• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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    • 专利摘要:
    INSERT FOR A CENTRIFUGATION TUBE, CENTRIFUGATION TUBE UNDERSTANDING THE SAME, METHOD FOR SEPARATING A TARGET CELL POPULATION FROM A SAMPLE AND KIT. An insert for a centrifuge tube suitable for use in density gradient separation is described. The insert includes a member dimensioned to fit inside the tube to divide the tube into a top portion and a bottom portion. Optionally, the insert has a support extending or hanging from the member for positioning the member inside the tube. At least two openings are located on the member so that a first opening is closer to a bottom end of the tube with respect to a second opening when the insert is positioned in the centrifuge tube. Also described are methods for separating a target population of cells from a sample using the insertion of a centrifuge tube.
    公开号:BR112013028522B1
    申请号:R112013028522-2
    申请日:2012-05-03
    公开日:2020-07-21
    发明作者:Steven M. Woodside
    申请人:Stemcell Technologies Inc；
    IPC主号:
    专利说明:

    [0001] This claim claims priority benefit from the copying interim order NO. US 61 / 482,886 deposited on May 5, 2011, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD
    [0002] This application relates to a device and method for separating cell populations and, more specifically, for an insert for a centrifuge tube and a method for separating cell populations using density gradient separation. INTRODUCTION
    [0003] In many applications it is desirable to enrich or, alternatively, deplete certain populations of cells in a biological sample. The fields of hematology, immunology and oncology rely on samples of peripheral blood and cell suspensions from related tissues, such as bone marrow, spleen, thymus and fetal liver. The separation of specific cell types from these heterogeneous samples is the key to research in these fields, as well as diagnostic and therapy agents for certain malignancies and immune / hematopoietic diseases.
    [0004] Purified populations of immune cells, such as T cells and cells presenting antigen are necessary for the study of immune function and are used in immunotherapy. Investigation of molecular, cellular and biochemical processes requires analysis of certain types of isolated cells. Numerous techniques have been used to isolate subsets of T cells, B cells, basophils, NK cells and dendritic cells.
    [0005] the isolation of hematopoietic stem cells has also been an area of great interest and pure populations of stem cells will facilitate studies on hematopoiesis. Transplantation of hematopoietic cells from peripheral blood, cord blood and / or bone marrow, is increasingly used in combination with the high dose of chemotherapy and / or radiotherapy for the treatment of a variety of disorders including malignant and non-malignant disorders genetic. Very few cells in such transplants are capable of long-term hematopoietic reconstitution, and thus there is a strong stimulus for developing techniques for purifying hematopoietic stem cells. In addition, serious complications and even the success of a transplant procedure is largely dependent on the effectiveness of the processes that are used to remove cells in the transplant that pose a risk to the transplant recipient. Such cells include T lymphocytes that are responsible for graft versus host disease (GVHD) in allogeneic grafts, and tumor cells in autologous transplants that can cause malignant tumor recurrence. It is also important to deflate the graft, removing unnecessary cells and thus reducing the volume of cyiropreserva to be infused.
    [0006] In certain cases, it is desirable to remove or destroy tumor cells from a biological sample, for example, in bone marrow transplants. Epithelial cancers of the bronchi, breast ducts and the gastrointestinal and urogenital tract represent an important type of solid tumors seen today. Migration of micrometastatic tumor cells is thought to be an important prognostic factor for patients with epithelial cancer. The ability to detect these metastatic cells is limited by the effectiveness of tissue or fluid sampling and the sensitivity of tumor detection methods. A technique to enrich circulating epithelial tumor cells in blood samples would increase the ability to detect metastatic disease and facilitate the study of such rare cells and the determination of biological changes that allow the disease to spread.
    [0007] Hematopoietic cells and immune cells have been separated based on physical characteristics, such as density and on the basis of susceptibility to certain pharmacological agents that kill dividing cells. The advent of monoclonal antibodies against cell surface antigens has greatly expanded the possibility of distinguishing and separating different types of cells. There are two basic approaches to separating cell populations from blood cell suspensions and the like using monoclonal antibodies. They differ in whether they are the desired or unwanted cells, which are distinct / labeled with the antibody (s).
    [0008] In positive selection techniques, the desired cells are labeled with antibodies and removed from the remaining unmarked / unwanted cells. In the negative selection, the unwanted cells are labeled and removed. Antibody / complement treatment and the use of immunotoxins are negative selection techniques, but the Fluorescence Activated Cell Classification (FACS) technique, and most immunosorbent batch-type techniques can be adapted for both positive and negative selection. In immunoadsorption techniques, cells are selected with monoclonal antibodies and preferably attached to a surface that can be removed from the remaining part of the cells, for example, column of granules, flasks, magnetic particles. Immunoadsorption techniques have gained favor clinically and in research because they maintain the high specificity of targeting cells with monoclonal antibodies, but unlike FACS, they can be extended to deal directly with the large number of cells in a clinical harvest and avoid the dangers of using cytotoxic reagents such as immunotoxins and complement. They do, however, require the use of a "device" or cell separation surface, such as a column of granules, panoramic bottle or magnet.
    [0009] Regardless of the specific method, current techniques for isolating specific cells from blood, such as hematopoietic stem cells, immune cells and circulating epithelial tumor cells, usually involve an initial step to remove red blood cells (RBC). Density separations are commonly used to remove RBC from peripheral blood. Ficoll-Paque ™ (Ficoll) (Amersham Pharmacia Biotech AB, Uppsala, Sweden), is the most widely used form of density separation (DSM) for this application. Others include Lymphoprep DSM ™ (Axis-Shield, Norway), and RosetteSep ™ DM-L (STEMCELL Technologies, Canada). In a Ficoll density separation, all the blood is placed on Ficoll and then centrifuged. The RBCs and granulocytes to sediment the cell grain and mononuclear cells remain at the Ficoll-plasma interface. the success of this technique is based on the difference in density between mononuclear cells, granulocytes and erythrocytes, whereby mononuclear cells are floating in Ficoll and RBCs and granulocytes are not.
    [0010] Ficoll density will be affected if blood is mixed with Ficoll, thus the need to carefully stratify the blood in the Ficoll layer before centrifugation. After centrifugation, if the interface between the plasma and Ficoll is disturbed during handling of the tube, it can be difficult to recover the mononuclear cells without also collecting some erythrocytes and granulocytes.
    [0011] Specific cells can be linked to red blood cells, for example, using RosetteSep ™ to form rosette immuno-formators ("immunorosettes") such that the target cells are pelleted with red blood cells during density gradient separation . Many populations of different specific lymphocytes and granulocytes can be isolated by density gradient separation in combination with RosetteSep ™. In addition, if all the blood is stored for more than 24 hours, the granulocytes change the density and will not granulate with the red cells. Using RosetteSep ™ to bind RBC to granulocytes can improve the separation of mononuclear cell density in these samples.
    [0012] It would be advantageous if the cell stratification and recovery process were reliable. There has been some tube development work to simplify the Ficoll process using inserts of various types in the test tubes. UniSep tubes (NovaMed, Israel) has an insert made up of a mesh disk held in place by a plastic holder. The tubes with this insert are difficult to load with Ficoll, as they require a centrifugation step, and the mesh can become clogged with cells. Vacutainer® CPT ™ tubes (BD, New Jersey) have a gel barrier along a Ficoll-like density separation medium. The tubes are expensive and the gel can become clogged by cells, especially if there is any immunorosetting of the erythrocytes. US Patent No. 5,840,502 describes a tube with a conical silicone insert that is snapped onto a centrifuge tube. These tubes generally require centrifugation to fill the tubes with density separation media.
    [0013] Accuspin ™ tubes (Sigma-Aldrich, St. Louis, MO) and Leucosep ™ (Greiner Bio-One, Monroe, NC) have a frit that separates the tube in an upper and lower section. A centrifugation step is necessary to fill the volume below the frit with medium density and during density separations this frit can be unobstructed. The frit can be coated with red blood cells (RBC) and sometimes clog, especially when separating immune-formed cells from the rosette ("immunorosetted").
    [0014] Thus, there is a need for an insert for a centrifuge tube that allows the tube to be loaded easily with Ficoll or another density separation medium. In addition, it would be advantageous that small volumes of trapped air upon insertion do not interrupt the use of the tube in the separation. It would also be advantageous if the insertion is not obstructed by small blood clots, aggregates, or rosettes of red blood cells and that it is possible to invert the tube to leak out the enriched sample without the liquid at the bottom of the tube, containing the grain, flowing to out. Finally, it is desirable to insert the insert securely in such a way that there is little chance of it being dislodged. SUMMARY
    [0015] The present description relates generally to an insert for a centrifuge tube useful for density gradient separation using density separation means (DSM). In one embodiment, insertion facilitates filling the centrifuge tube with DSM and stratifying a sample on top of the DSM in the centrifuge tube. The insert also facilitates the processing and handling of centrifuge tubes containing DSM during density gradient separation protocols. In one embodiment, insertion helps to avoid mixing between the DSM and an interface layer between the DSM and a remaining sample volume after density gradient separation.
