Cell recovery method and device

Abstract

The present disclosure provides a cell acquisition method and apparatus for efficiently settling and retaining cells from a liquid sample onto a solid support with low cell loss and low cellular morphology impact. The device has two configurations, one for use in a centrifuge to centrifuge a sample and the second for controlled release of liquid after centrifugation. The device comprises a base for holding a solid support, the solid support receiving cells on a top surface thereof, the base releasably holding a fluid chamber, the fluid chamber having a first opening, the first opening having a washer surrounding the opening, and the washer bearing against the top surface of the support. The opening receives a liquid sample, and the gasket defines an area into which target cells are deposited. The first cap closes the second opening during centrifugation. After centrifugation, the first cap is removed and a fluid absorbing element and cap are inserted into the fluid chamber, the fluid absorbing element and cap being designed to provide controlled removal of the liquid and to prevent the tip of the absorbing element from being positioned shorter than a predetermined distance from the solid support.

Description

[0001] field

[0002] The present disclosure relates to a cell recovery method and apparatus for efficiently sedimenting and retaining cells from a liquid sample onto a solid support with minimal cell loss and low impact on cell morphology.

[0003] background

[0004] There are many methods / devices for separating cells from liquid samples. Many involve the use of cytocentrifugation, which accelerates the sedimentation of cells to a desired collection point. The disadvantages of many of these methods / devices include significant cell loss and altered cell morphology, which may be detrimental to subsequent analysis.

[0005] In clinical applications, a variety of different fluid samples are routinely analyzed for the presence and characteristics of cells. These include blood, urine, cerebrospinal fluid, synovial fluid, and ascites. In the blood, a group of so-called "circulating rare cells" have been identified in some patients to represent cells that have disseminated from the tissue of their origin. The most popular type of circulating rare cells is the circulating tumor cell (CTC), which, if present, can provide important information about disease status and prognosis.

[0006] Efficient cell recovery in samples containing rare cells is important because even moderate cell losses during sample processing can lead to incorrect counts or false-negative results, where samples containing rare cells may be erroneously labeled as absent. A number of techniques have recently become available to enrich circulating rare cells from blood.

[0007] A variety of methods are used to extract rare cells (cell recovery) from patient samples, which usually produces fluid samples containing cells of interest combined with other cells (e.g., leukocytes). The cells obtained are important to researchers, and in most cases, these cells are subjected to microscopic studies and continuous molecular analysis. In order to make this analysis possible, the cells present in the sample should be precipitated (deposited) on the surface of a solid support that allows these cells to be examined under a microscope. Typically, the cells obtained are precipitated on a microscope slide, which can be a glass or plastic slide. One of the basic requirements of this process is to prevent cell loss. When the user works with rare cells, this becomes particularly important: in this case, the sample may contain very few such cells, and even the loss of a small amount of cells may make continuous analysis unreliable or impossible. The second major requirement is that the process of cell retention should cause minimal cell morphological changes to help cytological analysis.

[0008] For example, U.S. Pat. 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 conjunction with a filter card that is placed or ultrasonically welded between the microscope surface and the sample chamber. This method of fluid removal has the risk of accidental loss of sample to the surrounding absorbent filter card medium.

[0009] US Patent No. 7,628,955 discloses a chamber for fluid samples that can be sealed to the surface of a microscope slide using an integrally molded elastomeric gasket and oil film component. The device does not provide a means to remove liquid in a controlled manner to prevent removal of settled cells from the slide.

[0010] U.S. Patent No. 4,696,743 discloses a filter card placed between a sample chamber and a sediment receiving surface such as a microscope slide. Solids present in a fluid suspension are centrifuged onto the slide surface, while the surrounding fluid is drawn into an adjacent absorptive medium. This device fails to address the problem of cell loss by separating the fluid removal step from the centrifugation / sedimentation step.

[0011] U.S. Patent Publication No. 2003 / 0175850 discloses a method for enriching cells of interest from a suspension by selectively labeling the cells with biotin-conjugated antibodies and then binding them to the surface of a slide coated with an avidin / streptavidin substrate. The absorptive medium wicks away any unbound material or fluid, leaving a dry slide containing the sample of interest. The device does not account for the turbulence and shear stress generated by the lateral flow of liquid into the medium at an uncontrolled rate. While this confers the ability to remove unbound cells in suspension, it also leaves the risk of accidentally removing slide-bound target cells.

[0012] U.S. Patent No. 5,419,279 discloses a flanged hollow tube for sample deposition that engages a corresponding base to securely seal the tube to a microscope slide for staining purposes. The bottom of the tube features a groove so that an O-ring can be fitted between the interface of the slide and the tube to prevent leakage. The device relies on binding forces between the functionalized slide surface and the cells rather than centrifugal forces, in addition to functionalization to facilitate capture of target cells, and further, does not address the removal of supernatant throughout the cell sedimentation / binding step, which may provide a pathway for cell loss due to interference from the surrounding liquid medium.

[0013] U.S. Patent No. 5,480,484 discloses a fluid chamber that is centrally sealed (relative to a microscope slide) to a supportive backing plate by an elastomeric gasket and a metal clip. The number of fluid chambers can be varied to accommodate one or more samples, although the maximum permissible sample volume varies. The assembled centrifugal device floats freely within a specified rotor and deposits cells from a suspension onto a slide surface when centrifugal force is applied. The device does not describe a convenient and controlled method for removing supernatant. Instead, the chamber has a relatively narrow opening that is not inherently suitable for removal of fluids.

[0014] 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. Fig. 9 The invention relates to a device for preparing a liquid absorbing device for use in a rotor. The device is shown in Figure 2 and is placed in a separate compartment in the rotor, which protrudes into the same chamber so that the end of the plug contacts the surface of the slide. When centrifugal force is applied to the rotor, the particles suspended in the liquid are deposited on the surface of the slide. According to the patent, since the centrifugal force is high enough to overcome the capillary force, the absorption plug does not absorb the supernatant until the rotor slows down. On the other hand, the absorption element 3 is introduced into the system before centrifugation, and the absorption starts immediately and lasts until the centrifugal force exceeds the capillary force. This may lead to the loss of cells present in the solution. Although the concept of dividing the process of cell sedimentation and supernatant removal into mutually exclusive steps is similar to our device, there are key differences in the method of liquid absorption, which distinguishes the two designs. One of the main features of this prior art is that the absorption plug protrudes into the same chamber where the fluid containing particles is deposited. Although capillary action may not act on the fluid during centrifugation, it is still possible to cause the cell loss in the absorption plug only by the physical entanglement of the cells in the porous material, thereby causing cell loss. In addition, the absorption plug is specified as being in physical contact with the deposition surface when placed in the rotor. This close proximity and lack of control over the flow of fluid into the absorbent material may result in the unintended absorption of cells that are weakly adhered to the deposition surface.

[0015] U.S. Patent No. 4,344,562 discloses a device utilizing a conventional glass slide that can be locked into a housing by means of a holder. A portion of the housing is a reservoir in which a fluid sample can be received, the reservoir being closed at its bottom by the glass slide when the glass slide has been locked into the housing. The patent is intended to centrifuge small fluid samples without any cell loss, improving the accuracy of medical diagnosis and treatment. Rubber O-rings are used to provide a fluid seal between components.

[0016] Overview

[0017] The present disclosure provides a cell recovery device for settling and retaining target cells from a fluid sample. The device includes a base configured to releasably hold a solid support, wherein the solid support is configured to receive cells on the top surface of the solid support. The device includes a fluid chamber having a first opening, the first opening having a circumferential gasket around a bottom opening, wherein the base is configured to releasably hold the fluid chamber, while the gasket abuts against the top surface of the solid support to form a liquid-tight seal between the top surface and the solid support, and the fluid chamber has a second opening for receiving a liquid sample containing the acquired target cells. The gasket defines an area of ​​a selected size in which the target cells are deposited on the top surface of the solid support. The device includes a first removable cap, which is configured to close the second opening during centrifugation. The device also includes a fluid absorption element and a housing configured to receive an absorption element, and the fluid chamber is configured to receive the housing after centrifugation. The housing has a bottom opening, and when the fluid from the fluid chamber is introduced into the fluid chamber after centrifugation, it can be absorbed by the absorption element through the bottom opening. The fluid chamber includes a second removable cap configured to close the second opening and prevent positioning of the bottom end of the absorbent element less than a predetermined distance from the solid support. The cell acquisition device is configured to be received and releasably retained in a centrifuge.

[0018] The fluid-absorbing element can have a cross-sectional area, distribution along the axis of the absorbent element, and porosity within a certain range to provide control over the rate of absorption of the fluid and the end of the fluid-absorbing element is located at a certain distance from the surface of the solid support so that target cells deposited on the solid support are not detached from the surface of the solid support due to the flow of the fluid being absorbed by the absorbent element.

[0019] The device absorbent element may have a porosity in the range of about 1 micron to about 100 microns, alternatively, the porosity may be in the range of about 5 microns to about 50 microns, or alternatively, the porosity may be in the range of about 10 microns to about 20 microns. The end of the absorbent element is a predetermined distance above the surface of the solid substrate in the range of about 0.1 mm to about 3 mm.

[0020] The device is configured to be releasably retained in a centrifuge such that an axis normal to the surface of the solid support and preferably passing through the center of the area allocated for cell sedimentation intersects the axis of rotation and the long axis of the solid support is in the plane of rotation; or

[0021] An axis normal to the surface of the solid support intersects the axis of rotation, and the long axis of the solid support is perpendicular to the plane of rotation.

[0022] The region of selected size where the target cells are deposited on the top surface of the solid support can be functionalized with a treatment agent selected to change the interaction between the target cells and the surface so as to help the target cells adhere to the top surface of the solid support. These treatment agents can be selected from the group consisting of poly-L-lysine, silane coatings, gelatin, fibronectin, gold or silver coatings. For example, a slide coated with poly-L-lysine (Merck, USA) can be used.

[0023] These treatments are selected to provide different binding forces to the target cells and any other interfering cells, such that the target cells bind to the surface while any interfering cells do not bind to the top surface of the solid support.

[0024] The device absorbent element may be made of porous plastic, and the porous plastic may be coated with a hydrophilic coating. The device absorbent element may be made of non-porous absorbent material, such as paper tissue, cotton or other fibers that absorb fluid.

[0025] The porous plastic may be porous polyethylene coated with a hydrophilic coating.

[0026] The end of the absorbent material spaced apart from the top surface of the solid support is positioned at a distance in the range of about 0.3 mm to about 3 mm from the surface of the solid substrate.

[0027] The porosity and opening size of the fluid absorbent element are selected to provide a selected flow rate.

[0028] The selected flow rate provides a fluid force applied to target cells deposited on the surface that is below a detachment threshold level at which the target cells detach from the surface of the solid support.

[0029] The solid support can be a microscope slide.

[0030] The first removable cap may include a gasket to provide a fluid-tight seal during centrifugation.

