or as a method for increasing the probability of removing interfering cells (e.g., white blood cells, etc.) from the surface during the continuous washing step after centrifugation.

[0083] FIG. 17(h) shows the alignment of all components of the assembled device 40, which is important for the uniform sedimentation of cells and the clear visualization of the region of the slide on which the cells have sedimented.

[0084] FIGS. 18(a) - 18(f) show a series of diagrams demonstrating the importance of the proper alignment of the axis of the cell recovery device 40 with respect to the axis of rotation in a centrifuge. FIG. 18(a) shows a schematic view of the fluid chamber 44 with cells in the centrifuge, where the axis of the device is shifted from the axis of the centrifuge. FIG. 18(b) shows the force distribution, and FIG. 18(c) shows the non-uniform distribution of the sedimented cells. FIG. 18(d) shows a plot of the calculated displacement of the cells in a direction perpendicular to the axis of rotation from the original position as a function of the initial distance between the cells and the axis of the device for various displacements of the axis of the device with respect to the axis of rotation in the centrifuge (for a case of 10 mm). FIG. 18(e) shows the case of a displacement of 0.75 mm, and FIG. 18(f) shows the case of a displacement of 0 mm.

[0085] Figure 19 shows an elevation view of an example of the absorption element 46, which is composed of two porous components 82, 11 and a plastic housing 48 for this absorption element 46. The upper porous component 82 has a volume of 570 microliters, and the lower element 11 has a volume of 75 microliters. The total volume of the absorption element in this embodiment is equal to 645 microliters. Assuming that the porosity of the material used for the components of the absorption element 46 is about 30%, the total volume of the liquid absorbed will be at most 215 microliters, which is slightly higher than the typical volume (about 200 microliters) of the harvest generated by a cell separation device (e.g., Parsortix (registered trademark)). Using a porous material with a higher porosity allows for a higher wicking rate and a larger volume of liquid to be absorbed (if necessary).

[0086] Figure 20 is a plot showing the relationship between the cell loss rate (percentage) and the wicking rate for two different centrifugation speeds, 4400 rpm (circles) and 2200 rpm (triangles).

[0087] Figure 21 shows the cell loss rate as a function of the distance between the surface of the solid support 42 and the absorption element 46, which forms part of the present cell retention system. Panel (a) shows no addition of KCl, and panel (b) shows the addition of KCl to the solution.

[0088] (Optimization of wicking height) The previous optimization of the wicking rate was carried out on live SK - BR - 3 cells, which are more adhesive and more firmly attached than other possible cell types. Studies using a less "sticky" cell model, namely, pre - fixed SK - BR - 3 cells (ICT), revealed that at the previously set height of 0.5 mm, cell loss occurred during wicking. Therefore, the distance between the microscope slide and the wicking tip was increased to reduce cell loss.

[0089] At a wicking height of 1.0 mm, no cell loss occurred, but due to some changes in the position of the cells, the cells still experienced a certain form of turbulent flow, and it was found that the cells could be detached at 1.0 mm. Therefore, in the most gentle process, it was found that the recommended value of the wicking height should be 1.5 mm or more. By choosing a wicking height of 2.0 mm instead of 1.5 mm, the user-to-user variation when inserting the wicking tip into the supernatant was minimized, providing a larger buffer.

[0090] By optimizing the absorbent material and the distance between the surface of the slide and the tip of the absorbent material, the required flow rate of the fluid can be selected, which needs to be below the threshold defined by the shear stress on the cells, which may cause cell shedding during the fluid removal step. The flow rates were measured at a distance of 2 mm for two different porous absorbent materials (U200 and U210, PoreTechnology). The results are shown in Figures 22(a) and 22(b). Specifically, Figures 22(a) and 22(b) show the wicking rate as a function of time for the polyethylene porous material U200 (PoreTechnology) in panel 22(a) and U201 (PoreTechnology) in panel 22(b). The distance between the surface of the solid support 42 and the tip of the absorbent element 46 is approximately 2 mm.

[0091] The threshold level of cell detachment was determined experimentally and was equal to 500 μL / s for the selected shape of the fluid chamber 44 and the selected coating of the surface of the solid support 42 (glass slide). The distance of 2 mm between the solid support 42 and the tip of the absorbent element 46 is appropriate for the absorbent material U200 (see Figure 22(a)), but this distance needs to be increased for the absorbent material U201 (see Figure 22(b)).