    [0016] In one embodiment, the insert comprises a member sized to fit within a centrifuge tube and divides the tube into a top portion and a bottom portion. In one embodiment, the insert has at least two openings through the member so that a first opening is closer to a bottom end of the tube in relation to a second opening when the insert is positioned in the centrifuge tube. In one embodiment, the insert comprises a support extending or depending on the member for positioning the member within the tube. In one embodiment, the holder contacts the bottom end of the centrifuge tube and limits the position of the insert when the insert is pushed down into the centrifuge tube.
    [0017] In one embodiment, the first opening allows a fluid to flow into a space below the insert when positioned in a centrifuge tube. The second opening allows air to escape from the space below the limb when the centrifuge tube fills with fluid. Optionally, the insert has more than two openings. In one embodiment, the openings are sized to provide surface tension between each opening to prevent fluid contained below the limb from flowing through the openings when the tube is inverted.
    [0018] In one aspect, insertion is useful for the recovery or isolation of a target population separated from a sample. In one embodiment, the target population is a target population of cells and the sample is a biological sample. In one embodiment, the inserts described here are useful for separating and recovering leukocytes or mononuclear cells from blood samples using density gradient separation.
    [0019] The insert described here provides a number of advantages. In one embodiment, the insertion allows the sample to be poured into a centrifuge tube containing the DSM without mixing the sample and DSM, in order to impair the density gradient separation. In one embodiment, the insert allows the centrifuge tube to be easily filled with DSM. In one embodiment, the inserts described herein do not clog as with blood cells or other constituents of the sample during density gradient separation. In one embodiment, the sample volume in the top portion of the centrifuge tube can be poured out of the centrifuge tube, without pouring the liquid contained in the bottom portion of the centrifuge tube after density gradient separation. In one embodiment, the insertion allows the separated sample to be poured or decanted out of the centrifuge tube without disturbing the bottom portion of the separation medium and any sedimented material, such as the red blood cells contained in the bottom portion of the tube. The insert described here can also be easily and economically manufactured as a single piece using injection molding or other techniques known in the art and are easy to insert into centrifuge tubes.
    [0020] a) fornecer um tubo de centrifugação com uma inserção, tal como aqui descrito; b) encher uma porção do tubo de centrifugação com os meios de separação de densidade (DSM) , de tal forma que o DSM cobre o topo da inserção; c) adicionar um volume de amostra contendo a população alvo de células para o tubo de centrifugação de modo a formar uma interface entre o DSM e a amostra; d) girar o tubo de centrifugação para separar a população alvo de células a partir da amostra. In another aspect, a method is provided for separating a target population of cells from a sample comprising: a) provide a centrifuge tube with an insert, as described herein; b) filling a portion of the centrifuge tube with the density separation means (DSM), such that the DSM covers the top of the insert; c) adding a sample volume containing the target cell population to the centrifuge tube in order to form an interface between the DSM and the sample; d) rotate the centrifuge tube to separate the target cell population from the sample.
    [0021] In one embodiment, the method comprises recovering the target population of cells from a sample volume above the interface between the DSM and the sample after turning the centrifuge tube. In one embodiment, the target population of cells is recovered from the centrifuge tube by decanting a sample volume contained in the top portion of the centrifuge tube above the member. In one embodiment, the target cell population is recovered by using a volume transfer device such as a pipette to remove a volume of sample contained in the top portion of the centrifuge tube above the member. Optionally, the method includes binding dense particles to a second population of cells in the sample before spinning the centrifuge tube such as by immunorosetting (as described in US Patent No. 6,750,326, incorporated herein by reference) to improve the separation of a target population of cells from the rest of the sample. In one embodiment, the inserts described here can be used in a centrifuge with the brake on during deceleration, without impairing the recovery of target cells from the sample.
    [0022] In another aspect, a centrifuge tube is provided which comprises an insert for a centrifuge tube, as described herein. In one embodiment, the insertion is an integral part of the centrifuge tube.
    [0023] In another aspect, the present description provides a kit comprising an insert, as described herein. In one embodiment, the kit further comprises one or more of a centrifuge tube, a volume of DSM or printed instructions for carrying out any of the methods, as described herein.
    [0024] Other features and advantages of the present description will be evident from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred modalities of disclosure, are provided by way of illustration only, since various changes and modifications within the spirit and scope of the description will be evident to those experts in the field. art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
    [0025] For a better understanding of the modalities described here and to show more clearly how it can be carried out, reference will now be made, by way of example only, to the attached drawings showing at least one exemplary modality, and in which: Figure 1 shows a perspective view of an embodiment of an insert for a centrifuge tube with an inclination degree of 15 between the first opening and the second opening. Figure 1B is a top plan view of the insert shown in Figure 1A. Figure 1C is a sectional view of the insert shown in Figure 1B along line AA. Figure 2A shows a perspective view of an embodiment of an insert for a centrifuge tube with a concave top surface with a degree of inclination of 15 between the first opening and the second opening. Figure 2B is a top plan view of the insert shown in Figure 2A. Figure 2C is a sectional view of the insert shown in Figure 2B along line AA. Figure 3A shows a perspective view of an embodiment of an insert for a centrifuge tube with a concave top surface with an inclination degree of 25 between the first opening and the second opening. Figure 3B is a top plan view of the insert shown in Figure 3A. Figure 3C is a sectional view of the insert shown in Figure 3B, along line AA. Figure 4 shows a modality of a conical insertion with a central opening and three openings around the perimeter of the upper surface of the limb and a degree of inclination of 15 between the central opening and the perimeter of the limb. The central opening allows passage of cells during centrifugation and openings around the perimeter allow air to escape during filling of the tube with the density medium. Figure 5 shows a modality of a conical insert with a central opening and three openings around the perimeter of the upper surface of the limb and a degree of inclination of 40 between the central opening and the perimeter of the limb. The central opening allows the passage of cells during centrifugation and openings around the perimeter allow air to escape when filling the tube with the density medium. Figure 6 shows a cross-sectional view of an embodiment type as described here positioned inside a centrifuge tube Figure 7A shows a side sectional view of an embodiment as described herein positioned inside a centrifuge tube with the tube loaded with separation means and a stratified sample on top of the separation means. Figure 7B shows the tube after separating the sample into layers by density gradient centrifugation. Figure 7C shows the inverted tube with the separation means retained in the tube below the insert. DETAILED DESCRIPTION
    [0026] The insert described here is useful in combination with a centrifuge tube to separate a target population in a sample using density gradient separation. For example, in one embodiment, the insert can be positioned in a centrifuge tube and used to separate mononuclear cells from other cells in peripheral blood samples using density separation means by rotating the tube in a centrifuge. Cells and other constituents that have a density greater than the DSM will settle to the bottom of the tube, while cells and other constituents that have a density less than the DSM will settle at or above an interface between the sample and the DSM.
    [0027] In one embodiment, the insertion described here facilitates the filling of a centrifuge tube with the DSM when the insert is positioned in the centrifuge tube. Insertion is also useful for stratifying a sample and DSM in a centrifuge tube and avoiding interruption of an interface between the sample and DSM. In one embodiment, insertion is also useful for the recovery of a target population of cells in density gradient separation protocols.
    [0028] Several exemplary embodiments of an insert for a centrifuge tube according to the present invention are shown in Figures 1 to 5.
    [0029] Referring to Figure 1, an insert 10 for a centrifuge tube is provided. Insert 10 has a member 12 and support 14. Support 14 extends from member 12 to position the insertion with the centrifuge tube. As shown in Figure 1, support 14 can take the form of a cylindrical side wall. In some embodiments, the support may be in the form of rods or legs that extend or hang from the limb and serve to position and stabilize the insertion in a centrifuge tube.
    [0030] In one embodiment, the insert has at least two openings through the member. Referring to Figure 1, a first opening 16 is shown through the member 12 and a second opening 18 through the member 12. In one embodiment, the first opening is closer to the bottom of the centrifuge tube in relation to the second opening when the insert is positioned in the centrifuge tube. Optionally, as shown in Figure 1, the first opening 16 and / or the second opening 18 can include a cutout portion of the support 14. In one embodiment, the periphery of the opening includes a support cutout portion 14 and at least part side wall of a centrifuge tube. In one embodiment, the first and / or second openings are circular. In one embodiment, the first and / or second openings are non-circular and include straight or curved sides or a combination thereof. In one embodiment, the first and second openings have different shapes.