[0031] Areas on the surface of the solid support outside of the area of ​​selected size where target cells deposit onto the top surface of the solid support can be coated with a protective coating to prevent accidental spilled fluid from adhering to the surface of the support.

[0032] The housing configured to receive the fluid absorbent element includes overflow features to prevent overflow of fluid caused by the insertion of the fluid absorbent element into the fluid chamber. These overflow features may include at least one opening in the upper portion of the absorbent element housing, and the opening may have any of a square, circular or slot shape.

[0033] The present disclosure provides a method for retaining target cells present in a fluid medium on a solid support and post-processing, the method having an optimized retention rate for target cells at least a portion of which adhere to the surface of the solid support and retaining the morphology of the target cells. The method includes subjecting the fluid medium to centrifugation to induce target cells to settle on the surface of the solid support. After sedimentation, the fluid medium is removed, and it is characterized in that the removal rate of the controlled fluid medium is such that the target cells that settle on the surface do not detach from the surface by the fluid flow near the settled cells. The volume of the residual fluid is controlled in all stages of cell retention and post-processing. The target cells that settle are characterized in that the morphology of the target cells is substantially unchanged from their morphology when they float freely in the fluid medium.

[0034] The flow rate is controlled by the porosity of the material from which the absorbent element is made, the area of ​​the opening in the housing of the absorbent element, and the cross-section of the absorbent element.

[0035] At least a portion of the surface of the solid support on which the target cells are deposited has a selected size defined by the size of an opening in a fluid chamber in which a fluid containing the target cells is placed for centrifugation. The area of ​​the selected size can be functionalized with a treatment selected to alter the interaction between the target cells and the surface to aid in the adhesion of the target cells to the top surface of the solid support.

[0036] These treatments can be selected from the group consisting of poly-L-lysine, silane coatings, gelatin, fibronectin, gold or silver coatings. These treatments are selected to provide different binding forces to target cells and other cells, making it easier for target cells to bind to the surface while other cells bind less effectively to the top surface of the solid support.

[0037] The parameters of the absorbent element and absorbent element housing are selected so that the shear forces associated with the fluid removal rate are low enough that detachment of target cells is minimal, but the same shear forces are sufficient to remove other cells not of interest, thereby resulting in enrichment of the target cells of interest.

[0038] A further understanding of the functionality and advantages of the present disclosure may be realized by referring to the following detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Embodiments of the cell acquisition device disclosed herein will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0041] Figure 1 is a flow chart of the cell retention method disclosed herein.

[0042] FIG. 2( a ) is a perspective view of an assembled cell retention device 40 for cell sedimentation, wherein after centrifugation, cell loss is reduced (retention is increased) and the morphology of retained cells is improved.

[0043] FIG. 2( b ) shows an assembled cell retention device configured for centrifugation, ready for positioning in a centrifuge (not shown).

[0044] Figure 2(c) shows the assembled cell retention device positioned in a centrifuge (not shown) such that an axis 41 normal to the surface of the solid support 42 intersects the axis of rotation and the long axis of the solid support 42 is in the plane of rotation.

[0045] Figure 2(d) shows the assembled cell retention device positioned in a centrifuge (not shown) such that an axis 41 normal to the surface of the solid support 42 intersects the axis of rotation and the long axis of the solid support 42 is perpendicular to the plane of rotation.

[0046] Figure 3 is an exploded view of a cell retention device for cell sedimentation that reduces cell loss (increases retention) and improves the morphology of retained cells.

[0047] Figure 4(a) is a perspective view of an absorbent element 46 in a plastic housing 48 forming part of a cell retention device.

[0048] FIG. 4( b ) is a front view 46 of the absorbent element 46 of FIG. 4( a ) within the plastic housing 48 .

[0049] FIG. 5( a ) is a perspective view showing the end of the plastic housing 48 of the absorption element 46 having the rectangular opening 8 .

[0050] FIG. 5( b ) is a perspective view showing the end of the plastic housing 48 of the absorbent element 46 having a circular cross-sectional opening 8 a .

[0051] Figure 6(a) to Figure 6(e) Various embodiments of controlling the wicking rate during the fluid removal process after cell sedimentation is completed according to the proposed method are shown, wherein

[0052] FIG6( a ) shows that the fluid removal rate can be controlled by selecting the cross-sectional area of ​​the opening 8 in the plastic housing 48;

[0053] FIG. 6( b ) shows how this result can be achieved by selecting the distance between the plastic housing 48 containing and absorbing element 46 (not shown in FIG. 6 ); the above result can also be achieved by a combination of the above cross-sectional areas and distances;

[0054] FIG6( c ) shows the control of fluid flow by selecting the geometry of the absorbent element 46;

[0055] Figure 6(d) shows the control of fluid flow by an additional capillary 14 attached to the porous material 15 and the selection of the capillary length and inner diameter; and

[0056] FIG. 6( e ) shows a capillary 14 filled with a porous material having a gradually increasing cross-sectional area.

[0057] Figure 7(a) to Figure 7(e) There is shown in a stepwise manner further components forming an absorbent element as part of the device of the invention, including a flat tip 11, a cylindrical rod 82 and a hollow cylindrical rod 84, which allow increasing the wicking capacity of the absorbent element, wherein:

[0058] FIG. 7( a ) shows the end 46 of the absorbent element formed into a flat porous member 11 , which is placed adjacent to the solid support 42 ;

[0059] FIG. 7( b ) shows an exploded view of an absorbent element 46 comprising two porous components 11 and 82 ;

[0060] FIG. 7( c ) shows the assembled multi-component absorbent element 46 , which comprises three parts: the flat porous component 11 , the cylindrical porous part 82 , and the hollow cylindrical porous part 84 ;

[0061] FIG. 7( d ) shows a plastic housing for an absorbent element 46 (not shown in this figure) comprising a wicking chamber 48 and a cap 50 having a pleated embossed surface 51 ; and

[0062] FIG. 7( e ) shows an exploded view of the packaged multi-component absorbent element 90 .

[0063] FIG. 8( a ) shows the end of the absorbent element 46 in the housing 48 and a sealing gasket 54 attached to the surface of the substrate 42 , the gasket 54 forming a cavity for the reagent.

[0064] FIG. 8( b ) shows another embodiment of a gasket 60 in which a protruding element 62 is added to the gasket 54 .

[0065] Figure 8(c) to Figure 8(f) 8( c ) and 8( d ) and the other embodiment is shown in FIGS. 8( e ) and 8( f ) , respectively.

[0066] Fig. 9is a view of the absorbent element housing 48 showing the overflow protection feature 10 .

[0067] Figure 10(a) shows a first embodiment of an absorbent material shell 48 having a flow accelerating feature 17, which is a circular hole, for increasing the wicking rate without affecting cell loss.

[0068] Figure 10(b) shows a second embodiment of an absorbent material housing 48 having flow acceleration features 17a which are elongated slots.

[0069] Figure 11 (a) shows a perspective view of a solid support 42 in the form of a glass slide, in which an area of ​​the surface is functionalized with a fluid chamber 44, wherein the fluid chamber 44 is removed from the gasket 54 surrounding the functionalized area.

[0070] FIG. 11( b ) shows the reattached fluid chamber 44 .

[0071] FIG. 12( a ) is a perspective view showing the cell holding device partially disassembled at the stage of introducing a fluid into the fluid chamber 44 .

[0072] FIG. 12( b ) is a perspective view showing the cell holding device assembled with the screw cap 68 after the introduction of a fluid sample containing cells to be acquired.

[0073] FIG. 12( c ) is a perspective view showing a lower side view of the nut 68 having an O-ring 70 for leakage elimination during centrifugation.

[0074] Fig.13 The base 52 of the cell holding device is shown with an aperture 80 for visualization of the fluid and cells without detaching the fluid chamber 44 .

[0075] Fig.14 A solid support 42 of a cell retention device is shown having a section 86 of a surface functionalized for capturing cells and a polytetrafluoroethylene protective layer 82 surrounding the functionalized area 86 and area 84 for labeling.

[0076] FIG. 15( a ) shows a molded one-component absorbent element 46 having a conical tip 25 with a circular cross-section 28 .

[0077] FIG. 15( b ) shows a molded one-part absorbent element 46 having a flattened end with a triangular shape 27 and a rectangular cross-section 29 .

[0078] Figure 16(a) to Figure 16(d) is a photograph showing an exemplary embodiment of a cell recovery device 40 of the present invention, having a glued fluid chamber 44, wherein:

[0079] Figures 16(a) and 16(b) show another embodiment of the proposed device after the absorbent element is detached from the assembly (Figure 16(a)) and after the assembled device when the absorbent element is in contact with the fluid (Figure 16(b)); and

[0080] Figures 16(c) and 16(d) show another embodiment of the proposed device, where the fluid chamber 44 is glued directly to the surface of the slide 42. Figure 16(c) shows the cap 68 attached to the fluid chamber 44 before the centrifugation step of the process. Figure 16(d) shows the encapsulated absorbent element 46 made of absorbent tissue inserted into the fluid chamber 44 for fluid removal after the centrifugation stage is completed.

[0081] Figure 17(a) to Figure 17(f) are perspective views showing different methods of holding portions of components of a cell holding device in various examples.

[0082] FIG. 17( g ) is a bottom view showing the alignment of the slide 42 with the functionalized surface area 86 in the assembled device with the bottom opening 80 in the base 52 .

[0083] FIG. 17( h ) is a bottom view of the base 52 showing the alignment of the slide 42 with the functionalized surface area 86 in the assembled cell holding device with bottom openings in the base 52 and the fluid chamber 44 .

[0084] Figure 18(a) to Figure 18(f) A series of figures are shown which illustrate the importance of correct alignment of the cell retention device axis relative to the rotation axis of the centrifuge, wherein:

[0085] FIG. 18( a ) shows a schematic diagram of a fluid chamber 44 with cells in a centrifuge and the axis of the device offset from the axis of the centrifuge;

[0086] FIG. 18( b ) shows the force distribution during centrifugation.

[0087] Figure 18(c) shows the resulting uneven distribution of settled cells;

[0088] Figure 18(d) shows a graph of the calculated displacement of the cell from the original position in a direction perpendicular to the axis of rotation as the initial distance between the cell and the axis of the device varies for different displacements of the device axis relative to the axis of rotation of the centrifuge of 10 mm;

[0089] FIG. 18( e ) shows the same situation as FIG. 18 for a displacement of 0.75 mm; and

[0090] Figure 18(f) shows the same situation for a displacement of 0 mm.

[0091] Fig.19 A front view of an example of an absorption element 46 is shown, which includes two porous components 11 and 82 and a plastic housing for the absorption element.

[0092] Fig. 20 is a graph of percent cell loss versus wicking rate for two different centrifugation speeds, 4400 rpm (circles) and 2200 rpm (triangles).