[0092] (Results (photos of captured cells, evidence of reduced cell loss)) Pre-labeled SK-BR-3 cells were processed and counted through both systems. n = 10 replicates were performed with both the Cytospin® and the cell harvesting device. A non-parametric Wilcoxon rank sum test was performed with a p-value of 0.00018. The cell harvesting device had an average cell recovery rate of 94% ± 6% (SD).

[0093] Figure 23(a) is a micrograph of DAPI-stained cells that were injected into the fluid chamber 44 and sedimented by centrifugation using the apparatus according to the method, which shows the cells retained on the slide surface after centrifugation and subsequent fluid removal. Figure 23(b) is a magnified image (×100) of the micrograph of Figure 23(a). Figures 23(a) and 23(b) demonstrate an improvement in the retention (capture) rate in the proposed method and apparatus compared to the industry standard Cytospin® method. The improved form shows less severe "flattening" of the cells on the Cytospin®, less bleb formation, and reduced signal intensity compared to the cell harvesting device. No cytoplasmic diffusion is seen with the cell harvesting device. The staining intensity with the cell harvesting device is quite high.

[0094] Figure 24 shows the percentage cell capture efficiency for two cell capture methods, using the Cytospin® shown in dataset 1 and the cell retention method and device of the present invention shown in dataset 2. From Figure 24, it is clear that the controlled liquid removal rate, in combination with other features of the proposed method, enables an increase in the cell capture rate from approximately 30% (Cytospin® dataset in Figure 24) to over 90 - 95% for the proposed device (Harvest Chip dataset in Figure 24).

[0095] FIG. 25(a) shows a microscopic photograph of cells captured using Cytospin (registered trademark), and FIG. 25(b) shows a microscopic photograph of cells captured using the cell recovery device of the cell holding device of the present invention. It can be seen that the cells captured using Cytospin (registered trademark) are damaged, but FIG. 25(b) clearly shows that the morphology of the collected cells is preserved by the method of the present invention.

[0096] FIG. 26(a) shows a microscopic photograph showing the morphology of SK-BR-3 cells fixed in a state where no fluid is present around them (referred to as dry fixation), while FIG. 26(b) shows a microscopic photograph showing the morphology of SK-BR-3 cells fixed in a state where a controlled amount of fluid is present (referred to as wet fixation). The images were obtained using a magnification of 400 times and CK-FITC conjugate staining. As can be seen from FIGS. 26(a) and 26(b), control of the amount of residual fluid present in the device after a large amount of fluid has been removed enables improvement in the morphology of the retained cells. An additional advantage in this case of the controlled amount present in the device at the staining stage is that the background level is reduced. Fixation using a controlled amount of residual fluid produced the lowest background signal compared to Cytospin (registered trademark) or dry fixation methods when no fluid was present (FIG. 26(b)) or when there was no control of the residual fluid level (FIG. 26(a)).

[0097] It has been demonstrated that in Cytospin (registered trademark), a larger overall cell size of 15 - 25 μm is observed compared to 12 - 20 μm (dry fixation) and 12 - 15 μm (wet fixation) on the solid support of the cell recovery device. Also, the images obtained on slides processed using Cytospin (registered trademark) had a lower fluorescence signal. The signal from the cell recovery device using a controlled amount of residual fluid (wet fixation) showed the strongest intensity, and dry fixation was intermediate between Cytospin (registered trademark) and wet fixation.

[0098] Similar results have been obtained with Cytospin (registered trademark) and patient samples processed using this cell capture device (see Figures 27(a) and 27(b)). Comparison of Cytospin (registered trademark) and this device regarding the final form: The cells retained using Cytospin (registered trademark) in Figure 27(a) appear considerably larger and jagged compared to the cells retained using this device shown in Figure 27(b).