    [0031] Figure 2 shows another embodiment of an insert 200 for a centrifuge tube with member 212, support 214 and a first and second openings 216 and 218. Member 212 has a concave surface, with an inclination of 15 degrees from the first opening 216 for the second opening 218 located on the perimeter of the member. Figure 3 shows a similar insert 300 for a centrifuge tube. Support 314 extends from member 312, and has a concave surface with an inclination of 25 degrees from the first opening 316 to the second opening 318 located at the perimeter of the member.
    [0032] Figures 4 and 5 show other modalities of an insertion for a centrifuge tube with a conical member for separating the top and bottom portion of the tube. Referring to Figure 4, insert 400 has member 412, support 414, a first central opening 416 and a plurality of second openings 418 around the perimeter of the member. Member 412 has a conical top surface with an inclination of about 15 degrees from the first opening in the bottom of the member to the perimeter. Figure 5 shows a similar insert with a member 512, support 514, first opening 516 and a plurality of second openings 518 around the perimeter of the member 512. Member 512 has a conical top surface that forms a 40 degree inclination from from the first opening at the bottom of the limb to the perimeter. Optionally, the insert has one, two, three, four, five, or more than five second openings. In one embodiment, the second openings have different sizes. In one embodiment, the second openings are equally spaced around the periphery of the limb.
    [0033] In one embodiment, the member is sized to fit within a centrifuge tube to divide the tube into a top portion and a bottom portion. Referring to Figure 6, an insertion positioned inside the empty centrifuge tube 50 is shown and member 12 divides the centrifuge tube into a top portion and a bottom portion. As shown in Figure 6, the support 14 contacts the bottom conical portion of the centrifuge tube and prevents the insertion from going further down into the centrifuge tube. In one embodiment, the support extending or hanging from the second member also facilitates the placement of the insert within a centrifuge tube and prevents the insert from becoming dislodged during handling of the tube or during separation steps such as centrifugation.
    [0034] In one embodiment, the insertion described here facilitates filling the centrifuge tube with the DSM. For example, the first opening allows air or fluid to communicate between the space in a top portion of the centrifuge tube above the member and a space below the centrifuge tube in a bottom portion of the centrifuge tube. In one embodiment, one or more second openings also allow fluid or air communication between a space in a top portion of the centrifuge tube above the member and a space below the centrifuge tube in a bottom portion of the centrifuge tube. In one embodiment, the second opening allows air to escape from the space below the member when the bottom end of the tube is filled with liquid through the first opening. Optionally, the insert has one, two, three, four, five, or more than five second openings. In one embodiment, the second openings have different sizes. In one embodiment, the second openings are equally spaced around the periphery of the limb.
    [0035] In one embodiment, the first opening is closer to a bottom end of the tube in relation to a second opening when the insert is positioned in the centrifuge tube. The relative arrangement of the first and second openings allows the fluid to pass through the first opening and fill the bottom of the tube. When fluid fills the tube through the first opening, air is displaced out of the second opening positioned above the first opening which facilitates filling or loading the tube with fluid. In one embodiment, the first opening and the second opening are separated in the member by a distance of at least 1.5 times, 2 times, 3 times, 4 times, 5 times or more than 5 times the diameter of the first opening. In one embodiment, the first opening and second opening are separated in the member by a distance of at least 1.5 times, 2 times, 3 times, 4 times, 5 times or more than 5 times the diameter of the second opening. As used herein, "diameter" refers to the longest transverse distance defined by both opposite sides of the opening when the insert is placed in a centrifuge tube.
    [0036] In one embodiment, the first opening is located in a lower part of the top surface of the member when the insert is positioned in a centrifuge tube. For example, referring to Figure 6, the first opening 16 is located in the lower part of the member 12 in relation to the bottom end of the centrifuge tube 50. Likewise, it will be appreciated that when the inserts shown in Figures 2 -5 are positioned inside a centrifuge tube, a first opening will be located in the lower part of the top surface of the member in relation to the bottom of the centrifuge tube.
    [0037] In another embodiment, the second opening is located at the highest part of a top surface of the member when the insert is positioned in a centrifuge tube. For example, referring to Figure 6, second opening 18 is located at the highest part of the member 12 in relation to the bottom end of the centrifuge tube 50 and closest to the open end of the tube. In one embodiment, locating the second opening at the top of the limb helps to prevent air bubbles from becoming trapped under the insert when the tube is filled with fluid. In one embodiment, the first and / or second openings are located at or near the perimeter of the limb. In one embodiment, the first and / or second openings are located next to the side wall of the centrifuge tube when the insert is positioned in the centrifuge tube. For example, Figure 4 shows an insert with a first central opening and a plurality of second openings located on the perimeter of the member.
    [0038] In one embodiment, the openings are sized to provide surface tension between the openings to prevent liquid contained under the insert from flowing through the openings when the tube is inverted. For example, in one embodiment, each opening has an area of less than 15%, less than 10%, less than 5%, between 5% and 1%, or less than 1% in relation to the cross-sectional area of the pipe. centrifugation. A person skilled in the art will readily be able to determine the size of the first and second openings to allow a fluid, such as DSM, to be retained by the surface tension between the openings below the insert. In one embodiment, the first opening is larger than the second opening. For example, in one embodiment, the first opening has a diameter of about 1 mm to about 5 mm. In one embodiment, the first opening has a diameter of about 2 mm to about 4 mm. In one embodiment, the second opening has a diameter of less than about 2 mm.
    [0039] In one embodiment, the insert has a support extending or hanging from the member for positioning the member inside the tube. In one embodiment, the holder contacts the bottom of the centrifuge tube and limits how far the insert can be pushed down into the tube. In one embodiment, the support resists being compressed when the insert is pushed down towards the bottom end of the centrifuge tube such as when the insert is inserted down into the tube, or, when a tube containing the insert is centrifuged . In one embodiment, the support is a cylindrical sidewall extending from a lower surface of the member. In another embodiment, the support may include one or more legs extending from a bottom surface of the member to contact the bottom end of the centrifuge tube. In one embodiment, the centrifuge tube has a tapered bottom end and the support contacts at least part of the bottom tapered portion of the centrifuge tube.
    [0040] In one embodiment, at least a portion of a top surface of the member forms an angle between 5 degrees and 75 degrees with a plane perpendicular to a longitudinal axis of the centrifuge tube when the insert is positioned in the centrifuge tube. Optionally, at least a part of a top surface of the member forms an angle between 15 degrees and 65 degrees, approximately 15 degrees, 25 degrees or approximately about 50 degrees with a plane perpendicular to a longitudinal axis of the centrifuge tube when the insert is positioned in the centrifuge tube.
    [0041] In one embodiment, the inclined portion of the top member allows the first opening to be closer to the bottom end of the centrifuge tube in relation to the second opening when the insert is positioned in the centrifuge tube. For example, member 12 of the insert shown in Figure 1 constitutes an inclination of 15 degrees with a plane perpendicular to a longitudinal axis of the centrifuge tube when the insert is positioned in the centrifuge tube. Figure 4 shows an insertion with a conical member that forms a 15 degree inclination with a plane perpendicular to a longitudinal axis of the centrifuge tube when the insert is positioned in the centrifuge tube. Figure 5 shows an insertion with a conical member that forms a 40 degree inclination with a plane perpendicular to a longitudinal axis of the centrifuge tube when the insert is positioned in the centrifuge tube.
    [0042] In one embodiment, at least part of the top surface of the member has a concave feature when the insert is positioned in the centrifuge tube. In one embodiment, the concave feature has the shape of a cone or parabola or a section of a cone or parabola. Optionally, the concave feature is tilted from a point on the perimeter of the member for a first opening. In one embodiment, the first opening is at the bottom of the concave feature when the insert is positioned in the centrifuge tube. For example, Figures 2 and 3 show an insertion with a member that has a concave characteristic with an inclination from the second opening 218, 318 to the first opening 216, 316 diametrically opposite from the second opening. Figures 4 and 5 show an insertion with a member that has a concave feature in the form of a cone. Additional members allow a first opening to be positioned lower in relation to one or more second openings when the insert is positioned inside the tube is also included in the scope of the present description.
    [0043] In one embodiment, the member is sized to fit within a centrifuge tube and forms an interference fit with the side wall of the centrifuge tube when the insert is positioned in the tube. In one embodiment, the perimeter of the member extends radially outward after the support and forms a flange. In one embodiment, the interference fit between the member and the centrifuge tube prevents the passage of liquid between the member and the centrifuge tube. In one embodiment, the support is sized to fit within a centrifuge tube and forms an interference fit with the side wall of a centrifuge tube when the insert is positioned inside the tube. In one embodiment, the interference fit between the support and the centrifuge tube prevents the passage of liquid after the support and the centrifuge tube between the top portion and the bottom portion of the centrifuge tube.