[0093] Figure 21 (a) shows the percentage of cell loss as a function of the distance between the surface of a solid support 42 and an absorptive element 46 forming part of a cell retention system of the present invention, wherein no KCl is added.

[0094] Figure 21(b) shows the percentage of cell loss as a function of the distance between the surface of a solid support 42 and an absorptive element 46 forming part of the cell retention system of the present invention, and wherein KCl is added to the solution.

[0095] Figure 22(a) shows the wicking rate as a function of time for polypropylene porous material U200 (Pore Technology Inc.), and the distance between the surface of the solid support 42 and the end of the absorbent element 46 is about 2 mm.

[0096] Figure 22(b) shows the wicking rate as a function of time for polypropylene porous material U201 (Pore Technology Inc.), and the distance between the surface of the solid support 42 and the end of the absorbent element 46 is about 2 mm.

[0097] Figure 23(a) is a photomicrograph of DAPI stained cells incorporated into fluid chamber 44 and sedimented by centrifugation, and shows cells remaining on the slide surface after centrifugation with continuous fluid removal according to the methods of the present invention.

[0098] FIG. 23( b ) is an enlarged image (X100) of the micrograph of FIG. 23( a ).

[0099] Fig.24 The cell capture efficiency percentages of the two cell capture methods are shown: , and the cell retention methods and devices of the present invention shown in Dataset 2.

[0100] Figure 25(a) shows the use of Micrographs of captured cells.

[0101] FIG. 25( b ) shows a micrograph of cells captured using the cell recovery device 40 of the cell retention device of the present invention, which clearly shows that the method of the present invention retains the morphology of the acquired cells.

[0102] Figure 26(a) shows a micrograph showing the morphology of SK-BR-3 cells fixed in the absence of fluid surrounding them (referred to as dry fixation), wherein the image was obtained using 400x magnification and staining with CK-FITC conjugate.

[0103] Figure 26(b) shows a micrograph showing the morphology of SK-BR-3 cells fixed in the presence of a controlled volume of fluid (wet fixation), wherein the image was obtained using 400x magnification and staining with CK-FITC conjugate.

[0104] FIG. 27( a ) shows the use of Streck Biomedica mBC patient samples, Micrograph of retained circulating tumor cells (CT).

[0105] Figure 27(b) shows a micrograph of circulating tumor cells (CT) retained by the cell retention device of the present invention using a Streck Biomedica mBC patient sample, showing that the morphology of the settled cells is significantly better when using the methods disclosed herein.

[0106] Fig.28 A bar graph showing the capture efficiency of EDTA HNV blood spiked with pre-labeled SK-BR-3 cells that were captured on a cell recovery device 40 or Cytoslide™ of the cell retention device of the present invention. Separation, pooling, capture, fixation, and then staining. The experiment was performed by multiple trained users across multiple instruments / multiple days. The effects of pipetting variability were reduced (compare and STD of the cell recovery device 40).

[0107] Fig.29 The HyCEAD plotted for the following TM / Histogram of signal intensity: HyCEAD for continuous molecular analysis of retained cells TM Negative and positive controls for the operation, negative controls for the complete process of sample retention and sequential molecular analysis, and two modes of cell retention for cells containing samples for sequential molecular analysis.

[0108] Fig.30 The picking of SK-BR-3 cells deposited onto a solid support 42 with a controlled volume of residual fluid according to the proposed method is shown. The upper figure shows the retained cells with the capillary of the cell picking tool placed over the cells; the middle figure shows the cells being removed from their initial location by the cell picking tool, and the lower figure shows the cells being dispensed (repositioned) by the cell picking tool.

[0109] Fig.31 It shows that the Fig.31 % cell recovery at different stages of the procedure for the various protocols indicated.

[0110] Fig.32 The percentage of cell recovery for multiple consecutive wicking steps (a, b, c) is shown, wherein 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, and wherein the test is repeated three times, demonstrating that if the wicking rate is appropriately selected according to the method of the present invention, multiple wicking events do not result in cell loss.

[0111] Fig.33 is a photomicrograph of leukocytes that were sedimented and stained with DAPI using the methods disclosed herein, wherein the photo was taken with the fluid chamber 44 still attached to the solid support, demonstrating that the cells can be stained with the fluid chamber 44 attached.

[0112] Fig.34 Shown are micrographs (rows A, B) and photographs (row C) of cells that have settled using (Column I), solid support of a cell recovery device forming part of a cell harvesting device of the present invention (Columns I and II) where residual fluid is not controlled (Column II) and residual fluid volume is controlled according to the methods disclosed herein (Column III). Cell recovery device spiked with HNV cells Data - Cell recovery device using wet fixation method on live EDTA tube cells after staining. The cells presented the worst morphology (column I, rows A and B), with diffuse cytoplasm and decreased fluorescence intensity and increased background. The least amount of morphological changes were observed in the wet-fixed cell recovery device 40 condition (column III). Similar results were observed for CD45 staining, which suggests that The reduced cell density in the culture medium is also worrisome.

[0113] Detailed Description

[0114] Various embodiments and aspects of the present disclosure will be described with reference to the details discussed below. The following description and accompanying drawings are illustrations of the present disclosure and should not be construed as limiting the present disclosure. Many specific details are described to provide a thorough understanding of the various embodiments of the present disclosure. However, in some cases, in order to provide a concise discussion of the embodiments of the present disclosure, well-known or conventional details are not described.

[0115] As used herein, the terms "comprises" and "comprising" should be interpreted as inclusive and open-ended rather than exclusive. Specifically, when used in the specification and claims, the terms "comprises" and "comprising" and variations thereof mean including the specified features, steps or components. These terms should not be interpreted to exclude the presence of other features, steps or components.

[0116] As used herein, the term “exemplary” means “serving as an example, instance, or illustration,” and should not be construed as preferred or advantageous over other configurations disclosed herein.

[0117] As used herein, the terms "about" and "approximately" are intended to encompass variations that may exist in the upper and lower limits of a range of values, such as variations in properties, parameters, and dimensions. In a non-limiting example, the terms "about" and "approximately" mean plus or minus 10% or less.

[0118] Unless defined otherwise, 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.

[0119] As used herein, the phrase "target cells" means cells of interest separated by the device of the present invention. In some cases, if the specific intended use or the following post-processing method does not require selectivity in cell separation, all cells present in the sample may be of interest, and therefore all cells present in the sample will be considered "target cells". Although other cells may be present, so that there is a mixture of different types of cells, only "target cells" are separated or primarily separated.

[0120] As used herein, the phrase "accelerating features" refers to structural elements designed to increase the rate at which fluid is removed from the fluid chamber 44 after centrifugation is complete without loss of cells deposited on the surface of the solid support 42. This allows for a reduction in the time required for sample processing. These accelerating features are located on the housing 48, providing additional fluid connections between the fluid present in the fluid chamber 44 and the absorbing element 46. The accelerating features can be circular, or have the shape of a slot. Other shapes are also possible. The accelerating features are configured so that the opening of the feature is sufficient to increase the flow rate to a desired value, and the accelerating features are located in the housing 48 at a distance from the end of the housing 48 that the additional shear stress applied to the cells on the solid support 42 due to the increase in flow does not cause additional cell loss.

[0121] As used herein, the phrase "overflow protection features" refers to structural elements designed to prevent fluid from overflowing from the fluid chamber 44 when the absorbent element 46 disposed in the housing 48 is inserted into the fluid chamber 44. These overflow protection features are located on the housing 48 to provide additional fluid connections between the fluids present in the fluid chamber 44 in case the volume of the sample is excessive. These overflow protection features prevent turbulence from being generated in the fluid chamber 44, which may cause cells to detach from the surface of the solid support 42.

[0122] The present disclosure describes a device comprising a fluid chamber assembled with a microscope slide (or other substrate) which has been fluid-tightly sealed using an elastomeric gasket. The surface of the slide has been functionalized with a coating that allows for the capture and retention of cells and is held in a polypropylene holder that is clamped to the fluid chamber, thereby physically securing and positioning the chamber relative to the slide. In addition to the assembly containing the microscope slide, it is also characterized by a porous absorptive element contained within a polypropylene cover that improves usability while providing a means by which to control the flow of liquid entering the porous absorptive element. The absorptive element in the polypropylene cover is introduced into the assembly after the centrifugation stage is completed and can be replaced with the same absorptive element and cover if the process of cell post-processing requires multiple steps of fluid introduction and removal.

[0123] This device is intended to be used as a cytocentrifugal device, wherein the cells of interest suspended in the surrounding medium have a higher density than the surrounding medium, so when centrifugal force is applied to the device comprising a cell suspension, the cells of interest with a higher density are preferentially sedimented according to the direction of the centrifugal force. The cytocentrifugal device can be mainly divided into two groups based on its fluid removal method. In the "fluid removal at the same time" system, the absorption medium is located between the fluid chamber and the slide surface, which allows the liquid to be removed to the adjacent absorption medium while applying centrifugal force. In theory, before removing liquid, the cells of interest with a higher density must have been sedimented to the slide surface, because if not done so, the cells still suspended in the medium will be sucked into the adjacent absorption element and lost. The feature of another method of cytocentrifugal is the liquid-tight seal between the fluid chamber and the slide surface, which does not allow the removal of liquid during centrifugation. In the cytocentrifugal retained by fluid, the cells of interest are sedimented when surrounded by its supportive liquid medium (supportive liquid medium), and the removal of fluid is carried out after centrifugation. The cells of interest with a relatively higher density in the liquid medium are still preferentially sedimented to the slide surface.

[0124] The cell centrifugation that fluid retains provides many benefits, but also proposes some challenges from the perspective of sample handling.Because the cell is settled in the liquid medium that relative buoyancy (that is, resistance to sedimentation) is provided, rather than completely lacking buoyancy when centrifuged in air, the cell with higher density needs a larger amount of centrifugal force to overcome fluid resistance.The existence of the cell in the liquid medium provides advantages, because the settled cells are subjected to less force that presses them on the slide, helping to retain fine morphological features.There is no simultaneous fluid removal and also provides the benefit of effectively removing all approaches that may lose interested cells during the centrifugation process.According to the principle of liquid-tightness, the fluid comprising interested cells is limited to stay in the room and contact the slide surface-therefore, the cells suspended in the liquid medium are also limited to stay in the room and are deposited on the slide surface.

[0125] The limitation of the centrifugal fluid retention method of cells is that the process of fluid removal and cell sedimentation is separated. Therefore, a separate fluid removal method is needed. Considering that cells do not undergo such a large centrifugal force due to the buoyancy of the surrounding medium, the contact area of ​​cells and slides may not be so large, thereby adversely affecting the adhesion of cells to the slide surface. This requires the process of liquid removal as gently as possible and with as little turbulence as possible. This device and its use method provide a method in a controlled manner that fluid is removed from the sample chamber, and the controlled manner minimizes the detachment of cells from the slide surface, thereby maximizing the yield of the interested cells of precipitation.