[0099] Pre-labeled SK-BR-3 cells separated with Parsortix (registered trademark) were added to EDTA HNV blood, pooled, captured, fixed, and stained either on the solid support of the cell recovery device or on a Cytoslide (registered trademark) (Fisher Scientific). (The experiments were performed by multiple trained users over multiple devices / days). The effect of pipetting variation was reduced (comparing STD with Cytospin (registered trademark) and HS). The results are shown in Figure 28. Summary statistics: Cytospin (registered trademark): 38.8% ± 12.6%, fixation of the cell recovery device: 96.7% ± 3.3%. As can be seen, the final capture efficiency (count after staining) of this cell recovery device is significantly superior compared to Cytospin (registered trademark). Much of the cell loss seen with Cytospin (registered trademark) occurred during the initial centrifugation of the cells onto the slide using the filter card fluid chamber 44.

[0100] (Post-treatment of cells) Example of post-treatment of cells: Lysis of the retained cells using continuous molecular analysis is shown in Figure 29, which shows a histogram of the capture efficiency of EDTA HNV blood to which pre-labeled SK-BR-3 cells separated, pooled, captured, fixed, and then stained either on the cell recovery device of the cell retention device of the present invention or on a Cytoslide (registered trademark) were added. The experiments were performed by multiple trained users over multiple devices / days. The effect of pipetting variation was reduced (comparing the standard deviation (STD) of Cytospin (registered trademark) and the cell recovery device 40).

[0101] The pre-labeled SK-BR-3 cells can be seen inside the capillary micropipette on the surface of the solid support 42 of the cell recovery device (Image 1), then aspirated into the microcapillary (Image 2), and then moved and deposited at different locations on the slide surface (Image 3). The results of single cell collection using the cell recovery device of the present invention are shown in FIG. 30, which shows the collection of SK-BR-3 cells deposited on the solid support using a controlled amount of residual fluid according to the proposed method. The pre-labeled SK-BR-3 cells can be seen inside the capillary micropipette on the surface of the solid support 42 of the cell recovery device (upper panel of FIG. 30), then aspirated into the microcapillary (central panel of FIG. 30), and then moved and deposited at different locations on the slide surface (lower panel of FIG. 30).

[0102] FIG. 31 shows that cell loss may be high if the parameters are not properly controlled. FIG. 31 shows the collection of SK-BR-3 cells deposited on the solid support 42 using a controlled amount of residual fluid according to the proposed method. The upper panel shows the cells held with the capillary of the cell collection tool placed on top of the cells. The middle panel shows the cells being removed from their initial location by the cell collection tool. The lower panel shows the cells being dispensed (relocated) by the cell collection tool. When using a cell recovery device with a wicking height of 2.0 mm and a two-piece wicking cap, the amount of cell loss due to the wicking and resuspension steps is minimal.

[0103] By appropriately controlling the process parameters (wicking rate and residual fluid volume), cell loss is minimal even in multiple steps of the process. This is also confirmed in Figure 32, which shows the cell recovery rate (%) at various stages of the process using various protocols, as shown in Figure 32. As a post-treatment step, it is also possible to directly stain the cells within the fluid chamber 44 of the cell recovery device. This requires multiple wicking steps to exchange the reagent within the fluid chamber, but replaces multiple steps in the staining after slide fixation. As can be seen from Figure 32, it has been demonstrated that cell loss due to supernatant wicking and fluid exchange is minimal.

[0104] Achievement of good morphology and high recovery rate is shown in Figure 32, which shows the cell recovery rate for multiple consecutive wicking steps (a, b, c). The first wicking (a) is performed after cell sedimentation, the second wicking (b) is performed after resuspension, and the third wicking (c) is performed after staining. This test was repeated 3 times, demonstrating that multiple wicking events do not result in cell loss when the wicking rate is appropriately selected according to the method.

[0105] Data for Streck SKBR-3 samples counted between each wicking and buffer exchange step are shown in Figure 32, and an image of the surface with cells stained and retained by the proposed device is shown in Figure 34.

[0106] Staining of cells within the fluid chamber 44 using DAPI is shown in Figure 33, which is a micrograph of white blood cells sedimented and stained with DAPI using the method disclosed herein. This photograph was taken with the fluid chamber 44 still attached to the solid support, demonstrating that cells can be stained with the fluid chamber 44 attached.