    [0044] In one embodiment, the inserts described here are made by injection molding to form an insert made of a single piece of material. Alternatively, the support member e can be made separately and joined to form the insert. In one embodiment, all or part of the insert is made of silicone, plastic or rubber or a combination of these. In one embodiment, all or part of the insert is made of high density polypropylene, copolyester, polycarboant, polytetrafluoroethylene or a combination of these. Optionally, the perimeter or flange of the member is made of a resilient material suitable to form an interference fit with a centrifuge tube. In one embodiment, the insertion is preferably made of a material with a density greater than the density of the DSM useful for separating a target population of cells. Commercial DSM having different densities include Ficoll ™ (GE Healthcare, density = 1.077 g / ml), RosetteSep DM-L ™ (Stemcell Technologies, Canada, density = 1.081 g / ml), RosetteSep DM-M ™ (Stemcell Technologies, Canada, density = 1.085 g / ml), ™ Lymphoprep (Axis-Shield, Norway, density = 1.077 g / ml), OptiPrep ™ (Axis-Shield, Norway, density = 1.32 g / ml) Histopaque ™ (Sigma-Aldrich, St Louis, MO, density = 1.077, 1.019, 1.083 g / ml). Suitable plastics that have a density greater than the normal DSM useful for separating a target cell population include, but are not limited to Tritan (Eastman, Kingsport, Tennessee, density = 1.18 g / cm3), polycarbonate (Lexan ®, SABIC Innovative, Saudi Arabia, a density = 1.2 g / cm3) polytetrafluoroethylene (Teflon ®, Du Pont, Delaware, USA, density = 2.2 g / cm3) and other plastic materials that have a density greater than about 1.08 g / cm3, one or more, preferably greater than 1.1 g / cm3. Inserts made of a material that has a density greater than that of DSM will not float in DSM. When the centrifuge tube using an insert is centrifuged at high speed in a centrifuge, the insert will experience a buoyant force that opposes the force of the interference fit between the tube and the insert and may cause the insert to float. A floating insert will not be correctly positioned in the tube, making it useless for density gradient separation. In one embodiment, the insertion density is greater than 1.08 g / cm3. In one embodiment, the insertion density is greater than 1.1 g / cm3. In one embodiment, the entire part of the insert is made of polycarbonate, copolyester, polytetrafluoroethylene or high density polypropylene.
    [0045] While centrifuge tubes with conical bottom ends are generally preferred in the art, other tubes, such as flat bottom or round bottom tubes, can also be used with the inserts described herein. Examples of centrifuge tubes commonly used in the art include 15 ml or 50 ml Falcon ™ conical tubes (available from Becton Dickson, Franklin Lakes, NJ USA), 0.5 ml round bottom Falcon ™ tubes and 0.5, 1.5 and 2.0 ml microcentrifuge tubes (Eppendorf AG, Hamburg, Germany). Other suppliers of centrifuge and microcentrifuge tubes include Corning Lifescience (Lowell, MA) and Greiner Bio-one (Frickenhausen, Germany). In one embodiment, the inserts described here are sized to fit 15 ml or 50 ml conical bottom centrifuge tubes. In another embodiment, the inserts are sized to fit 5 ml tubes or 0.5, 1.5 and 2.0 ml microcentrifuge tubes.
    [0046] In another aspect of the present disclosure, a centrifuge tube is provided comprising an insert as described herein. In one embodiment, the insertion is an integral part of the centrifuge tube. In one embodiment, the insert is attached to the side wall of the centrifuge tube. In one embodiment, the insertion is made from the same material as the centrifuge tube.
    [0047] Figure 7 shows a modality of an insertion positioned in a centrifuge tube in different phases of a density gradient separation protocol. With reference to Figure 7A, the insert of Figure 1 is shown positioned in the centrifuge tube 50 with the member 12, support 14, first opening 16 and second opening 18. Tube 50 is shown filled just above the top portion of the insert with DSM 60. A sample 70 containing a target population is stratified on top of DSM 60. In one embodiment, sample 70 is added to the tube and mixed completely or partially with a portion of the DSM that is located above the insert. Mixing the sample with the DSM above the upper surface of the insertion member does not significantly affect the separation of the target population during centrifugation or the formation of an interface between the sample and the remaining DSM. Figure 7B shows the tube after density gradient separation and volume formation 74 containing the target population from above the insert at the DSM 60 interface and maintaining sample volume 72. In one embodiment, the described inserts allow for easy removal of a volume above the insertion from the centrifuge tube to recover the target population. Referring to Figure 7C, inverting the tube causes any volume previously contained above said insert to spill out of the tube, while DSM 60 and any non-target cells are retained in a 50 centrifuge tube. While Figure 7C shows the tube inverted at 180 degrees, it is also possible to invert the tube at smaller angles such as greater than about 90 degrees, about 90 to about 135 degrees or about 112-157 degrees to remove the volume of fluid contained above the insert.
    [0048] a) fornecer um tubo de centrifugação com uma inserção, tal como aqui descrito; b) encher uma porção do tubo de centrifugação com os meios de separação de densidade (DSM) , de tal forma que o DSM cobre o topo da inserção; c) adicionar um volume de amostra contendo a população alvo de células para o tubo de centrifugação de modo a formar uma interface entre o DSM e a amostra, e d) girar o tubo de centrifuga para separar a população alvo de células a partir da amostra. In another aspect, a method is provided for separating a target population of cells from a sample. In one embodiment, the method comprises: a) provide a centrifuge tube with an insert, as described herein; b) filling a portion of the centrifuge tube with the density separation means (DSM), such that the DSM covers the top of the insert; c) add a sample volume containing the target cell population to the centrifuge tube to form an interface between the DSM and the sample, and d) rotate the centrifuge tube to separate the target cell population from the sample.
    [0049] In one embodiment, the target population comprises cells that are suitable for separation using density gradient separation. For example, in one embodiment, the target cell population includes at least one cell type selected from breast cells, stem cells, ES cells, tumor cells, cancer cells, immune cells, hematopoietic stem cells and peripheral blood mononuclear cells. In one embodiment, the target cell population is leukocytes. In one embodiment, the target cell population has an average density less than the density of the DSM. A person skilled in the art will readily be able to select a DSM suitable for use in the methods described herein in order to separate a specific target cell population.
    [0050] In one embodiment, the sample volume containing the target cell population is added to the centrifuge tube through layers of the sample in the DSM to form an interface between the DSM and the sample. For example, the sample can be immersed in the DSM with the centrifuge tube using a pipette or a similar volume transfer device. In one embodiment, the sample volume containing the target cell population is added to the centrifuge tube so that the sample mixes with the DSM above the insertion in the centrifuge tube. For example, the sample can be poured into the centrifuge tube. In one embodiment, the sample is poured down the side wall of the centrifuge tube to minimize sample mixing and DSM. An expert will appreciate that while insertion helps to avoid mixing between the sample and the DSM below the insertion, mixing between the sample and the DSM above the insertion will not prevent the formation of an interface or the separation of a target population from cells from the sample. In one embodiment, the sample mixes fully or partially with the DSM above the insert and an interface forms between the sample fully or partially mixed with the DSM and the DSM remaining in the centrifuge tube.
    [0051] In one embodiment, the sample is a biological sample that contains one or more types of cells. In one embodiment, the sample is a liquid such as whole peripheral blood, mucus or cerebrospinal fluid. In one embodiment, the sample is a solution that contains one or more types of cells. For example, in one embodiment the sample is a sample of tissue or bone marrow, in which the cells have been dissociated and are present in solution.
    [0052] In one embodiment, the method involves recovering the target cell population. For example, in one embodiment, the target cell population is recovered from a sample volume above the interface between the DSM and the sample after turning the centrifuge tube to separate the target cell population from the sample.
    [0053] In one embodiment, the target cell population is recovered by inverting the centrifuge tube to remove a sample volume above the insertion in the centrifuge tube. In one embodiment, the DSM, and any material that has settled under the insert, remains in the centrifuge tube when the tube is inverted and does not flow out through the openings in the member. Optionally, in one embodiment the sample is a blood sample and a volume of plasma is removed from above an interface layer before removal of the target cell population either by aspiration or inversion of the tube to pour the interphase layer between and the DSM and the plasma layer.
    [0054] In one aspect, the methods described herein can be used in conjunction with methods known in the art to improve the separation of target populations by density gradient separation. For example, in one embodiment the methods include binding dense particles to a second population of cells in the sample before spinning the centrifuge tube, such as by immunorosetting as described in US Patent No. 6,750 .326. In one embodiment, the second population of cells bound to the particles separates below the interface between the DSM and the rest of the sample while the target population of cells separates to the interface between the DSM and the sample. In one embodiment, the dense particles are selected from red blood cells, silica particles, metal particles, metal oxide particles, polymer particles and glass particles. In one embodiment, the second population of cells is defined by specific surface proteins and dense particles are linked to the second population of cells by means of specific antibodies to cell surface proteins.