[0126] Upon removal of the liquid, the cells settle in a designated area of ​​the slide within the sample chamber. It is beneficial to make the sample chamber removable in order to aid in further downstream processing where a variety of reagents may be used to incubate or wash the cells, or where it is necessary to image the settled cells with subsequent post-processing. The device of the present invention comprises a polymer base with protruding hooks that clip into corresponding positions on the sample chamber located on top of the slide. The clips are designed to be easy to assemble, but also provide a convenient means by which the end user can remove the chamber without the use of specialized tools or methods.

[0127] To facilitate manipulation of reagents in downstream processes such as immunofluorescence staining and fluorescence in-situ hybridization (where minimizing fluid volumes to reduce assay costs is optimal), the device can include hydrophobic printing on surfaces other than exposed bioadhesive areas. The hydrophobicity of the printed surface serves as a convenient reagent well that can accommodate small volumes of liquid (wells have a capacity of 10 μL to 200 μL), but by retaining the liquid in a controlled area, cells of interest are also confined to designated areas, allowing for tight localization of the sample and minimizing losses due to downstream processes.

[0128] In order to maximize the ability of the cells of interest to adhere to the slide surface and resist detachment, the slide surface is functionalized with a bioadhesive coating to modify the interaction of the settled cells with the surface of the slide. The design of the device in which the slide is held in a polymer base allows the device to be compatible with almost any microscope slide assembled in the base, and the polymer base is clamped together with a chamber sealed on the slide via a gasket. Therefore, many functionalized slides were tested in the device and their cell adhesion properties were evaluated. Since the slide can be easily detached from the rest of the device components, this allows the device to be compatible with almost any microscope, including upright and inverted microscopes. In addition, slides with settled cells are compatible with other types of standard equipment such as, for example, slide staining devices. Using CellTracker TMGreen pre-labeled cultured SK-BR-3 cells were incorporated into 1X PBS to produce a cell suspension and added to the sample chamber. After centrifugation, the supernatant was removed, and the cells were fixed and then detached from the sample chamber by incubating in ice-cold methanol for 5 minutes. The resulting slide containing the settled cells was washed 3 times by running 1X PBS on the sample surface using a micropipette to induce cell detachment. The subsequent slide surface was imaged and cell density was evaluated under FITC. As a result, poly-L-lysine showed the highest cell density. Therefore, this surface modification was selected as the main slide surface of the device of the present invention. Other types of coatings can replace poly-L-lysine for surface functionalization, such as proteins, silanes, organic polymers, inorganic metals. For example, other forms of polylysine, silane coatings, gelatin, fibronectin, gold or silver coatings and other coatings that enhance the adhesion of cells to solid supports. Other methods that enhance the adhesion of cells to the surface of solid supports can be used, such as surface structure modification, etching, plasma treatment.

[0129] Different materials can be used as solid supports, such as glass, different types of plastics, or quartz.

[0130] Reference Figure 1 and Figure 2(a), Figure 1 A flow chart of a cell retention method using a cell acquisition device disclosed herein is shown, indicated by 100. In step 12, a fluid sample having cells being separated is introduced into a fluid chamber 44 connected to a solid support 42 (Fig. 2 (b)), both of which form a part of the device described below. In step 14, the fluid sample is then subjected to centrifugation, which forces the cells to move toward the solid support 42, which induces the sedimentation of the cells and promotes the adhesion of the cells to the solid support. After centrifugation, in step 16, the fluid is removed under controlled conditions by carefully controlling the fluid removal rate in step 18 and actively controlling the remaining fluid in step 20. Careful control of the fluid is advantageous because it prevents cells from detaching from the solid support due to excessive forces that may occur when the flow rate is high and when the stress level exceeds the adhesion force that keeps the cells on the surface of the solid support during centrifugation or after centrifugation is completed. In addition, the removal of the fluid after the centrifugation step is completed eliminates the removal of cells that may still be present in the solution (not bound to the surface) if the centrifugation is not completed. In addition, the proposed method requires control of the volume of fluid left in the fluid chamber after the fluid removal step is completed. This is necessary to preserve cell morphology because the cells remain wet during all steps of the process. The volume of the remaining fluid depends on the fluid parameters (surface tension and contact angle) as well as the inner diameter of the fluid chamber 44 and the distance between the ends of the solid support 42 and the absorbent element 46. The volume is essentially controlled by appropriately selecting the above distance between the ends of the absorbent element 46.

[0131] If the cell post-treatment requires multiple steps (e.g., dyeing, washing, etc., as shown in step 24), the addition of reagents is performed at a controlled fluid delivery rate. This allows preventing the detachment and removal of cells that adhered to the solid support in the previous steps of the method. The following fluid removal steps are performed under the control of the above-mentioned controlled fluid removal rate and the remaining fluid volume, which are illustrated in step 26.

[0132] After the preparation step 24 , the prepared cells are then subjected to the desired analysis, whether molecular analysis in step 28 or imaging in step 30 .

[0133] Refer to Figure 2 and Figure 3 , an assembled cell retention device is generally shown at 40, which is used to perform cell sedimentation with reduced cell loss (increased retention rate) and improved morphology of retained cells, and includes a substrate 42, a fluid chamber 44, and includes a wicking cap 50 shown in Figure 2 (a) or a protective cap 68 shown in Figure 2 (b) and an absorbent element 46, which extends downward from the cap 50 into the housing 48 and is in fluid communication with a liquid containing cells placed in the fluid chamber 44, and wherein the absorbent element extends within the absorbent element rod 46, the wicking cap 50, the base 52 and the fluid chamber gasket 54. The absorbent element 46 can be made as a single-piece element, as further shown in Figure 15, or can be composed of multiple parts, as shown in Figures 7 (b), 7 (c) and 7 (e). In the latter case, the parts are placed in contact so that fluid flow between these parts is possible. The absorbent element 46 is assembled with the cap 50 and the housing 48 so that when they are mounted in the fluid chamber 44, the end of the absorbent 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 that once the cells are collected, the substrate 42 can be easily detached. 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.

[0134] The fluid chamber 44 is releasably attached to the base, as will be described below. Different methods of attaching the fluid chamber 44 to the slide or the base that holds the slide can be based on the use of an adhesive ( Figure 16(a) to Figure 16(b) ) or Figure 17(a) to Figure 17(f) , which is achieved by mechanical retention as shown in the figure, using retention feature 30 when the fluid chamber is pressed in, or using retention feature 31 when the top of the element is melted and retention feature 32 is formed; or by hooks 34 and 35 being aligned through rotation to properly retain the fluid chamber 44.

[0135] FIG4( a) is a perspective view of an absorbent element 46 in a plastic housing 48, and FIG4( b) is a front view of the absorbent element 46 in the plastic housing 48 of FIG4( a). The plastic housing 48 may be pressed into the cap 50 or may be held by an adhesive. The plastic housing 48 is provided with an overflow protection feature 10 as shown in FIG6( a). The cap 50 is provided with a riffle 51 on the side surface for ease of use, and a hole 9 on the top of the cap 50 to allow for release of pressure differentials that may be generated during the wicking process.

[0136] Fig. 5(a) is a perspective view of the end of a plastic housing 48 with an absorbent element 46 having a rectangular opening 8 at the end of the housing 48, and Fig. 5(b) shows the end with a circular cross-sectional opening indicated at 8a. Other shapes of openings are possible as long as the cross-sectional area combined with other parameters of the absorbent element 46 provides the desired fluid flow during the wicking phase of operation.

[0137] Figure 6(a) to Figure 6(e) Various embodiments of controlling the wicking rate during the fluid removal process after cell sedimentation is completed according to the proposed method are shown. FIG. 6( a) shows that control of the fluid removal rate can be achieved by selecting the cross-sectional area of ​​the opening 8 in the plastic shell 48, the opening 8 having a rectangular shape as shown in FIG. 5( a). FIG. 6( b) shows how this result can be achieved by selecting the distance between the plastic shell 48 containing the absorbent element 46 and the substrate 42. Note that the above results can also be achieved by a combination of the above cross-sectional areas and distances. FIG. 6( c) shows that the fluid flow rate can be controlled by selecting the geometry of the absorbent element 46.

[0138] Figure 6(d) is a photograph showing that control of fluid flow can be achieved by using additional capillaries 14 attached to the porous material 15 and selecting the capillary length and inner diameter. Figure 6(e) shows a capillary 14 attached to the porous material 15 with a gradually increasing cross section.

[0139] Figure 7(a) to Figure 7(e)More components related to the absorbent element 46 that form part of the device of the present invention are shown step by step. Figure 7(a) shows the end of the absorbent element 46 placed above the functionalized surface of the solid support 42. The figure shows a porous flat component 11 adjacent to the surface for retaining cells. As further described, the shape, type of porous material and the distance between the end of the absorbent element and the solid support 42 define the wicking rate, and all these parameters are optimized to reduce cell losses that may be caused by excessive flow. Figure 7(b) shows an absorbent element 46, which includes a flat absorbent end 11 and a cylindrical porous element 82 above the absorbent element 11 for increasing the wicking capacity (the maximum liquid volume that can be absorbed by the element 46). Other configurations of these components are also possible: for example, the element 11 can be made into a cone with different cross-sections; the element 82, which is called a cylindrical element, can be made with a square or rectangular cross-section.

[0140] Elements 11 and 82 are placed in contact with each other to ensure uninterrupted fluid flow. Therefore, from the perspective of wicking, these parts act as a single combined absorbent element 46. Figure 7 (c) shows that the wicking capacity is additionally increased by a hollow cylindrical porous element 84, into which part 82 is inserted. In a multi-component absorbent element, not only the shape of the elements can be selected, but also their porosity can be selected so that the wicking rate and total volume provide the required performance. Figure 7 (d) shows a plastic shell for an absorbent element 46 comprising a plastic cavity 48 and a cap 50, which allows good contact between the parts of the absorbent element 46 and only allows liquid wicking through a controlled opening 8 (not shown) at the bottom 48 of the plastic cavity. The cap 50 is also equipped with embossing on the side of the cap for ease of use.

[0141] Figure 7(e) shows an exploded view of an encapsulated multi-component absorbent element 90. Another embodiment of an encapsulated absorbent element 90 includes a plastic cavity 48, a cap 50, and a molded single-component porous absorbent element 46, which is further shown in Figures 15(a) and 15(b).

[0142] Fig. 8(a) shows the end of the absorbent element 46 in the housing 48 and the sealing gasket 54 attached to the surface of the solid support 42 or substrate 42, the sealing gasket 54 forming a cavity for the reagent. Fig. 8(b) shows an embodiment of a gasket 60 in which a protruding element 62 is added to the gasket 54. The purpose of the protruding element 62 is to simplify the separation of the gasket 54 from the slide when the operation stage before microscopic imaging is completed.