[0107] Figure 34 shows photomicrographs (rows A and B) and photographs (row C) of cells sedimented using Cytospin® (column I) and cells on the solid support 44 of the cell recovery device 40 that forms part of the cell collection device of the present invention (columns I and II), showing the state without controlling the residual fluid (column II) and the state using control of the amount of residual fluid according to the method disclosed herein (column III). HNV cells were injected into the fluid sample placed in the cell recovery device 40 using the following wet fixation method for live EDTA tube cells after staining. Using the same sample, cell sedimentation was performed using Cytospin®. Cytospin® showed the worst morphology (columns I, rows A and B) with cytoplasmic diffusion, decreased fluorescence intensity, and increased background. The least amount of morphological change was seen under the conditions of the wet-fixed cell recovery device 40 (column III). Similar results were seen for CD45 staining showing decreased cell density in Cytospin®, which is also a concern.

[0108] The following are non-limiting and exemplary protocols for the use of the device.

[0109] (Cell Recovery Device 40 EDTA Protocol (Staining on Slide)) 1. Deposit the contents of the Parsortix® harvest directly into the fluid chamber 44 of the cell recovery device 40. 2. Centrifuge the sample in a StatSpin® CytoFuge® (the following is a reference to this device. https: / / www.fishersci.ca / shop / products / statspin-cytofuge-2-personal-cytocentrifuge-system-3 / 220011) (rotate at 600 rpm for 2 minutes, then at 4400 rpm for 2 minutes). 3. Tighten the wicking cap 50 and wick (suction) the device for 5 minutes. 4. Pipette 150 μL of ice-cold acetone along the tube wall into the liquid chamber 44 and incubate in a freezer (-20 °C) for 5 minutes to fix the cells. 5. After fixation, transfer the remaining acetone to an appropriate waste container. 6. Remove the fluid chamber 44 from the cell recovery device 40. 7. Evaporate the remaining acetone and dry the slide, then (for 10 minutes) proceed to staining or storage. (Cell Recovery Device 40 Streck Protocol (Staining on Slide)) 1. Deposit the contents of the Parsortix® harvest directly into the fluid chamber 44 of the cell recovery device 40. 2. Centrifuge the sample in a StatSpin® CytoFuge® at 600 rpm for 2 minutes, followed by 4400 rpm for 2 minutes. 3. Tighten the wicking cap and allow the device to wick (absorb) for 5 minutes. 4. Prepare a dry buffer consisting of a 200 μL solution of 10% serum (horse or fetal bovine) in 0.083 M KCl (i.e., 20 μL serum + 180 μL KCl solution). 5. Pipette 200 μL of the dry buffer along the tube wall into the liquid chamber 44. 6. Centrifuge the sample in a StatSpin® CytoFuge® at 600 rpm for 2 minutes. 7. Use the second wicking cap 50 to remove the supernatant. 8. Remove the fluid chamber 44 from the cell recovery device 40. 9. Disassemble the cell recovery device and pull out the PTFE slide from the base. 10. Place the PTFE slide with the sample on a hot plate set at 37 °C and evaporate the liquid remaining in the target area for 30 minutes. 11. After confirming that the slide surface is completely dry, proceed to fixation. 12. Using a pipette, introduce 50 μL of ice-cold acetone in droplet form onto the target area on the slide. 13. Fix the cells by placing the slides in a freezer (-20 °C) for 5 minutes. 14. Transfer the remaining acetone to a separate container (decant), and dry the slides (10 minutes) before proceeding with staining or storage.