    [0055] In another aspect, the present description provides a kit comprising an insert for a centrifuge tube, as described herein. In one embodiment, the kit further includes a centrifuge tube, a volume of density separation medium, an antibody composition for binding cells specific to dense particles and / or printed instructions for carrying out the methods described here.
    [0056] The following non-limiting examples are illustrative of the present invention; EXAMPLES EXAMPLE 1; DENSITY GRADIENT SEPARATION
    [0057] 1. Encher um tubo de centrifugação aproximadamente 1 /3 cheio com solução de gradiente de densidade (por exemplo, Ficoll-Paque, Histopaque). 2. Suavemente suspensão das células de camada (por exemplo, sangue total ou suspensão de células) no topo de uma solução de gradiente de densidade. É crítico minimizar a mistura entre a suspensão de células e uma solução com gradiente de densidade. Para sangue total, recomenda-se diluir o sangue com um volume igual de meio tamponado tal como salina tamponada de fosfato (PBS). 3. Centrifugar durante 15 minutos a 400xg com o freio desligado para limitar interrupção para a interface. 4. Retire cuidadosamente as células enriquecidas a partir da interface Ficoll-plasma usando uma pipeta. Às vezes, é preferível remover o plasma primeiro para minimizar o volume total da amostra antes da etapa de lavagem (5). 5. Lave as células enriquecidas 1 a 2 vezes com 5-10 x volume de PBS + 2 % FBS com uma centrifuga. Density gradient separation is a well-known process in which cells are separated into one or more fractions based on differences in cell density. An exemplary protocol for a single batch density gradient separation is described below: 1. Fill a centrifuge tube approximately 1/3 full with density gradient solution (eg Ficoll-Paque, Histopaque). 2. Gently layer cell suspension (eg, whole blood or cell suspension) on top of a density gradient solution. It is critical to minimize the mixture between the cell suspension and a density gradient solution. For whole blood, it is recommended to dilute the blood with an equal volume of buffered medium such as phosphate buffered saline (PBS). 3. Centrifuge for 15 minutes at 400xg with the brake off to limit interruption to the interface. 4. Carefully remove the enriched cells from the Ficoll-plasma interface using a pipette. Sometimes, it is preferable to remove the plasma first to minimize the total sample volume before the washing step (5). 5. Wash the enriched cells 1 to 2 times with 5-10 x volume of PBS + 2% FBS with a centrifuge.
    [0058] 1. Adicionar 50 µL de composição de anticorpo por ml de sangue periférico inteiro. 2. Incubar durante 20 minutos à temperatura ambiente. 3. Diluir a amostra com um volume igual de solução salina tamponada de fosfato (PBS) + 2 % de soro bovino fetal (FBS) e misturar suavemente. 4. Estratificar a amostra diluída no topo de Ficoll-Hypaque ou estratificar o Ficoll sob a amostra diluída. 5. Centrifugar durante 20 minutos a 1200xg, temperatura ambiente, com o freio desligado. 6. Remover as células enriquecidas da interface Ficoll: plasma. 7. Lavar as células enriquecidas com 5-10 x volume de PBS + 2 % FBS. Opcionalmente, enriquecimento de monócitos e outras células aderentes, 1 mM de EDTA pode ser adicionado à amostra de sangue total e para todas as soluções de lavagem / diluição. An exemplary negative selection protocol for immunorosetting of whole peripheral blood cells using Ficoll-Hypaque is presented below: 1. Add 50 µL of antibody composition per ml of whole peripheral blood. 2. Incubate for 20 minutes at room temperature. 3. Dilute the sample with an equal volume of phosphate buffered saline (PBS) + 2% fetal bovine serum (FBS) and mix gently. 4. Stratify the diluted sample on top of Ficoll-Hypaque or stratify the Ficoll under the diluted sample. 5. Centrifuge for 20 minutes at 1200xg, room temperature, with the brake off. 6. Remove the enriched cells from the Ficoll: plasma interface. 7. Wash the enriched cells with 5-10 x volume of PBS + 2% FBS. Optionally, enrichment of monocytes and other adherent cells, 1 mM EDTA can be added to the whole blood sample and for all wash / dilution solutions.
    [0059] Accuspin ® density centrifuge tubes (available from Sigma-Aldrich), Leucosep ® (available from Greiner Bio-One GmbH, Germany), UniSep (available from Novamed, Jerusalem, Israel), and ACT Dendreon (previously available from MICRA Scientific, Inc. and described in US Patent No. 5,840,502) each contains an insert intended to facilitate sample loading without interrupting the layer between the separating means and the sample.
    [0060] These tubes must be loaded first with the appropriate DSM volume based on the size of the tube and the location of the insert. Generally, the tubes are then centrifuged for a short period to drive the DSM below the insert. The tubes are then ready to be used in a standard density gradient separation protocol as defined in Example 1. With these tubes, the step 2 layer of Example 1 is simplified, allowing the sample to be quickly added to the DSM once which only mixes with the DSM volume above the insert. In addition, the layer removal step (i.e., step 4 of Example 1) is simplified, allowing cells at the interface to be recovered by pouring out of the tube. As with the standard method, it may be preferable to remove most of the plasma first before recovering the cells. EXAMPLE 4: IMMUNOROFIXATION ("IMMUNOROSETTING") USING PREVIOUS ART TUBES WITH INSERTIONS
    [0061] 1. Adicionar o volume recomendado de DSM para o tubo. 2. Centrifugar durante 5 minutos em 400 g, o Ficoll-Paque deve preencher o volume abaixo da inserção. 3. Preparar amostra de sangue para seleção negativa por imuno-formação de roseta ("immunorosetting") como estabelecido no Exemplo 2: 4. Adicionar 50 µL de composição de anticorpo por ml de sangue periférico inteiro 5. Incubar durante 20 minutos à temperatura ambiente. 6. Diluir a amostra com um volume igual de solução salina tamponada de fosfato (PBS) + 2 % de soro bovino fetal (FBS) e misturar suavemente. Adicionar o sangue ao tubo e separar células: 7. Despejar ou estratificar a amostra de sangue diluída em topo de Ficoll-Paque. 8. Centrifugar durante 20 minutos a 1200xg, temperatura ambiente, com o freio ligado. 9. Remover as células enriquecidas a partir da interface Ficoll-plasma pipetando para fora interface ou vertendo para fora volume acima de inserção de tubo. 10. Lavar as células enriquecidas com 5-10 x volume de PBS + 2 % FBS. As células enriquecidas estão agora prontas para uso posterior. The Dendreon ACT centrifuge tube described in US Patent No. 5,840,502 is a standard 50 ml polypropylene centrifuge tube with an insert specially designed for use with density gradient separation that allows the user to remove cells at the Ficoll interface -plasma by pouring out a part of the sample containing the cells from the tube. An exemplary protocol for immuno-formation of the rosette ("immunorosetting") using Dendreon tubes includes the following steps: Prepare tube for density gradient separation as needed: 1. Add the recommended volume of DSM to the tube. 2. Centrifuge for 5 minutes at 400 g, Ficoll-Paque must fill the volume below the insert. 3. Prepare blood sample for negative selection by immuno-formation of rosette ("immunorosetting") as set out in Example 2: 4. Add 50 µL of antibody composition per ml of whole peripheral blood 5. Incubate for 20 minutes at room temperature. 6. Dilute the sample with an equal volume of phosphate buffered saline (PBS) + 2% fetal bovine serum (FBS) and mix gently. Add blood to the tube and separate cells: 7. Pour or stratify the diluted blood sample on top of Ficoll-Paque. 8. Centrifuge for 20 minutes at 1200xg, room temperature, with the brake on. 9. Remove the enriched cells from the Ficoll-plasma interface by pipetting out the interface or pouring out the volume above the tube insert. 10. Wash the enriched cells with 5-10 x volume of PBS + 2% FBS. The enriched cells are now ready for later use.
    [0062] Vacutainer ® CPT ™ tubes (BD, New Jersey) have a gel barrier along a Ficoll-like density separation medium. Separation of cells using CPT tubes follows the same general procedure, except that steps 1 and 2 are not necessary since the density separation medium is already in the tube kept below a gel layer. EXAMPLE 5: ENRICHMENT OF CD8 + T CELLS BY IMMUNOROFIXATION ("IMMUNOROSETTING") USING FICOLL-PAQUE
    [0063] This example demonstrates the enrichment of T + CD8 cells from whole peripheral blood, using the methods described in Examples 1 and 2, using standard 50 ml tubes, Dendreon tubes and CPT tubes. RosetteSep ™ CD8 + T cell enrichment cocktail (available from STEMCELL Technologies) was used to compare cell enrichment in different tubes. The results shown in Table 1 demonstrate that there is no significant difference in purity and recovery observed for separations performed in standard 50 ml tubes, CPT or Dendreon tubes. CPT and Dendreon tubes were originally designed to provide the most convenient density gradient separation by eliminating two time-consuming steps: the need to stratify the blood in Ficoll and the need to pipette the interfacial cells after centrifugation. the RosetteSep ™ immunorosetting reagent is useful for the selection of specific cell populations by density gradient separation. Thus, improvements in the density gradient separation provided by these tubes are also applicable to RosetteSep ™ enrichments.