[0143] The gasket 54 may have a circular, rectangular or square cross section. They may be attached to the bottom of the fluid chamber 44 by gluing it to the bottom of the fluid chamber 44, with the gasket 54 extending circumferentially around the area where the target cells settle. Alternatively, the gasket 54 may be held in a groove complementary to the size and shape of the gasket built into the bottom of the chamber 44. Alternatively, the gasket may be held on the surface of the solid support 42 without being attached to the chamber 44. It should be understood that the gasket may be optional, as there are other ways of forming a liquid-tight seal between the chamber 44 and the solid support 42 known to those skilled in the art.

[0144] Figure 8(c) to Figure 8(f) FIG. 8( c ) is a bottom view of the fluid chamber 44 showing the recess 66 ( FIG. 8( c ) ) having dimensions for receiving the gasket 54 ( FIG. 8( d )). FIG. 8( e ) shows the fluid chamber 44 with an additional recess 68 added to the bottom of the fluid chamber 44 to receive the protruding element 62 of the gasket 54 (where the gasket is absent in FIG. 8( e )). FIG. 8( f ) shows the gasket 54 with its protruding element 62 installed in the fluid chamber 44.

[0145] Once the target cells have settled on the top surface of the solid support 42, the fluid chamber 44 is filled with liquid without cells. At this point, the absorbent element 46 mounted in the housing 48 is inserted into the fluid chamber 44 together with the wicking cap 50 and fixed therein by the leakproof wicking cap 50. The purpose of the absorbent element 46 is to draw liquid from the fluid chamber 44 in such a way that the cells are not damaged, deformed or moved away from the surface of the support 42 during the removal of the liquid. This is achieved primarily by the design parameters of the absorbent 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 absorbent element 46 and its end and the surface of the support 42.

[0146] With respect to the absorbent element 46, the fluid absorbent element is designed to have a cross-sectional area, distribution along the axis of the fluid absorbent element 46, and porosity within a certain range to provide control over the rate at which fluid is absorbed into the absorbent element 46 along the length of the absorbent element 46 and upward through the end spaced from the top surface of the solid support 42, so as to control the removal of liquid from the fluid chamber 44. The mechanical design of the housing 48 is also optimized to aid in the optimal removal of liquid. The first is the design of the housing 48 and the cap 50 so as to keep the end of the absorbent element 46 spaced from the top surface of the solid support 42, thereby positioning the end of the fluid absorbent element at a distance shorter than a predetermined distance from the surface of the solid support 42. The distance at which the end of the absorbent element 46 is positioned from the top surface of the solid substrate 42 on which the target cells have been deposited is important for the following reasons.

[0147] The detachment of the target cells attached to the surface of the solid support 42 is mainly caused by the shear stress derived from the fluid flow. The shear stress on the target cells produced by the fluid flow is defined by the derivative of the fluid velocity to the distance. The absolute value of the velocity is defined by the flow rate of the fluid, and the flow rate of the fluid depends on the design parameters of the selected porous material of the absorption element 46 and the absorption element 46. The increase in the distance between the end of the solid support 42 and the absorption element 46 allows the reduction of shear stress and reduces the loss of target cells. On the other hand, this also slows down the removal of fluid, and can prevent the complete removal of fluid from the fluid chamber 44, which makes the process undesirable or inefficient. The optimization of all the above-mentioned design parameters and the implementation of the flow increase or acceleration features 17 and 17a (shown in Figure 10 (a) and Figure 10 (b)) provide a reduction in the wicking time for removing fluid from the fluid chamber 44 and allow fluid removal with reduced cell loss under an acceptable fluid removal time. The fluid flow rate provided by the accelerating features 17 and 17a shown in Figures 10(a) and 10(b) can be significantly higher than the flow rate provided by the absorbent element 46 through the end 8 of the housing 48, but, by the time the absorbent element is introduced into the fluid chamber 44, there are no cells present in the majority of the fluid and the distance between the feature 17 or 17a and the support 42 is selected to be sufficiently large that there is no cell detachment associated with the fluid flow.

[0148] In addition to the distance from the top surface of the base 46 where the tip is located, the housing 48 may be provided with an overflow protection feature. Fig. 9 A view of an absorbent element housing 48 is shown showing overflow protection features 10 that include openings in the upper wall of the housing 48 to facilitate fluid flow into the housing 48. The protection features are located at the top of the housing 48 and they are very close to the portion of the absorbent element 46 that has a large cross-sectional area. In addition, the cross-sectional area of ​​the overflow features is large and serves to direct the fluid flow to the portion of the absorbent element 46 that has a large volume of porous material. Thus, excess fluid that may initially be present in the fluid chamber 44 will be absorbed and will not overflow the fluid chamber 44. In addition, these features are away from the surface of the solid support 42 where the cells settle during the centrifugation stage. This ensures that the increase in flow due to the overflow features does not cause cells to detach from the surface and does not result in cell loss. Examples of these overflow feature openings in the housing 48 are shown in FIG. Fig. 9 Shown in.

[0149] FIG. 10( a) shows a first embodiment of an absorbent housing fluid chamber 48 having a flow acceleration feature 17 that is an elongated slot. FIG. 10( b) shows a second embodiment of an absorbent housing 48 having a feature 17a that is a circular hole. The presence of feature 17 or 17a provides an increase in wicking rate without a negative impact on cell retention. This is achieved by arranging feature 17 or 17a away from the end of housing 48 and therefore at a great distance from the surface 42 where the cells settle. In order to reduce sample processing time, it may be desirable to increase the wicking rate.

[0150] FIG. 11 (a) shows a perspective view of a solid support 42 in the form of a glass slide, wherein the functionalized area of ​​the surface of the glass slide is surrounded by a gasket 54 while the fluid chamber 44 is removed, and FIG. 11 (b) shows the reattached fluid chamber 44. The ability to remove and reattach the fluid chamber 44 allows visual control at different stages of the process, including microscopy (if necessary). In addition, reattachment of the fluid chamber 44 may be required for post-processing of cells after the microscopy stage is completed.

[0151] Figure 12(a) to Figure 12(c) The cell retention device at the stage of introducing fluid into the fluid chamber 44 is shown, wherein the first nut 68 is removed and the fluid sample flows into the fluid chamber 44, after which the nut 68 is screwed onto the fluid chamber 44. Nut 68 is used in the centrifugal stage of the process to prevent accidental leakage of the liquid containing cells. This is achieved by using a gasket 70 in the form of an O-ring shown in Figure 12 (c), which provides a fluid-tight connection between the fluid chamber 44 and the nut 68. The gasket 70 can have other shapes (e.g., flat), as long as a fluid-tight connection is achieved. As mentioned above, cap 68 is used in the centrifugal stage, and the purpose is to prevent the fluid containing cells from leaking from the fluid chamber 44. It is expected that there is no fluid removal during the centrifugal step, which allows cells to be kept in the fluid, thereby preventing the cell damage present in the device described in other patents or the device existing on the market. After completing centrifugation, cap 68 is replaced with a second nut 50 that accommodates an absorption element 46. At this stage, fluid removal occurs and is removed in a controlled manner due to the appropriate choice of the porosity of the material used to make the absorbent element 46, the absorbent element configuration / cross-section, and the distance between the end of the absorbent element and the surface of the solid support 42. This distance is defined by the design of the housing 48 as well as the design of the cap 50.

[0152] 2(c) and 2(d), the device 40 is configured to be releasably retained in a centrifuge so that the axis 41 intersects the axis of rotation 49 of the centrifuge, the axis 41 is orthogonal to the surface of the solid support 42 and passes through the center of the area where the target cells are deposited on the top surface of the solid support 42. The orientation of the device in the centrifuge can vary depending on the specific centrifuge used, and can be placed in the centrifuge so that the long axis of the solid support 42 is in the plane of rotation (as shown in FIG. 2(b)), or the long axis of the solid support 42 can be perpendicular to the plane of rotation, as shown in FIG. 2(c). Other orientations are also possible due to the axial symmetry of the lumen of the fluid chamber 44.

[0153] Fig.13 The base 52 of the cell holding device is shown with a hole 80 for visualizing the fluid and cells without detaching the fluid chamber 44, and Fig.14 A solid support 42 of a cell retention device is shown having a section 86 of the surface functionalized for capturing cells and a polytetrafluoroethylene protective layer 82 surrounding the functionalized area 86 .

[0154] FIG. 15( a ) shows a molded absorbent element 46 having a conical end 25 with a circular cross-section 28 , and FIG. 15( b ) shows a molded absorbent element 46 having a flat end with a triangular shape 27 and a rectangular cross-section 29 .

[0155] Figure 16(a) to Figure 16(b) Another embodiment of the proposed device is shown, FIG. 16( a ) showing the absorbent element removed from the assembly, and FIG. 16( b ) showing the assembled device when the absorbent element is in contact with the fluid. Figure 16(c) to Figure 16(d) Another embodiment of the proposed device is shown, where the fluid chamber 44 is glued directly to the surface of the slide 42. Figure 16(c) shows the cap 68 attached to the fluid chamber 44 before the centrifugation step of the process. Figure 16(d) shows the encapsulated absorbent element 46 made of absorbent tissue, inserted into the fluid chamber 44 for fluid removal after the centrifugation stage is completed.

[0156] Figure 17(a) to Figure 17(f) Examples of different methods of retaining parts of an assembly are shown. The perspective view in Figure 17(a) shows a base 52 provided with hooks 30 integrally formed with the base 52 along two opposing long edges for retaining the fluid chamber assembly 44. Centrally located in the base 52 is a hole 80. Tabs 37 are located along two opposing long edges of the base 52, which tabs 37 are used for slide retention.

[0157] FIG17( b) shows a base 52 that uses a pin 31 integrally formed therewith instead of the hook 30 in FIG17( a). The pin 31 is heat-pressable so that when the fluid chamber 44 is engaged with the base 52, the feature 32 shown in FIG17( c) (which is produced by pressurizing the pin 31 under heating) prevents the fluid chamber 44 from being separated from the base 52. When the pin 31 is pressurized under heating, a bulge 32 of plastic is produced that is larger than the opening through which the pin protrudes, which holds the fluid chamber 44 in place.

[0158] Figures 17(d) and 17(e) show another embodiment in which the retention of the fluid chamber is achieved by a retainer 33 engaged with a hook 34, which is attached to the fluid chamber 44 when the fluid chamber 44 is rotated. A stopper 35 is added to the hook 34 to prevent the base 52 from being separated from the fluid chamber 44. Figure 17(d) shows the assembly before the fluid chamber 44 is attached to the base 52, and Figure 17(e) shows the assembled device.

[0159] Figure 17(f) shows a fluid chamber assembly 44, which is locked on the base 52 using a retainer 33 and a hook 34 when the fluid chamber 44 is engaged by sliding the fluid chamber 44 in the direction of the long side of the base 52, which is opposite to the embodiment shown in Figure 17(e), in which the engagement is achieved by rotating the fluid chamber 44.