[0110] (Cell Recovery Device 40 Streck Protocol (Intracellular Staining) 1. Deposit the contents of the Parsortix® harvest directly into the fluid chamber 44 of the Cell Recovery Device 40. 2. Centrifuge the sample at 4400 rpm for 2 minutes in a StatSpin® CytoFuge®. 3. Tighten the wicking cap 50 containing the absorption element 46 and allow the device to wick (absorb) for 5 minutes. 4. Prepare X μL of staining solution (20 - 50 μL of solution has been previously tested) in a permeabilization reagent (e.g., Inside Perm). Preferably, since it is desirable that all antibodies are conjugated to a fluorophore, allow a single antibody incubation rather than multiple incubations. 5. Introduce 50 μL of the staining solution into the fluid chamber 44 of the Cell Recovery Device 40 by pipetting along the walls of the device. 6. Incubate the solution in the dark at room temperature for 45 minutes. 7. After the antibody incubation, pipette along the walls and gently introduce 200 μL of 1× PBS into the fluid chamber 44. 8. Centrifuge the sample at 4400 rpm for 2 minutes in a StatSpin® CytoFuge®. 9. Tighten the wicking cap 50 containing the absorption element 46 and allow the device to wick (absorb) for 5 minutes. 10. Repeat the addition of 1× PBS and wicking twice to completely wash away the remaining debris. 11. For the final wash before covering with a coverslip, prepare 200 μL of a 5% glycerol solution with deionized water. 12. After wicking, use a P1000 pipette to introduce a 5% glycerol solution into the liquid chamber 44. Gently mix the solution within the liquid chamber 44 to resuspend the cells and ensure a uniform dispersion. 13. Centrifuge the sample in a StatSpin® CytoFuge® at 600 rpm for 2 minutes, followed by centrifugation at 4400 rpm for 2 minutes. 14. Tighten the wicking cap 50 containing the absorption element 46 and allow the device to wick (absorb) for 5 minutes. 15. Use a removal tool to remove the fluid chamber 44 and remove the PTFE slide containing the sample from the base. 16. Place the PTFE slide on a hot plate set at 37 °C and incubate for 30 minutes. Protect the slide from light to prevent photobleaching. 17. After drying, the glycerol remaining in the target area of the PTFE slide still appears glassy. 18. Add one drop of mounting medium onto the target area and place a coverslip on top. Apply pressure from one end of the coverslip to prevent air bubbles.

[0111] (Cell Recovery Device 40 Protocol - Alternative Protein Buffer) 1. FBS is time-consuming to prepare (heat-inactivate) and difficult to ship. 2. Consider alternatives (Stabilguard, BSA, horse serum, Hank’s) with performance equivalent to FBS. 3. 10% horse serum (the same reagent used in the blocking step) was performed in the same manner as FBS. Also, blocking before staining becomes unnecessary.

[0112] (Optimization of Liquid Removal Rate by Selection of Absorbing Material, Distance between Slide and Tip of Absorbing Element, and Cross-Section of Absorbing Element)

[0113] It has been found that the use of the absorbent element housing 48 is extremely beneficial for limiting the contact area between the liquid medium and the porous material, since the porous material without the housing 48 has too high an absorption rate, resulting in cell detachment. In one embodiment, the housing 48 can be made of polypropylene. However, it will be understood that the housing 48 can be made of other materials including, but not limited to, polycarbonate, polystyrene, other types of plastics, rubber, glass, metal, etc.

[0114] Experiments were conducted using live pre-labeled SK-BR-3 as a model system. These cells were allowed to settle on the surface of the solid support 42, and then the liquid was removed using a syringe pump at different suction rates. Cell loss was evaluated at each removal rate. As a result, cell loss associated with an increased liquid removal rate was found. Tests were conducted on cells sedimented at two different centrifugal forces (265×g / 2200 rpm and 1060×g / 4400 rpm) to determine the maximum allowable liquid removal rates for cells loosely adhered and strongly adhered to the slide surface.

[0115] For both cells adhered at low (265×g) and high (1060×g) centrifugal forces, the maximum allowable rate of supernatant aspiration was found to be 500 μL / min. Cell dissociation from the slide surface was observed more dramatically at rates exceeding 500 μL / min for cells sedimented at 265×g compared to cells sedimented at 1060×g. To minimize the potential for cell loss during the wicking process, the maximum allowable wicking rate was determined using lower sedimentation force (265×g) conditions.