    [0064] Inserts with an inclined top member as shown in Figure 1 without a separate cylindrical support were made from a silicone stop. The insert had a thickness of about 5 mm and an angle of about 15 ° to the horizontal when placed in a vertical 15 ml conical bottom centrifuge tube. The insertion was kept in place by an interference fit with the side wall of the centrifuge tube. The insert was placed in the tube so that when about 5 ml of Ficoll-Paque was added, the level of the liquid covered the highest point of the insert. The openings were cut at the highest and lowest points at the stop. The openings were in the shape of a semicircle with a diameter of about 2 mm with the open end of the semicircle facing outwardly from the member.
    [0065] The effectiveness of the insert was tested in a standard density gradient separation with a standard 15 ml tube or a 15 ml tube with the insert described above. In each case, 5 ml of Ficoll and 3 ml of blood diluted 1: 1 with PBS + 2% FBS was used. The centrifuge rotation was 20 min at 400xg, with the brake off.
    [0066] 1. Estratificar sangue cuidadosamente sobre Ficoll, rotação em centrifugação, remover camada de tamponamento contendo células cuidadosamente com uma pipeta. 2. Estratificar sangue cuidadosamente sobre Ficoll, rotação de centrífuga, agitar tubo para misturar camada de tamponamento contendo célula e verter para fora. No caso do tubo padrão, não verter para fora grão. 3. Verter sangue em tubo, rotação em centrifuga, agitar tubo para misturar camada de tamponamento contendo células e verter para fora. The following three different methods were used to prepare the samples for density separation: 1. Stratify blood carefully over Ficoll, spin in centrifugation, remove buffer layer containing cells carefully with a pipette. 2. Stratify blood carefully over Ficoll, centrifuge rotation, shake tube to mix buffer layer containing cell and pour out. In the case of the standard tube, do not spill out grain. 3. Pour blood into a tube, spin in a centrifuge, shake tube to mix buffer layer containing cells and pour out.
    [0067] Using method 3 above, almost no cells were recovered in the standard tube. This is not surprising, since the blood mixed with Ficoll does not form a different density layer for the cells to settle during centrifugation. In all other cases, there was a similar number of cells recovered as shown in Table 2.
    [0068] The phenotype of the recovered cells was analyzed based on the scattering of light in a flow cytometer. Using methods 2 and 3, where the cell sample was poured over Ficoll, there was about 10% granulocyte contamination of the recovered cells compared to 1% for the standard method. This is independent of the presence of the insert. The insertion, therefore, allows density gradient separation without careful sample stratification and removal of the buffer layer. the higher granulocyte content of the recovered sample observed for methods 2 and 3 may be due to some cells adhering to the insertion surface or becoming stuck in the surface irregularities. The irregularities in the surface exist because the inserts were cut manually from silicone stops using a knife. This would not be expected for smooth insertion.
    [0069] Inserts as shown in Figures 1 to 4 were made with resin using stereolithography methods designed for rapid prototyping. All four inserts are designed to have a shape compatible with injection molding, to fit into a standard 50 ml centrifuge tube (eg Falcon brand tube) such that the bottom of the insert fits over the top portion of the conical section of the tube and to allow filling of the volume under insertion with density gradient medium without the need for centrifugation. Each of the inserts has a small opening at the highest point of the insertion so that air can escape when liquid flows through the large lower opening located in the insertion. Each insert is designed so that it is not completely submerged when the tube is filled with approximately 20 ml of Ficoll.
    [0070] The inserts were housed inside the tubes and filled with about 20 ml of Ficoll. The conical insert shown in Figure 4 does not fill up with Ficoll easily. To obtain the Ficoll to fill the volume below the insertion, it was necessary to apply an additional downward force, such as holding the tube and swinging an arm in a circular motion. The angled insert as shown in Figure 1 also requires some additional centrifugal force to fill. The inserts emptied with a concave top surface shown in Figures 2 and 3 were easy to load with Ficoll under normal gravity (that is, without additional agitation). Therefore, the emptied inserts were the easiest to load with density gradient material.
    [0071] Each type of insertion was tested for the leak of Ficoll when the tube was inverted. First, the tubes with the inserts were centrifuged briefly at 300 xg so that all bubbles were removed from all inserts. Then, each tube was inverted to 45 ° from the vertical and then completely upside down. In all cases, after the inversion to 45 ° in relation to the vertical the entire Ficoll above the insertion poured out, but the Ficoll under the insertion remained in place. After completely inverting the tube, the Ficoll under the insertion slowly poured into the inserts shown in Figures 1 to 3. Therefore, the conical insertion (as in Figure 4) provided the most robust Ficoll retention under the insertion.
    [0072] Each insertion was then tested in a density gradient separation. About 20 ml of Ficoll was added to each tube in such a way that the Ficoll level was about 2 mm above the top of the insert. 30 ml of whole peripheral blood diluted 1: 1 with PBS was then added to each tube. the blood was added quickly, without taking into account gently stratifying about Ficoll. A standard tube was also prepared following the method described in Example 1 and using 30 ml of the same diluted blood stratified over 20 ml of Ficoll.
    [0073] The tubes were then centrifuged at 400x g for 20 min with the brake off. After centrifugation, the plasma layer was removed from all tubes and the tubes with the inserts were shaken in such a way that the buffer layer mixed with the liquid above the insert and the liquid were decanted by inverting the tube to 45 ° in relation to to the vertical. The buffer layer was carefully removed with a pipette from the standard tube. The number of cells recovered from each tube is shown in Table 3. Granulocytes were counted as a subset of the total cells using a hemocytometer. The results show that the standard tube appeared to give a higher total cell recovery than any of the tubes with an insert. However, this may be due to the cells attached to the inserts. The red blood cells appeared to cling to the insertion surfaces and, presumably, some nucleated cells. The inserts in the present example were made from a resin that is not biocompatible, while inserts made from injection molded plastic are likely to exhibit less cell adhesion.
    [0074] Inserts as shown in Figure 4 were injection molded from polypropylene and then inserted into 50 ml Falcon centrifuge tubes such that the bottom of the insert rested on the conical portion at the bottom of the tube. The flange on the top portion of the insert serves to hold the insert in place and provide some conformity to adjust if the diameter of the tube is not uniform. The height of the inserts in the tube was such that 17 ml of DSM filled the tube to a level slightly above the top portion of the insert. The volume of the tube below the bottom of the central orifice at the insertion was at least 10 ml so that if the entire volume above the DSM is filled with blood (that is, about 35 ml) and then the blood can have a hematocrit of 25% without fully filling the volume below the insert. Since blood is normally diluted 1: 1 with a diluent such as PBS prior to density gradient separation, the initial hematocrit sample can be up to 50%, which exceeds the normal range.
    [0075] The DSM was introduced under the insert by placing the tip of the serological pipette over the opening at the bottom of the conical section and distribution, the DSM flows through the orifice and under the insert, while air is displaced through the holes around the top edge of the insert. . Once all the air is displaced, the DSM volume that exceeds the volume below the insert is displaced through the holes at the top end of the insert. Alternatively, the DSM is added above the insert, the tube cap is replaced and the tube is shaken with a vigorous downward motion to drive the DSM into the insert.
    [0076] 1. Despejar ou estratificar a amostra de sangue diluído no topo de Ficoll-Paque 2. Centrifugar por 10, ou 20 minutos em 1200xg, temperatura ambiente, com o freio ligado. 3. Remover as células enriquecidas da interface Ficoll:plasma pipetando para fora interface ou vertendo para fora volume acima de inserção de tubo. 4. Lavar células enriquecidas com 5-10 x volume de PBS + 2 % de FBS. Once the DSM is loaded, the tubes are ready for use in density gradient separations with or without the addition of RosetteSep ™. When using RosetteSep ™, the reagent is incubated with the sample, as described in Example 4. The density separations then proceed as follows: 1. Pour or stratify the diluted blood sample on top of Ficoll-Paque 2. Centrifuge for 10, or 20 minutes at 1200xg, room temperature, with the brake on. 3. Remove the enriched cells from the Ficoll interface: plasma by pipetting out the interface or pouring out the volume above the tube insert. 4. Wash cells enriched with 5-10 x volume of PBS + 2% FBS.
    [0077] The effects of changes in spin speed are shown in Table 4. 10 min of spin was sufficient for effective separation.