[0160] FIG. 17( g) shows a bottom view of a base 52 with a slide 42 retained on the top surface of the base 52, while the slide 42 includes an area 86 functionalized with a selected treatment agent. Surface functionalization is known to improve cell adhesion to a surface. The selection of a particular type of functionalization allows for increased retention of a particular type of cell, which can also be used as a method for enriching collected cells or increasing the likelihood of removing interfering cells (e.g., leukocytes) from the surface during a series of washing steps following centrifugation.

[0161] Figure 17(h) shows the alignment of all components of the assembled device 40, which is important for uniform sedimentation of the cells and clear visualization of the area of ​​the slide where the cells have settled.

[0162] Figure 18(a) to Figure 18(f)A series of figures are shown inclusively, illustrating the importance of proper alignment of the axis of the cell recovery device 40 relative to the axis of rotation in the centrifuge. Figure 18(a) shows a schematic diagram of the fluid chamber 44 with cells in the centrifuge and the axis of the device offset from the axis of the centrifuge, Figure 18(b) shows the force distribution, and Figure 18(c) shows the resulting uneven distribution of settled cells. Figure 18(d) shows a graph of the calculated displacement of the cell from the original position in a direction perpendicular to the axis of rotation as a function of the initial distance between the cell and the axis of the device for different displacements of the axis of the device relative to the axis of rotation of the centrifuge, this figure is for a displacement of 10 mm, Figure 18(e) shows the same for a displacement of 0.75 mm; and Figure 18(f) shows the same for a displacement of 0 mm.

[0163] Fig.19 A front view of an example of an absorbent element 46 is shown, comprising two porous components 82 and 11, wherein the upper porous component 82 has a volume of 570 microliters and the lower component 11 has a volume of 75 microliters, and a plastic housing 48 for the absorbent element 46. In this embodiment, the total volume of the absorbent element is equal to 645 microliters. Assuming that the porosity of the material used for the components of the absorbent element 46 is ~30%, the total volume of liquid absorbed can be as high as 215 microliters, which is slightly higher than that of a cell separation device (e.g., ) produces a typical volume of acquisition (about 200 microliters). It may be advantageous to use a porous material with a higher porosity, as this will allow for a higher wicking rate and a larger volume of absorbed fluid (if desired).

[0164] Fig. 20 is a graph of percent cell loss versus wicking rate for two different centrifugation speeds, 4400 rpm (circles) and 2200 rpm (triangles).

[0165] Figure 21 shows the percentage of cell loss as a function of the distance between the surface of a solid support 42 and an absorptive element 46 forming part of a cell retention system of the present invention, where (a) no KCl is added and (b) KCl is added to the solution.

[0166] Optimization of Wicking Height

[0167] Previous optimization of the wicking rate was performed on live SK-BR-3 cells, which are more adherent and more firmly attached than other potential cell types. Using a less "sticky" cell model: pre-fixed SK-BR-3 cells (ICT) revealed that the previously set height of 0.5 mm resulted in cell loss during wicking. Therefore, the distance between the microscope slide and the wicking tip was extended in an effort to reduce cell loss.

[0168] It was found that while no cell loss occurred at 1.0 mm wicking height, some variation in cell position indicated that the cells were still experiencing some form of turbulence resulting in cell detachment at 1.0 mm. Therefore, for the gentlest operation, the recommended value for wicking height was found to be 1.5 mm or greater. A wicking height of 2.0 mm was chosen rather than 1.5 to provide a larger cushion to minimize user-to-user variability when introducing the wicking tip into the supernatant.

[0169] The optimization of the distance between the end of the absorbent material and the surface of the slide and the absorbent material allows the selection of required fluid flow, which should be below the threshold value defined by the shear stress on the cell, which may cause the detachment of the cell during the fluid removal step. For a distance of 2 mm, the flow of two different porous absorbent materials (U200 and U210, Pore Technology, Inc.) was measured. The result is shown in Figure 22 (a) and Figure 22 (b). Specifically, Figure 22 (a) and Figure 22 (b) illustrate the wicking rate as a function of time for the polyethylene porous material U200 (Pore Technology Inc.) shown in Figure 22 (a) and the U201 (Pore Technology Inc) shown in Figure 22 (b). The distance between the end of the solid support 42 surface and the absorbent element 46 is about 2 mm.

[0170] The threshold level for cell detachment was determined experimentally and was equal to 500 uL / s for the selected geometry of the fluid chamber 44 and the selected coating of the surface of the solid support 42 (glass slide). A distance of 2 mm between the end of the solid support 42 and the absorbent element 46 was suitable for absorbent material U200 (see FIG. 22( a)), but for material U201, this distance should be increased (see FIG. 22( b)).

[0171] Results (pictures of captured cells, evidence of reduced cell loss)

[0172] Pre-labeled SK-BR-3 cells were processed and counted by both systems. n = 10 replicates were performed for and the cell recovery device. A non-parametric Wilcoxon rank sum test was performed with a p value of 0.00018. The cell recovery device had a mean of 94% ± 6% (SD) cell recovery rate.

[0173] FIG. 23( a) is a photomicrograph of DAPI-stained cells incorporated into a fluid chamber 44 and sedimented by centrifugation using a device according to the method of the present invention, the photomicrograph showing cells remaining on the slide surface after centrifugation with continuous fluid removal. FIG. 23( b) is an enlarged image (X100) of the photomicrograph of FIG. 23( a). FIG. 23( a) and FIG. 23( b) demonstrate the similarity with industrial Improved retention (capture) rates of the proposed method and device compared to the standard in the method. Improved morphology showing less severe "flattening" of cells and less foaming, cells in the cell recovery device compared to the standard in the method. No cytoplasmic spread was observed using the cell recycler. The staining intensity was much higher in the cell recycler.

[0174] Fig.24 The cell capture efficiency percentages of the two cell capture methods are shown: and the cell retention methods and devices using the present invention shown in Dataset 2. Fig.24 It is clear from the results that the controlled fluid removal rate combined with the other features of the proposed method allows the capture rate of cells to be increased from about 30% ( Fig.24 In dataset) to more than 90%-95% in the proposed device ( Fig.24 The Harvest Chip dataset in .

[0175] Figure 25(a) shows the use of FIG25( b ) shows a micrograph of cells captured by a cell recovery device using the cell retention device of the present invention. The captured cells were damaged, while Figure 25(b) clearly shows that the method of the present invention preserves the morphology of the acquired cells.

[0176] Figure 26 (a) shows a micrograph showing the morphology of SK-BR-3 cells fixed in the absence of fluid around them (known as dry fixation), while Figure 26 (b) shows a micrograph showing the morphology of SK-BR-3 cells fixed in the presence of a controlled volume of fluid (known as wet fixation). Images were obtained using 400x magnification and staining with CK-FITC conjugate. As can be seen in Figures 26 (a) and 26 (b), controlling the residual volume of fluid present in the device after removing a large amount of fluid allows to improve the morphology of the retained cells. During the staining stage, an additional advantage of the controlled volume present in the device is that the background level is reduced in this case. Compared to Fixation with a controlled volume of residual fluid produced the lowest background signal compared to dry fixation methods either when no fluid was present ( FIG. 26( b ) ) or without controlling the residual fluid level ( FIG. 26( a ) ).

[0177] It was confirmed that compared with 12 μm to 20 μm (dry fixation) and 12 μm to 15 μm (wet fixation) on the solid support of the cell recovery device, Larger total cell sizes, ranging from 15 μm to 25 μm in diameter, were observed in The images obtained with the treated slides had a lower fluorescence signal. The signal using the cell recovery device with a controlled volume of residual fluid (wet fixation) showed the strongest intensity, and the dry fixation was between and wet fixation.

[0178] use Similar results were obtained with patient samples treated with the cell capture device of the present invention, see Figures 27(a) and 27(b). Comparison of the final form between the device of the present invention and the device of the present invention: In FIG. 27( a ), the The retained cells appear larger and more jagged than the cells retained using the device of the present invention shown in Figure 27(b).

[0179] EDTA HNV blood spiked with pre-labeled SK-BR-3 cells was passed Separate, pool, capture, immobilize, and then store on a solid support or Cytoslide in a cell recovery device. TM (Fisher Scientific). (Experiments performed by multiple trained users across multiple instruments / days). The effects of pipetting variability were reduced (compare and HS STD). The results are Fig.28 Summary statistics: 38.8% ± 12.6%, cell recovery device fixed: 96.7% ± 3.3%. As can be seen, with In comparison, the final capture efficiency (counting after staining) on ​​the cell recovery device of the present invention is significantly better. Most of the cell loss observed in RT-PCR occurred during the initial spin down of the cells onto the slide using the filter card fluid chamber 44.

[0180] Post-treatment of cells

[0181] Example of cell post-processing: Fig.29 Lysis of retained cells and sequential molecular analysis are shown in FIG. , which shows a bar graph of capture efficiency of EDTA HNV blood spiked with pre-labeled SK-BR-3 cells that were Separate, collect, capture, fix, and then in the cell recovery device or Cytoslide of the cell retention device of the present invention. TM The experiment was performed by multiple trained users across multiple instruments / days. The effects of pipetting variability were reduced (compare and the standard deviation (STD) of the cell recovery device 40).

[0182] Pre-labeled SK-BR-3 cells can be seen on the surface of the solid support 42 of the cell recovery device within a capillary micropipette (Image 1), then aspirated into the microcapillary (Image 2), after which the cells are moved to a different location on the slide surface and deposited (Image 3). Fig.30 The single cell picking results using the cell recovery device of the present invention are shown in , which shows the picking of SK-BR-3 cells deposited onto a solid support in the presence of a controlled volume of residual fluid according to the proposed method. The pre-labeled SK-BR-3 cells can be seen within the capillary micropipette on the surface of the solid support 42 of the cell recovery device ( Fig.30 ), and then aspirated into the microcapillary tube ( Fig.30 The cells were then moved to different locations on the slide surface and deposited ( Fig.30 ).

[0183] Fig.31 The figure shows that if the parameters are not properly controlled, cell loss can be high. Fig.31The picking of SK-BR-3 cells deposited onto a solid support 42 with a controlled volume of residual fluid according to the proposed method is shown. The upper figure shows the retained cells with the capillary of the cell picking tool placed over the cells; the middle figure shows the cells being removed from their initial position by the cell picking tool, and the lower figure shows the cells being dispensed (repositioned) by the cell picking tool. When using the cell recovery device apparatus with a 2.0 mm wicking height and a two-piece wicking cap, the amount of cell loss due to the wicking and resuspension steps is minimal.

[0184] With proper control of the operating parameters (wicking rate and volume of residual fluid), cell loss is minimal even with multiple operating steps. Fig.32 It is confirmed that the use of Fig.32 % of cell recovery at different stages of the operation as indicated in the various protocols. It is possible to stain the cells directly in the cell recovery device fluid chamber 44 as a post-processing step. This would require multiple wicking steps to exchange reagents within the fluid chamber, however this replaces multiple steps in staining after slide fixation. Fig.32 As can be seen in Figure 3, cell loss due to wicking of supernatant and exchange of fluids has been shown to be minimal.