Claims

Claim 1 A cell recovery device for sedimentation and retention of target cells from a fluid sample, comprising: a) a base configured to removably hold a solid support, the solid support being configured to receive cells on an upper surface thereof; b) a fluid chamber having a first opening, the fluid chamber providing a liquid-tight seal between the upper surface and the fluid chamber, the fluid chamber having a second opening for receiving a liquid sample containing target cells to be collected, the first opening defining a region of a selected size on the surface of the solid support on which the target cells accumulate, and including a first removable cap configured to close the second opening during centrifugation; d) a fluid absorption element and a housing configured to receive the fluid absorption element; the housing having a bottom opening through which fluid can pass, the fluid chamber being configured to receive the housing after centrifugation, the fluid chamber including a second removable cap configured to close the second opening, and the combination with the housing preventing positioning of the tip of the fluid absorption element at a distance shorter than a predetermined distance from the surface of the solid support; e) the cell recovery device being configured to be received in and removably held by a centrifuge. Claim 2 The fluid absorption element has a certain cross-sectional area, a distribution along the axis of the fluid absorption element, and a range of porosities providing control of the fluid absorption rate; The tip of the fluid absorption element is provided at that distance from the surface of the solid support, so that the target cells fixed on the solid support are not detached from the surface of the solid support by the flow of the fluid absorbed by the fluid absorption element. The device according to claim 1. Claim 3 The fluid absorption element is a porous material having a porosity in the range of about 1 to about 100 microns; The predetermined distance of the tip of the absorption element on the surface of the solid substrate is in the range of about 0.1 mm to about 4 mm. The device according to claim 1 or 2. Claim 4 The fluid absorption element has a porosity in the range of about 5 to about 50 microns; The predetermined distance of the tip of the absorption element on the surface of the solid substrate is in the range of about 0.1 mm to about 3 mm. The device according to claim 1. Claim 5 The liquid absorption element has porosity in the range of about 10 to about 20 microns, The device according to any one of claims 1 to 4, wherein the predetermined distance from the surface of the solid substrate to the tip of the absorption element is in the range of about 0.1 mm to about 3 mm.

6. The device is configured to be removably held within the centrifuge, such that an axis perpendicular to the surface of the solid support passing through the center of the section of the surface capturing the cells intersects the axis of rotation. The device according to any one of claims 1 to 5.

7. The device is configured to be removably held within the centrifuge, such that the long axis of the solid support is within the plane of rotation, or the long axis of the solid support is perpendicular to the plane of rotation. The device according to claim 6.

8. The selected size of the region on the upper surface of the solid support where the target cells are deposited is selected to facilitate the interaction between the target cells and the surface enabling the attachment of the target cells to the upper surface of the solid support. The device according to any one of claims 1 to 7.

9. The selected size of the region on the upper surface of the solid support where the target cells are deposited is modified to facilitate the interaction between the target cells and the surface and assist in the attachment of the target cells to the upper surface of the solid support. The device according to any one of claims 1 to 8.

10. The selected size of the region on the upper surface of the solid support where the target cells are deposited is selected to modify the interaction between the target cells and the surface and assist in the attachment of the target cells to the upper surface of the solid support, and is functionalized with a selected agent. The device according to claim 1.

11. The agent is selected from the group consisting of poly-L-lysine, silane coating, gelatin, fibronectin, gold or silver coating. The device according to claim 10.

12. The agent is selected to provide differential binding affinity to cells present that are not the target cells to be isolated, such that non-target cells do not bind to the surface of the upper surface of the solid support. The device according to claim 10.

13. The fluid absorption element is made of porous plastic. The device according to any one of claims 1 to 12.

14. The porous plastic is treated to modify its absorption characteristics. The device according to claim 13.

15. The apparatus according to claim 13, wherein the porous plastic is porous polyethylene coated with a hydrophilic material.

16. The apparatus according to any one of claims 1 to 15, wherein the tip of the fluid absorption material spaced apart from the upper surface is disposed at a distance in the range of about 0.1 mm to about 3 mm from the surface of the solid substrate.

17. The apparatus according to claim 2, wherein the porosity of the fluid absorption element and the size of the opening are selected to provide a selected flow rate.

18. The apparatus according to any one of claims 1 to 17, wherein the solid support is in the form of a transparent microscope slide.

19. The apparatus according to any one of claims 1 to 18, wherein the first removable cap includes a gasket that provides a liquid-tight seal during centrifugation.

20. A region on the surface of the solid support outside the region of the selected size where the target cells are deposited on the upper surface of the solid support is coated with a protective coating that limits the spread of fluid present within the region of the selected size where the target cells are deposited on the rest of the surface of the solid support. The apparatus according to any one of claims 1 to 19.

21. The apparatus according to any one of claims 1 to 20, wherein the housing configured to receive the fluid absorption element includes an overflow mechanism for preventing overflow of fluid caused by insertion of the fluid absorption element into the fluid chamber.