    [0078] The effect of putting the centrifuge brake on is shown in Table 5. Surprisingly, it was observed that the brake on can be when using these inserts because it does not matter if the plasma interface: DSM is disturbed at the end of the centrifuge step, in contrast with a standard density separation.
    [0079] After density separation, some red coloration of the upper surface of the insert was observed. This stain covered 0 to 80% of the insert surface and can be easily removed by washing the insert with a fresh volume of buffer. The resulting suspension was shown to be almost entirely red blood cells, by first counting the cells on a hemocytometer and then counting the cells after adding 1% acetic acid that smooths the outer cell membrane, but not the nuclear membrane. There were essentially no visible cells after the addition of acetic acid. Thus, cells maintained for insertion are not expected to affect cell enrichment. The use of biocompatible materials that resist cell adhesion will further improve the performance of the inserts.
    [0080] The effect of different bubble volumes under the insertion shown in Figure 4 on cell separation performance is shown in Table 6. Blood samples were incubated with RosetteSep ™ CD3 enrichment cocktail prior to density separation. The smallest bubble (about 1 ml in volume) spreads around the circumference of the insert, but it was not entirely around the tube. The 3 ml volume bubble runs all the way around the tube. The medium sized bubble is the largest bubble one would expect to see below the insert even with relatively careless addition of the DSM. The data show that the volume of air under the insertion can be quite significant (greater than about 8 ml) without affecting cell separation. This is probably because the air escapes in a relatively short time after the start of the centrifugation and the density of the DSM under the insert is only slightly modified and perhaps only in the top portion of the DSM, whose portion is then displaced back above the inserts like the RBC rests during separation.
    [0081] Commercially available Lymphoprep ® tubes (available from Axis-Shield, Norway), and Leucosep ® tubes (available from Greiner Bio-One, Monroe, NC) 50 ml tubes were compared to tubes containing the inserts shown in Figure 4, using standard density gradient separation (as set out in Example 3) and with the addition of RosetteSep ™ (as defined in Example 8). The only difference in treatment between the tubes was that the tubes containing the inserts described here were only centrifuged for 10 min, instead of the standard 20 for commercially available tubes. This was done to reduce the total separation time.
    [0082] The density separation was performed using Ficoll-Paque Plus (GE Healthcare). Nucleous cell recovery was determined for each type of tube and then normalized to the recovery obtained in the standard 50 ml control separation tube. The data in Table 8 shows that recovery is similar across all tubes and no different than for a standard density separation.
    [0083] For RosetteSep ™ separations, the purity of the desired cell population was determined at the beginning and the samples enriched by flow cytometry, while the recovery of the desired cells was determined from the measured purities and cell counts for the initial and enriched samples. RosetteSep ™ cocktails were used to enrich NK cells, B cells, monocytes, CD4 + T cells, CD8 + T cells and CD3 + T cells. The data in Table 9 shows that the purity and recovery are comparable for all tubes. The tubes containing the inserts as described here in Figure 4 exhibited similar recovery and purity compared to competing tubes, despite a shorter centrifugal separation time.
    [0084] A model system was used to evaluate the effectiveness of the present invention used with RosetteSep ™ to enrich tumor cells circulating in the blood. The CAMA breast adenocarcinoma cell line was seeded in about 1% or 0.1% whole blood. The samples were then incubated with a RosetteSep ™ CD45 depletion cocktail for 10 or 20 minutes and then either 1) carefully stratified on Ficoll-Paque Plus in a standard 50 ml centrifuge tube, centrifuged at 1200xg for 20 minutes with the brake off, and carefully removed, or 2) quickly pipetted into a 50 ml centrifuge tube with an insert as shown in Figure 4, which contains Ficoll-Paque Plus, and centrifuged at 1200xg for 10 minutes with the brake on, and simply poured out. CTC purity was assessed by baseline flow cytometry analysis and enriched fractions for nucleated cells expressing EpCAM, and recovery was determined using purity and cell counts. The results presented in Table 10 demonstrate that the enrichment of the CAMA cells was equivalent, or better, using centrifuge tubes with inserts of the invention when comparing equal cocktail incubation times. Purity and recovery are generally higher for separations using the tube + insert compared to the tube only with the same cocktail incubation time.
    [0085] In addition, similar results can be achieved using the tube + insert with a shorter cocktail incubation time (10 min) compared to using the tube alone with a higher incubation time (20 min.)
    [0086] Inserts as shown in Figure 4 made of polypropylene (density = 0.90 g / cm3), Tritan ™ (density = 1.18 g / cm3), Lexan ™ (density = 1.2 g / cm3) and a high-grade polypropylene density (density> 1.1 g / cm3), were inserted into 50 ml conical centrifuge tubes (Greiner Bio-one, Germany). The tubes were filled with 15 ml of Ficoll-Paque ™ (GE Healthcare) as described in Example 8. Ficoll-Paque ™ has a density of 1.077 g / cm3. The tubes were then completely filled by adding an additional volume of about 35 ml of water using a serological or pouring pipette. To test whether the inserts had any effect on the centrifuge tubes and whether the inserts performed differently at different speeds of rotation, the tubes were centrifuged at speeds ranging from 2300-3500 rpm in a Beckman Coulter centrifuge (with an angular acceleration resulting from about 1200 to 2400 times the acceleration of gravity). Under normal conditions for density gradient separation (ie 2300 rpm or less), there were no adverse events with the tubes. However, when centrifuging at higher speeds, polypropylene inserts have sometimes been observed to float on top of the centrifuge tube.
    [0087] Table 11 shows the frequency of floating inserts observed for polypropylene inserts at different spin speeds. Floating inserts were also observed if the centrifuge tubes with the inserts were heated to 50 ° C for 1-3 days before centrifugation. Inserted tubes were stored at -20 ° C, 4 ° C, room temperature and 50 ° C for 1-3 days and then allowed to reach room temperature for at least one day. Table 12 shows the frequency of the floating inserts observed for polypropylene after being stored at different temperatures. It is believed that polypropylene inserts float during high speed centrifugation because under the high pressure generated, the tube expands and the interference fit between the insert and the tube is lost. Storing the tubes at 50 ° C can exacerbate this effect because the plastic tube relaxes a little, reducing the amplitude of the interference fit with the insert. This could be remedied by expanding the diameter of the flange around the circumference of the insert, however, this can also make insertion and / or placing the insert in the centrifuge tube more difficult.
    [0088] The inserts made of Tritan, Lexan and high density polypropylene (density> 1.1 g / ml) were placed in centrifuge tubes and then stored at 50 ° C for 1-3 days. As shown in Table 13, none of the inserts floated after centrifugation, regardless of the spin speed. Inserts made of materials with a density higher than DSM, therefore, are more stable under a variety of separation conditions.
    [0089] Although the present description has been described with reference to what are presently considered to be the preferred examples, it should be understood that the disclosure is not limited to the disclosed examples. On the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
    [0090] All publications, patents and patent applications are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
    权利要求:
    Claims (15)
    [0001]
    Insert (10) for a centrifuge tube (50), the insert (10) comprising: a member (12) sized to fit within the tube to divide the tube into a top portion and a bottom portion, and at least two openings through the member characterized by the fact that a first opening (16) is closer to a bottom end of the tube in relation to a second opening (18) when the insert is positioned in the centrifuge tube, in which the first opening (16) allows fluid communication between a space in the top portion of the tube above the member (12) and a space in the bottom portion of the tube below the member (12) when the insert is positioned in the centrifuge tube and the second opening (18) allows air to escape from the space below the member (12) when the bottom end of the tube is filled with liquid through the first opening (16), wherein the at least two openings (16, 18) are sized to provide surface tension through the openings to prevent liquid contained below the insert from flowing through the openings when the tube is inverted.
    [0002]
    Insertion, according to claim 1, characterized by the fact that: a) the first opening (16) is located in a lower part of a top surface of the member (12) when the insert is positioned in the centrifuge tube; b) the second opening (18) is located in a higher part of a top surface of the member (12) when the insert is positioned in the centrifuge tube; c) the at least two openings (16, 18) are located on a perimeter of the member (12); d) each of the at least two openings (16, 18) has an area of less than 15%, less than 10%, or less than 5% with respect to a cross-sectional area of the centrifuge tube; e) the first opening (16) is larger in relation to the second opening (18); and / or f) the first opening (16) has a diameter of about 1 mm to about 5 mm and the second opening (18) has a diameter less than about 2 mm.
    [0003]
    Insertion according to either of claims 1 or 2, characterized by the fact that it further comprises a support (14) extending or hanging from the member (16) for positioning the member inside the tube, in which: a) the support (14) contacts the bottom end of the tube when the insert is positioned in the centrifuge tube; b) the centrifuge tube has a conical bottom end and the support (14) contacts the conical bottom end when the insert is positioned in the centrifuge tube; c) the support (14) limits a distance between the member (12) and the bottom of the tube when the insert is pushed down towards the bottom end of the centrifuge tube; d) the support (14) resists being compressed when the insert is pushed down towards the bottom end of the centrifuge tube; e) the support (14) comprises a cylindrical side wall extending from a bottom surface of the member (12); and / or f) the support (14) comprises one or more legs extending from a bottom surface of the member.