[0185] exist Fig.32 The achievement of good morphology and high recovery rate is shown in , which shows the cell recovery percentage for multiple consecutive wicking steps (a, b, c), wherein 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, and wherein the test is repeated three times, demonstrating that if the wicking rate is appropriately selected according to the method of the present invention, multiple wicking events do not result in cell loss.

[0186] Data for Streck SKBR-3 samples counted between each wicking and buffer exchange step are given in Fig.32 , and an image of a surface where cells are retained and stained in the proposed device is shown in Fig.34 Shown in.

[0187] exist Fig.33 4 shows the staining of cells with DAPI in the fluid chamber 44, Fig.33 is a photomicrograph of leukocytes sedimented using the inventive method disclosed herein and stained with DAPI, wherein the photo was taken with the fluid chamber 44 still attached to the solid support, demonstrating that the cells can be stained with the fluid chamber 44 attached.

[0188] Fig.34Shown are micrographs (rows A, B) and photographs (row C) of cells that have settled using (Column I), and on a solid support 44 of a cell recovery device 40 forming part of a cell harvesting device of the present invention (Columns I and II), wherein the residual fluid was not controlled (Column II) and the residual fluid volume was controlled according to the methods disclosed herein (Column III). HNV cells were spiked into a fluid sample placed in a cell recovery device 40 after staining using the following wet fixation method on live EDTA tube cells. Using The same samples were used for cell sedimentation. The cells presented the worst morphology (column I, rows A and B), with diffuse cytoplasm and decreased fluorescence intensity and increased background. The least amount of morphological changes were observed in the wet-fixed cell recovery device 40 condition (column III). Similar results were observed for CD45 staining, which suggests that The reduced cell density in the culture medium is also worrisome.

[0189] The following are non-limiting and exemplary protocols for the device.

[0190] Cell Recovery Device 40 EDTA Protocol (Staining on Slides)

[0191] 1. The contents of the harvest are deposited directly into the fluid chamber 44 of the cell recovery device 40.

[0192] 2. Make samples in StatSpin TM CytoFuge TM In the spin drop (this is the reference of the device https: / / www.fishersci.ca / shop / products / statspin-cytofuge-2-personal-cytocentrifuge-system-3 / 220011), spin at 600 rpm for 2 min, followed by 4400 rpm for 2 min.

[0193] 3. Screw on the wicking cap 50 and allow the device to wick for 5 min.

[0194] 4. Pipette 150 μL of ice-cold acetone into the fluid chamber 44 against the tube wall and incubate in a freezer (-20° C.) for 5 min to fix the cells.

[0195] 5. After fixation, pour remaining acetone into an appropriate waste container.

[0196] 6. Disconnect the fluid chamber 44 from the cell recovery device 40.

[0197] 7. Allow any residual acetone to evaporate and leave slides dry (10 min) before proceeding to staining or storage.

[0198] Cell Recovery Device Streck Protocol (Staining on Slides)

[0199] 1. The contents of the harvest are deposited directly into the fluid chamber 44 of the cell recovery device 40.

[0200] 2. Make samples in StatSpin TM CytoFuge TM The spin was lowered to 600 rpm for 2 min, followed by 4400 rpm for 2 min.

[0201] 3. Screw on the wicking cap and allow the device to wick for 5 min.

[0202] 4. Prepare a drying buffer consisting of 200 μL of a 10% serum (horse serum or fetal bovine serum) solution in 0.083 M KCl (ie, 20 μL serum + 180 μL KCl solution).

[0203] 5. Pipette 200 μL of drying buffer into the fluid chamber 44 close to the tube wall.

[0204] 6. Run the sample in StatSpin TM CytoFuge TM Spin down at 600 rpm for 2 min.

[0205] 7. Use the second wicking cap 50 to wick away the supernatant.

[0206] 8. Disconnect the fluid chamber 44 from the cell recovery device 40.

[0207] 9. Disassemble the cell recovery device - pull the PTFE slide out of the base.

[0208] 10. Place the PTFE slide with the sample on a hot plate set to 37°C and allow any remaining liquid on the target area to evaporate for 30 minutes.

[0209] 11. After confirming that the surface of the slide is completely dry, fix it.

[0210] 12. Using a pipette, introduce 50 μL of ice-cold acetone in a drop-wise manner onto the target area on the slide.

[0211] 13. Place the slides in a freezer (-20°C) for 5 min to fix the cells.

[0212] 14. Decant any remaining acetone and allow slides to dry (10 min) before proceeding forward to staining or storage.

[0213] Cell Recovery Device 40Streck Protocol (In-Solution Staining)

[0214] 1. The contents of the harvest are deposited directly into the fluid chamber 44 of the cell recovery device 40.

[0215] 2. Make samples in StatSpin TM CytoFuge TM Medium spin down, spin at 4400 rpm for 2 min.

[0216] 3. Screw on the wicking cap 50 containing the absorbent element 46 and allow the device to wick for 5 minutes.

[0217] 4. Prepare x μL of staining solution (20 μL to 50 μL of solution has been tested previously) in a permeabilization reagent (eg, Inside Perm). Preferably, all antibodies will be fluorophore conjugated, thus allowing for a single antibody incubation rather than multiple.

[0218] 5. Introduce 50 μL of staining solution into the fluid chamber 44 of the cell recovery device 40 by pipetting against the wall of the device.

[0219] 6. Incubate the solution at room temperature in the dark for 45 min.

[0220] 7. After the antibody incubation, gently introduce 200 μL of IX PBS into the fluid chamber 44 while pipetting against the wall.

[0221] 8. Run the sample in StatSpin TM CytoFuge TM Medium spin down, spin at 4400 rpm for 2 min.

[0222] 9. Screw on the wicking cap 50 containing the absorbent element 46 and allow the device to wick for 5 minutes.

[0223] 10. Repeat the procedure of adding IX PBS and wicking 2 more times to completely wash away any remaining debris.

[0224] 11. For the final wash before cover-slipping, prepare 200 μL of a 5% glycerol solution in deionized water.

[0225] 12. After wicking, use a P1000 pipette to introduce the 5% glycerol solution into the fluid chamber 44. Gently mix the solution within the fluid chamber 44 to resuspend the cells to ensure even distribution.

[0226] 13. Make samples in StatSpin TM CytoFuge TM The spin was lowered to 600 rpm for 2 min, followed by 4400 rpm for 2 min.

[0227] 14. Screw on the wicking cap 50 containing the absorbent element 46 and allow the device to wick for 5 minutes.

[0228] 15. Use the Detacher Tool to disengage the fluid chamber 44 and remove the PTFE slide containing the sample from the base.

[0229] 16. Place the PTFE slide on a hot plate set to 37°C and incubate for 30 min. Protect the slide from light to prevent photobleaching.

[0230] 17. After drying, any remaining glycerin on the target area of ​​the PTFE slide will still appear glassy.

[0231] 18. Add 1 drop of mounting media onto the target area and place a coverslip on it. Apply pressure from one edge of the coverslip to prevent air bubbles.

[0232] Cell Recovery Device 40 Protocol—Alternative Protein Buffers

[0233] 1. The preparation of FBS (heat inactivation) is labor intensive and FBS is difficult to transport.

[0234] 2. Alternatives with comparable performance to FBS were explored (Stabilguard, BSA, horse serum, Hank's).

[0235] 3. 10% horse serum (the same reagent used in the blocking step) performs the same as FBS. In addition, it avoids the need for pre-staining blocking.

[0236] The fluid removal rate is optimized by selecting the absorbent material, the distance between the slide and the end of the absorbent element, and the cross-section of the absorbent element.

[0237] It was found that the porous material without the housing 48 itself gave an absorption rate that was too fast to cause cell detachment, so it was found to be very beneficial to use an absorbent element housing 48 in order to limit the contact area between the liquid medium and the porous material. In an embodiment, the housing 48 can be made of polypropylene. However, it should 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 or metal.

[0238] Live pre-labeled SK-BR-3 cells were used as a model system for the experiment. These cells were settled on the surface of a solid support 42, and then fluid was removed using a syringe pump with different extraction rates. The cell loss for each removal rate was assessed. Therefore, the cell loss associated with the increased liquid removal rate was determined. Cells settled at two different centrifugal forces (265xg / 2200rpm and 1060x g / 4400rpm) were tested to determine the maximum permissible fluid removal rate for cells that were more loosely and more firmly adhered to the slide surface.

[0239] It is determined that the maximum permissible supernatant extraction rate is 500 μL / min for both cells adhered at low centrifugal forces (265 x g) and cells adhered at high centrifugal forces (1060 x g). Compared with cells sedimented under 1060 x g conditions, for cells sedimented under 265 x g conditions, it is observed that cells dissociate more violently from the surface of the slide at a rate higher than 500 μL / min. In order to minimize the chance of losing cells during the wicking process, the maximum permissible wicking rate is determined using a lower sedimentation force (265 x g) condition.

Claims

1. A cell recovery device for settling and retaining target cells from a fluid sample, comprising: a) a base configured to releasably hold a solid support configured to receive cells on a top surface of the solid support; b) a fluid chamber having a first opening, the fluid chamber providing a fluid-tight seal between the top surface and the fluid chamber, the fluid chamber having a second opening for receiving a liquid sample containing target cells being acquired, the first opening defining an area of ​​a selected size on the surface of the solid support where the target cells are deposited, the device comprising a first removable cap configured to close the second opening during centrifugation; d) a fluid-absorbing element and a housing configured to receive the fluid-absorbing element, the housing having a bottom opening through which fluid can enter the housing, the fluid chamber configured to receive the housing after centrifugation, the fluid chamber comprising a second removable cap configured to close the second opening and in combination with the housing to prevent the end of the fluid-absorbing element from being positioned less than a predetermined distance from the surface of the solid support; and e) The cell recovery device is configured to be received and releasably retained in a centrifuge.

2. The device according to claim 1, wherein: The cross-sectional area, distribution along the axis of the fluid-absorbing element, and porosity of the fluid-absorbing element are within a certain range to provide control over the absorption rate of the fluid, and the end of the fluid-absorbing element is located at a certain distance from the surface of the solid support so that the target cells deposited on the solid support are not detached from the surface of the solid support due to the flow of the fluid being absorbed by the fluid-absorbing element.

3. The device according to claim 1 or 2, wherein: The fluid absorbent element is a porous material having a porosity in a range from about 1 micron to about 100 microns, and wherein the predetermined distance of the end of the absorbent element above the surface of the solid substrate is in a range from about 0.1 mm to about 4 mm.

4. The device according to claim 1, wherein: The fluid absorbent element has a porosity in the range of about 5 microns to about 50 microns, and wherein the distal end of the absorbent element is a predetermined distance above the surface of the solid substrate in the range of about 0.1 mm to about 3 mm.