22. The apparatus according to claim 21, wherein the overflow mechanism includes any of the following. a) At least one opening at the upper part of the absorption element housing. b) The opening configured to have any of a square, circular, or slot shape.

23. The apparatus according to any one of claims 1 to 22, wherein the housing configured to receive the fluid absorption element includes at least one flow acceleration mechanism for increasing the fluid removal rate without increasing the possibility of removing cells sedimented from the surface of the solid support.

24. The apparatus according to claim 23, wherein two or more of the flow acceleration mechanisms are provided on the absorption element housing.

25. The apparatus according to claim 23, wherein the flow acceleration mechanism is provided on the absorption element and is configured to have a circular shape or a slot shape.

26. The device according to claim 23, wherein the flow acceleration mechanism on the absorption element housing is installed at a distance in the range of about 5 mm to about 30 mm from the surface of the solid support. **Claim 27** The solid substrate is substantially transparent, The base includes an opening aligned with the region of the selected size on the surface of the solid support where the target cells are deposited, and provides an observation port for visual observation of the region of the selected size where the target cells settle without removing the fluid chamber from the solid substrate. The device according to any one of claims 1 to 26. **Claim 28** The device according to any one of claims 1 to 27, wherein a liquid-tight seal is provided by a gasket. **Claim 29** The device according to claim 28, wherein the gasket is fixed to the fluid chamber, and the gasket remains attached to the fluid chamber when the fluid chamber is removed from the solid substrate. **Claim 30** The device according to claim 29, wherein the gasket has a circular, rectangular or square cross-section and is fixed to the fluid chamber by being frictionally fitted into a groove at the bottom of the fluid chamber, and the gasket remains attached to the fluid chamber when the fluid chamber is removed from the solid substrate. **Claim 31** The device according to claim 29, wherein the gasket has a circular, rectangular or square cross-section and is fixed to the fluid chamber by being attached to the bottom of the fluid chamber using an adhesive, and the gasket remains attached to the fluid chamber when the fluid chamber is removed from the solid substrate. **Claim 32** The device according to claim 28, wherein the gasket is fixed to the surface of the solid support, and the gasket remains attached to the surface of the solid support when the fluid chamber is removed from the solid support. **Claim 33** The gasket is not fixed to the fluid chamber, and the gasket remains on the surface of the solid substrate when the fluid chamber is removed from the surface of the solid substrate. The device according to claim 28, wherein the gasket includes an outwardly projecting tab configured to be gripped by a user to remove the gasket from the surface of the solid substrate. **Claim 34** The gasket is a) After centrifugation and after liquid removal, a residual amount of fluid remains to completely cover the sedimented target cells and prevent them from drying, and b) The volume defined by the upper part of the gasket and the surface of the solid support provides a sufficient volume for the reagents used in cell post-treatment, The device according to claim 28, having a selected thickness.

35. The region of the surface of the solid substrate not exposed to the cell solution during cell sedimentation is functionalized with a coating of a hydrophobic material different from the functionalized surface region, When the solid support with the functionalized region is removed from the device and the retained cell region is exposed to the small amount of fluid in the functionalized region, the target cells are retained by the hydrophobic coating surrounding the region where the target cells are present, the device according to any one of claims 1 to 34.

36. A method for optimal retention of target cells attached to at least a part of the surface of a solid support and for the retention and post-treatment of target cells on the solid support in a fluid medium in the preserved form of the target cells, comprising: subjecting the fluid medium to centrifugation to induce sedimentation of the target cells onto the surface of the solid support; removing the sedimented fluid medium after sedimentation, characterized by a controlled rate of fluid medium removal so that the sedimented target cells are not detached from the surface by fluid flow in the vicinity of the sedimented cells; ensuring control of the amount of residual fluid at all stages of cell retention and post-treatment, wherein the sedimented target cells are substantially unchanged in form from the form when the form of the target cells is freely floating in the fluid medium.

37. The method according to claim 36, wherein the flow rate is controlled by the porosity of the material from which the absorption element is made, the area of the openings in the housing of the absorption element, and the cross-sectional area of the absorption element.

38. At least a part of the surface of the solid support on which the target cells sediment is of a selected size defined by the opening of the fluid chamber in which the fluid containing the target cells is placed for centrifugation, The surface with the selected size region is functionalized with a selected agent to change the interaction between the target cells and the surface to assist in the attachment of the target cells to the upper surface of the solid support, the method according to claim 36 or 37.