    [0004]
    Insertion according to any one of claims 1 to 3, characterized in that at least part of a top surface of the member (12) forms an angle between 5 degrees and 75 degrees, optionally between 15 degrees and 65 degrees, with a plane perpendicular to a longitudinal axis of the centrifuge tube when the insert is positioned in the centrifuge tube, and optionally where the top surface of the member (12) from the first opening (16) to the second opening (18) forms an angle between 5 degrees and 75 degrees with a plane perpendicular to a longitudinal axis of the centrifuge tube when the insert is positioned in the centrifuge tube.
    [0005]
    Insertion according to any one of claims 1 to 4, characterized in that at least part of a top surface of the member (12) has a concave characteristic when the insertion is positioned in the centrifuge tube.
    [0006]
    Insertion according to claim 5, characterized by the fact that the concave characteristic has the shape of a cone or parabola or a section of a cone or parabola and / or the concave characteristic is inclined from a point on the perimeter of the member (12) for a first opening (16).
    [0007]
    Insertion according to any one of claims 1 to 6, characterized in that the member (12) forms an interference fit with a side wall of the centrifuge tube when the insertion is positioned in the centrifuge tube, optionally in which the member (12) extends radially outwardly from the support to form a flange.
    [0008]
    Insertion according to any one of claims 1 to 7, characterized in that the insertion density is greater than 1.08 g / cm3, or greater than 1.1 g / cm3, and optionally in which the entire part of the insert is made of polycarbonate, copolyester, polytetrafluoroethylene or high density polypropylene.
    [0009]
    Insert according to any one of claims 1 to 8, characterized in that the centrifuge tube is a 50 ml centrifuge tube or a 15 ml centrifuge tube.
    [0010]
    Centrifuge tube characterized by the fact that it comprises the insert as defined in any one of claims 1 to 9, optionally in which the insert is integral to the centrifuge tube.
    [0011]
    Method for separating a target population of cells from a sample, characterized by the fact that it comprises: a) provide a centrifuge tube with the insert, as defined in any one of claims 1 to 10, b) filling a portion of the centrifuge tube with the density separation means (DSM), such that the DSM covers the top of the insert; c) adding a sample volume containing the target cell population to the centrifuge tube in order to form an interface between the DSM and the sample; d) rotate the centrifuge tube to separate the target population of cells from the sample, optionally rotate the centrifuge tube in a centrifuge with a centrifuge brake on during deceleration.
    [0012]
    Method according to claim 11, characterized by the fact that the target population comprises at least one cell type selected from mammary cells, stem cells, ES cells, tumor cells, cancer cells, immune cells, hematopoietic stem cells, leukocytes and peripheral blood mononuclear cells, and / or the sample comprises whole peripheral blood.
    [0013]
    Method according to claim 11 or 12, characterized by the fact that the target cell population has an average density less than the DSM density, and the method further comprises recovering the target cell population from a sample volume above the interface between the DSM and the sample, optionally by reversing the centrifuge tube to remove a sample volume comprising the target population of cells from the centrifuge tube, where the DSM below the insert remains in the centrifuge tube.
    [0014]
    Method according to any one of claims 11 to 13, characterized in that it further comprises the connection of dense particles to a second population of cells in the sample before turning the centrifuge tube, wherein the second population of cells connected to the particle separates below the interface between the DSM and the sample, and the target cell population separates towards the interface between the DSM and the sample, optionally where the dense particles are selected from red blood cells, silica particles, metal particles, metal oxide particles, polymer particles and glass particles, and / or the second population of cells is defined by specific surface proteins and dense particles are linked to the second population of cells by means of specific antibodies to cell surface proteins.
    [0015]
    Kit, characterized by the fact that it comprises the insert as defined in any one of claims 1 to 9, and a centrifuge tube, optionally further comprising a volume of density separation medium, and / or printed instructions for carrying out the method as per defined in any one of claims 11 to 14.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US5248480A|1992-05-28|1993-09-28|Diasys Corporation|Apparatus for drawing fluid sample and components thereof|
    US5455009A|1993-09-14|1995-10-03|Becton, Dickinson And Company|Blood collection assembly including clot-accelerating plastic insert|
    US5663051A|1994-08-31|1997-09-02|Activated Cell Therapy, Inc.|Separation apparatus and method|
    US5840502A|1994-08-31|1998-11-24|Activated Cell Therapy, Inc.|Methods for enriching specific cell-types by density gradient centrifugation|
    CN101185918A|2007-12-12|2008-05-28|湖南大学|Mechanism using simple glass centrifuge tube to implement solid-liquid high-speed centrifugation separation|
    ES2754033T3|2008-07-21|2020-04-15|Becton Dickinson Co|Density phase separation device|US9625360B2|2012-11-30|2017-04-18|Rarecyte, Inc.|Apparatus, system, and method for collecting a target material|
    US9539570B2|2012-11-30|2017-01-10|Rarecyte, Inc.|Apparatus, system, and method for collecting a target material|
    US9956555B2|2012-11-30|2018-05-01|Rarecyte, Inc.|Apparatus, system, and method for collecting a target material|
    US9039999B2|2012-11-30|2015-05-26|Rarecyte, Inc.|Apparatus, system, and method for collecting a target material|
    US9533303B2|2012-11-30|2017-01-03|Rarecyte, Inc.|Apparatus, system, and method for collecting a target material|
    US10054524B2|2012-11-30|2018-08-21|Rarecyte, Inc.|Apparatus, system and method for collecting a target material|
    US9513291B2|2012-11-30|2016-12-06|Rarecyte, Inc.|Apparatus, system, and method for collecting a target material|
    US9945839B2|2012-11-30|2018-04-17|Rarecyte, Inc.|Apparatus, system, and method for collecting a target material|
    CN104994818B|2012-12-21|2020-04-17|波士胶公司|Method for dosing super absorbent particles|
    DE102013103153B4|2013-03-27|2016-09-08|Andreas Hettich Gmbh & Co. Kg|Support device for a Zentrifugigutbehälter|
    ES2646301T3|2013-11-14|2017-12-13|Greiner Bio-One Gmbh|Receiving device, procedure for making it available and procedure for separating a mixture|
    CN104031831B|2014-07-01|2019-10-15|北京圣浦博大生物科技有限公司|A kind of separation method of haemocyte separating pipe and mononuclearcell|
    GB201418382D0|2014-10-16|2014-12-03|Ge Healthcare Uk Ltd|Device and method for separating cells|
    US20160223557A1|2015-02-04|2016-08-04|Oregon Health & Science University|Method of isolating circulating tumor cells|
    US20180250669A1|2015-09-15|2018-09-06|Spark-Tech Sp. Z O.O.|Device and method for fluids separation by density gradient|
    PL237582B1|2015-09-15|2021-05-04|Spark Tech Spolka Z Ograniczona Odpowiedzialnoscia|Insert and method for separation of fluids, using the density gradient|
    AU2016359598B2|2015-11-24|2021-10-07|Royal Biologics|Methods and apparatus for separating fluid components|
    CN107398358B|2016-05-19|2020-04-21|阿尔法韦士曼公司|Centrifugal rotor core with partial channels|
    EP3575788A4|2017-01-27|2020-10-14|Foundation for Biomedical Research and Innovation at Kobe|Mononuclear cell separating device and mononuclear cell separating method|
    WO2018231960A1|2017-06-13|2018-12-20|Veterinary Diagnostics Institute, Inc.|System and procedure for stabilizing, storing and recovering blood samples|
    EP3844478A1|2018-08-31|2021-07-07|Thermogenesis Corp.|Methods for isolating target cells from blood|
    CN110935570A|2019-11-07|2020-03-31|曾彩霞|Inclined biological centrifuge for multistage separation of light substances|
    CN111974475B|2020-08-20|2021-09-21|四川行之智汇知识产权运营有限公司|Blood component separator|
    法律状态:
    2018-12-11| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2019-10-22| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|
    2020-05-05| B09A| Decision: intention to grant|
    2020-07-21| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 03/05/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    2020-09-15| B09W| Decision of grant: rectification|Free format text: REFERENTE A RPI 2574 DE 05/05/2020 |
    2021-03-30| B16C| Correction of notification of the grant|Free format text: REF. RPI 2585 DE 21/07/2020 QUANTO AO RD. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201161482886P| true| 2011-05-05|2011-05-05|
    US61/482,886|2011-05-05|
    PCT/CA2012/000418|WO2012149641A1|2011-05-05|2012-05-03|Method and insert for density gradient separation|
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