5. The device according to any one of claims 1 to 4, wherein: The fluid absorbent element has a porosity in the range of about 10 microns to about 20 microns, and wherein the distal end of the absorbent element is a predetermined distance above the surface of the solid substrate in the range of about 0.1 mm to about 3 mm.

6. The device according to any one of claims 1 to 5, wherein: The device is configured to be releasably retained in the centrifuge such that an axis passing through the center of a segment of the surface for capturing cells and normal to the surface of the solid support intersects the axis of rotation.

7. The device according to claim 6, wherein: The device is configured to be releasably retained in 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.

8. The device according to any one of claims 1 to 7, wherein: The area of ​​selected size on the top surface of the solid support where target cells are deposited is selected to promote interaction between target cells and the surface, thereby allowing target cells to adhere to the top surface of the solid support.

9. The device according to any one of claims 1 to 8, wherein: The region of selected size on the top surface of the surface of the solid support on which the target cells are deposited is modified to promote interaction between the target cells and the surface, thereby facilitating adhesion of the target cells to the top surface of the solid support.

10. The device according to claim 1, wherein: The area of ​​selected size on the top surface of the solid support where target cells are deposited is functionalized with a treatment agent selected to modify the interaction between the target cells and the surface, thereby facilitating adhesion of the target cells to the top surface of the solid support.

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

12. The device according to claim 10, wherein: The treatment agent is selected to provide a differential binding force to any cells present that are not the target cells to be isolated, such that any non-target cells do not bind to the surface of the top surface of the solid support.

13. The device according to any one of claims 1 to 12, wherein: The fluid-absorbent element is made of porous plastic.

14. The device according to claim 13, wherein: The porous plastic is treated to modify its absorbent properties.

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

16. The device according to any one of claims 1 to 15, wherein: The end of the fluid absorbent material spaced apart from the top surface is positioned at a distance in a range of about 0.1 mm to about 3 mm from a surface of the solid substrate.

17. The device according to claim 2, wherein: The porosity of the fluid absorbent element and the size of the openings are selected to provide a selected flow rate.

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

19. The device according to any one of claims 1 to 18, wherein: The first removable cap includes a gasket to provide a fluid-tight seal during centrifugation.

20. The device according to any one of claims 1 to 19, wherein: The area on the surface of the solid support outside the area of ​​the selected size where the target cells are deposited on the top surface of the solid support is coated with a protective coating to limit the diffusion of fluid present in the area of ​​the selected size on the surface of the solid support where the target cells are deposited to the remaining portions.

21. The device according to any one of claims 1 to 20, wherein: The housing configured to receive the fluid-absorbent element includes an overflow feature to prevent overflow of fluid caused by insertion of the fluid-absorbent element into the fluid chamber.

22. The device according to claim 21, wherein The overflow characteristics include any of the following: a) at least one opening in an upper portion of the absorbent element housing; and b) The opening is configured to have any one of a square, a circle or a slot shape.

23. The device according to any one of claims 1 to 22, wherein: The housing configured to receive the fluid-absorbing element includes at least one flow acceleration feature to increase the rate of fluid removal without increasing the probability of removing settled cells from the surface of the solid support.

24. The device according to claim 23, wherein: There are two or more of said flow accelerating features located on said absorbent element housing.

25. The device according to claim 23, wherein: The flow acceleration features are configured to have a circular shape or a shape of a slot located on the absorbent element.

26. The device according to claim 23, wherein The flow acceleration features on the absorbent element housing are located at a distance in the range of about 5 mm to about 30 mm from the surface of the solid support.

27. The device according to any one of claims 1 to 26, wherein: The solid substrate is substantially transparent, and the base includes an opening that is aligned with the area of ​​selected size on the surface of the solid support where the target cells are deposited to provide a viewing port for visually observing the area of ​​selected size of the deposited target cells on the solid support without separating the fluid chamber from the solid substrate.

28. The device according to any one of claims 1 to 27, wherein: A fluid seal is provided by a gasket.

29. The device according to claim 28, wherein The gasket is attached to the fluid chamber such that when the fluid chamber is detached from the solid substrate, the gasket remains attached to the fluid chamber.

30. The device according to claim 29, wherein: The gasket has a circular, rectangular or square cross-section and is attached to the fluid chamber by being received in a groove in the bottom of the fluid chamber with a friction fit so that when the fluid chamber is disengaged from the solid substrate, the gasket remains attached to the fluid chamber.

31. The device according to claim 29, wherein: The gasket has a circular, rectangular or square cross-section and is attached to the fluid chamber by attaching to the bottom of the fluid chamber using an adhesive so that when the fluid chamber is detached from the solid substrate, the gasket remains attached to the fluid chamber.

32. The apparatus of claim 28, wherein: The gasket is attached to the surface of the solid support such that when the fluid chamber is detached from the solid support, the gasket remains attached to the surface of the solid support.

33. The apparatus of claim 28, wherein: The gasket is not attached to the fluid chamber such that when the fluid chamber is disengaged from the surface of the solid substrate, the gasket remains on the surface of the solid substrate, and wherein the gasket includes an outwardly protruding tab configured to be grasped by a user to remove the gasket from the surface of the solid substrate.

34. The apparatus of claim 28, wherein: The gasket has a thickness selected such that: a) after centrifugation and after fluid removal, the remaining volume of fluid remains to completely cover the settled target cells to prevent them from drying out, and b) The volume defined by the top of the gasket and the surface of the solid support provides sufficient volume for reagents used in post-processing of cells.

35. The device according to any one of claims 1 to 34, wherein: Areas of the surface of the solid substrate that are not exposed to the cell solution during cell sedimentation are functionalized with a coating of a hydrophobic material in a manner different from the functionalized surface areas, so that when the solid support having the functionalized areas is detached from the device and the areas retaining cells are exposed to small volumes of fluids contained in the functionalized areas, these fluids are retained by the hydrophobic coating surrounding the areas where the target cells are present.

36. A method for retaining and post-processing target cells present in a fluid medium on a solid support, the method having an optimized retention rate for target cells adhered to at least a portion of the surface of the solid support and retaining the morphology of the target cells, the method comprising: The fluid medium is subjected to centrifugation to induce target cells to settle onto the surface of the solid support, and the fluid medium is removed after sedimentation, characterized in that the controlled removal rate of the fluid medium is such that target cells settled on the surface are not detached from the surface by fluid flow near the settled cells, and that the volume of residual fluid is controlled during all stages of cell retention and post-processing, characterized in that the morphology of these settled target cells is substantially unchanged from their morphology when freely floating in the fluid medium.

37. The method of claim 36, wherein: The flow rate is controlled by the porosity of the material from which the absorbent element is made, the area of ​​the opening in the housing of the absorbent element and the cross-section of the absorbent element.

38. The method according to claim 36 or 37, wherein: At least a portion of the surface of the solid support on which the target cells are deposited has a selected size defined by an opening in a fluid chamber in which a fluid containing the target cells is placed for centrifugation, and wherein the surface having an area of ​​selected size is functionalized with a treatment agent selected to alter the interaction between the target cells and the surface to aid in the adhesion of the target cells to the top surface of the solid support.

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

40. The method of claim 38, wherein: The treatment agent is selected to provide different binding forces to target cells and other cells, such that target cells bind more readily to the surface while other cells bind less effectively to the top surface of the solid support.

41. The method according to any one of claims 36 to 40, wherein: The parameters of the absorbent element and the absorbent element housing are selected so that the shear forces associated with the fluid removal rate are low enough that detachment of target cells is minimal, but the same shear forces are sufficient to remove other cells, resulting in enrichment of the cells of interest.

42. The method according to any one of claims 36 to 41, further comprising post-processing the target cells adhered to the surface of the solid support by the steps of continuously introducing and removing fluid reagents into the compartments, so that the flow rate is controlled in each step so that the settled cells are not detached and lost, and the residual volume of the fluid in each consecutive step is controlled to avoid the removal of fluid below a predetermined volume, so that the morphology of the target cells is not affected by centrifugation under dry conditions.

43. A method for obtaining target cells from a fluid sample, comprising: a) placing a fluid sample in a fluid chamber, the fluid chamber being configured to be releasably retained in a centrifuge, the fluid chamber being in fluid communication with a 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, the solid support being detachable from the fluid chamber; b) releasably attaching the fluid chamber with the solid support attached thereto to a centrifuge, and centrifuging the fluid sample to induce sedimentation of target cells and promote cell adhesion to the surface of the solid support; c) removing the device from the centrifuge; d) removing the first cap and inserting a fluid absorbent element such that an end of the fluid absorbent element is spaced apart from the surface of the solid support by a predetermined distance, and sealing the fluid chamber with a second cap configured to be compatible with the fluid absorbent element; e) the cross-sectional area, distribution along the axis of the fluid-absorbent element, and porosity of the fluid-absorbent element are within a range to provide a controlled flow rate of the fluid leaving the fluid chamber, and the end of the fluid-absorbent element is located at a predetermined distance from the surface of the solid support so that target cells deposited on the solid support are not detached from the surface of the solid support due to the flow rate of the fluid being absorbed by the fluid-absorbent element; as well as f) controlling the residual volume of fluid to avoid removal of fluid below a predetermined volume, so that target cells remain in some fluid, so that the morphology of the target cells is not affected by centrifugation under dry conditions.

44. The method of claim 43, wherein: The controlled flow rate is controlled by the porosity of the material from which the absorbent element is made, the area of ​​the opening in the housing of the absorbent element and the cross-section of the absorbent element.

45. The method according to claim 43 or 44, wherein: The area on the surface of the solid support where the target cells are deposited has a selected size defined by an opening in a fluid chamber through which the target cells flow during sedimentation, and wherein the surface having the area of ​​selected size is functionalized with a treatment agent selected to alter the interaction between the target cells and the surface to aid in the adhesion of the target cells to the top surface of the solid support.

46. ​​The method of claim 45, wherein: The treating agent is selected from the group consisting of poly-L-lysine, silane coating, gelatin, fibronectin, gold or silver coating.

47. The method of claim 45, wherein: The treatment agent is selected to provide different binding forces to the target cell and any other cells, such that the target cell binds to the surface while other cells do not bind to the surface of the top surface of the solid support.

48. A method according to any one of claims 43 to 47, wherein: The parameters of the absorbent element and the absorbent element housing are selected so that the shear forces associated with the fluid removal rate are low enough that detachment of target cells does not occur, but the same shear forces are sufficient to remove other cells, resulting in enrichment of target cells.

49. The method according to any one of claims 43 to 48, further comprising post-processing the target cells adhered to the surface of the solid substrate by the steps of continuously introducing and removing fluid reagents into the fluid chamber, so that the flow rate is controlled in each step so that the settled cells are not detached and lost, and the residual volume of the fluid in each consecutive step is controlled to avoid the removal of fluid below a predetermined volume, so that the morphology of the target cells is not affected by centrifugation under dry conditions.

Images