39. The method according to claim 38, wherein the agent is selected from the group consisting of poly-L-lysine, silane coating, gelatin, fibronectin, and gold or silver coating.

40. The method according to claim 38, wherein the agent is selected to provide differential binding affinity to target cells and another cell, such that the target cells are more readily bound to the surface, while the binding of the other cells to the upper surface of the solid support is less efficient.

41. The parameters of the absorption element and the absorption element housing are selected such that the shear force associated with the fluid removal rate is low enough so that detachment of the target cells is minimized, while the same shear force is sufficient for the removal of the other cells, resulting in the enrichment of the cells of interest, according to any one of claims 36 to 40.

42. further comprising post-treating the target cells attached to the surface of the solid support by continuous introduction and removal of a fluid reagent into the compartment, and in each step, the flow rate is controlled so that the sedimented cells are not detached and lost, and the remaining amount of fluid in each successive step is controlled to avoid removal of less than a predetermined amount of fluid, such that the morphology of the target cells is not affected by centrifugation in the dry state, according to any one of claims 36 to 41.

43. A method for collecting target cells from a fluid sample, comprising: a) placing the fluid sample in a fluid chamber configured to be removably held within a centrifuge, the fluid chamber being in fluid communication with the surface of a solid support, having a sealed connection between the fluid chamber and the solid support, sealing the fluid chamber with a first cap, and the solid support being removable from the fluid chamber; b) removably attaching the fluid chamber with the solid support attached thereto to a centrifuge, centrifuging the fluid sample to induce sedimentation of the target cells and promote cell attachment to the surface of the solid support; c) removing the device from the centrifuge; d) removing the first cap, inserting a fluid absorption element such that the tip of the fluid absorption element is spaced a predetermined distance from the surface of the solid support, and sealing the fluid chamber using a second cap configured to fit the fluid absorption element. e) the fluid absorption element has a cross-sectional area, a distribution along the axis of the fluid absorption element, and a porosity within a range that provides a controlled flow rate of fluid from the fluid chamber; the tip of the fluid absorption element is provided at a predetermined distance from the surface of the solid support, so that target cells fixed on the solid support are not detached from the surface of the solid support by the flow rate of the fluid absorbed by the fluid absorption element; f) controlling the remaining amount of liquid to avoid removal of fluid less than a predetermined volume, so that the target cells remain in a small amount of fluid and the morphology of the target cells is not affected by centrifugation in a dry state; a method including the step of **Claim 44** The method according to claim 43, wherein the controlled flow rate is controlled by the porosity of the material from which the absorption element is fabricated, the area of the opening in the housing of the absorption element, and the cross-sectional area of the absorption element. **Claim 45** The region on the surface of the solid support where the target cells settle is a selected size defined by an opening in the fluid chamber through which the target cells pass during settlement; The surface with the region of the selected size is functionalized with a selected agent to alter the interaction between the target cells and the surface to assist in the attachment of the target cells to the upper surface of the solid support, according to the method of claim 43 or 44. **Claim 46** The method according to claim 45, wherein the agent is selected from the group consisting of poly-L-lysine, a silane coating, gelatin, fibronectin, a gold or silver coating. **Claim 47** The method according to claim 45, wherein the agent is selected to provide differential binding affinity to the target cells and another cell, so that the target cells are bound to the surface while the other cells are not bound to the surface of the upper surface of the solid support. **Claim 48** The parameters of the absorption element and the absorption element housing are selected such that the shear force associated with the fluid removal rate is low enough that detachment of the target cells does not occur, while the same shear force is sufficient for the removal of other cells, resulting in enrichment of the target cells, according to the method of any one of claims 43 to 47. **Claim 49** further comprising post-treatment of the target cells attached to the surface of the solid substrate by continuous introduction and removal of a fluid reagent into the fluid chamber; And, in each step, the flow rate is controlled so that the sedimented cells do not detach and are not lost, and the remaining amount of fluid in each successive step is controlled to avoid removal of less than a predetermined amount of fluid, such that the morphology of the target cells is not affected by centrifugation in the dry state, the method according to any one of claims 43 to 48.

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