JP6364946B2 - Separation structure and separation method - Google Patents

Description

  The present invention relates to a separation structure that separates a target component from a liquid sample using centrifugal force and a separation method using the separation structure, and particularly preferably a body fluid, a dispersed tissue specimen, or a cultured cell. The present invention relates to a separation structure and a separation method for separating and concentrating cells from a sample containing such cells.

  Research is being carried out in which only cells are separated from biological fluids such as body fluids and cell culture fluids, or from dispersed tissue specimens in which tissues are suspended and dispersed, and the separated cells are applied to clinical diagnosis and treatment. For example, morphological analysis, tissue type analysis, and genetic analysis are performed on tumor cells separated from blood and tissues, and therapeutic agents are selected based on the findings.

  As a method for separating cells from blood or the like, a density gradient centrifugation method is known in which cells are separated based on their density (Patent Document 1). This method is unnecessary by overlaying a solution containing cells such as blood on a solution having a density gradient (hereinafter referred to as a density gradient solution) and centrifuging, and collecting the layer containing the target cells. Cell fractions free from other cells and other components contained in blood. In addition, a centrifuge tube having a contraction region installed in the tube is used as a centrifuge container, and the upper liquid is collected without mixing the bottom liquid when decantation is performed after centrifugation. A container (Patent Document 3) that achieves the same effect as Patent Document 2 by a centrifugal partition tube (Patent Document 2), a partition wall plate, a porous partition plate such as a filter or a sieve, and a laminate of an upper and lower chamber and a filter membrane. The filtration device (patent document 4) containing is known.

  As the density gradient solution, a commercially available product (for example, Ficoll-Paque (manufactured by GE Healthcare Bioscience), a density of about 1.077 g / mL, a synthetic product having a molecular weight of 400,000 obtained by copolymerizing sucrose and epichlorohydrin) However, by using a density gradient solution with a density larger than that of the target cells, viruses, organelles, membrane-bound granules, etc., the cells are maintained on the density gradient solution. Can be separated from other, more dense components. Ficoll itself has an agglutinating action and can aggregate and polymerize and precipitate red blood cells and granulocytes contained in blood, so that cells and the like can be more effectively separated from blood. .

International Publication No. 1997/021488 Japanese Patent No. 339795 Special table 2002-536635 International Publication No. 2012/154257

  For the density gradient centrifugation, a separation container having a size and shape that matches the rotor shape of the centrifugal separation means to be used is used, but the separation container is usually closed at one end to form the bottom, and the other end is sealed at the opening. It is generally cylindrical. For this reason, when the cells that have moved in the density gradient solution according to the density are collected from the separation container after the centrifugation operation is completed, the tip of the pipette is inserted through the open end of the sealed container, Although a thin fraction will be collected, this operation requires skill, and the recovery rate of the target cells is likely to fluctuate. The tip of the pipette is inserted into the density gradient solution, and the target cells, etc. There was a problem that cells other than those may be collected by mistake.

  Therefore, in order to solve such a problem, the object of the present invention is to separate the unnecessary components from the separated cells together with the structure, thereby improving efficiency regardless of the skill level of the operator and / or the target cell density. An object of the present invention is to provide a separation structure (separation solution) that can be recovered automatically.

Specifically, after separating cells and the like with a structure employing the configuration of the present invention, the target cells are located in one of the upper layer / middle layer / lower layer of the separation liquid depending on their density. In other words, since the structure of the present invention is composed of two or more cylindrical members that can be separated by the separation part, this structure can be used regardless of the skill level of the operator regardless of the layer in which the target cell is located. It can be recovered by a simple operation of separating the structure.
More specifically, in the example of separating blood with Ficoll-Paque (density about 1.077 g / mL), unnecessary cells (red blood cells) are located in the lower layer, and target cells (cancer cells, white blood cells, etc.) are It will be located in the upper layer. In other words, the separation structure adopted as a conventional method for concentrating / separating blood cells is composed of one cylindrical member, and the idea of the separation structure that separates the upper layer and the lower layer by a partition structure or the like is basically It is limited to the collection of cells located in the upper layer.

  The present invention completed in view of the above object comprises a cylindrical structure having one end closed to form a bottom and the other end opened, and a cap that seals the opening. The structure has two or more cylinders. It is a separation structure characterized in that it is composed of a member and can be separated at the separation portion. The present invention also relates to a separation method for separating a target component from a liquid sample, particularly a method for isolating a target type of cell from two or more types of cells having different densities, wherein the density gradient solution has the structure described above. Filling from the bottom of the body to the vicinity of the separation part, centrifuging with the sample overlaid and sealing the opening, and separating the structure after centrifugation while maintaining the sealed state of the opening, This is a cell separation method comprising a step of collecting a fraction containing the target cells and the like. Hereinafter, the present invention will be described in detail based on the drawings.

  FIG. 1 illustrates one embodiment of the present invention. The separation structure 1 of this example is composed of two cylindrical members 2 and 3. A cylindrical member (upper side) 2 (sometimes referred to as “cylindrical member 2”) constituting the upper portion of the separation structure has an opening (in this figure, the opening is sealed by a sealing cap 4). As shown, the cap 4 may be attached to the separation structure 1 so as to be detachable. In addition to the cap type (type to be inserted into the cylindrical member 2) shown in FIG. The cylindrical member (lower side) 3 (which may be referred to as “cylindrical member 3”) is closed at one end to form the bottom portion 5.

  The cylindrical members 2 and 3 are each provided with a communication opening end (separation part) at the opposite end of the opening or the bottom, and when the both members are connected, the internal spaces of both the cylindrical members communicate with each other. One isolation structure is formed. This connection is only required to be able to separate the respective cylindrical members. For example, in addition to a mode in which the respective communication openings are fitted to each other, one (cylindrical member 2) as shown in FIG. For example, it can be inserted into the other (tubular member 3), a screw or the like is provided, or a joint member for fixing and holding both is attached.

  The communication opening end (separation part) 6 itself is large enough to pass a high-density component in the sample layered on the density gradient solution, and separates the cylindrical member while maintaining the sealed state by the cap 4. It is sufficient that the liquid held in the cylindrical member 2 does not flow out when it is done. Note that a filter or a mesh-shaped filter medium in which a vertical through hole is formed can be installed in either of the communicating openings of the tubular member 2 or the tubular member 3. By installing such a filter or the like, for example, when the structure collapses after the centrifugation operation, or when vibration is applied along with the transfer to the centrifugation means, the density gradient solution and the sample are mixed, or after the centrifugation operation It is possible to prevent the low density component maintained in the cylindrical member 2 and the high density component moved to the cylindrical member 3 from being mixed. In this example, the shape of the portion of the tubular member 2 reaching the communication opening end (separation portion) 6 is tapered toward the tubular member 3 (the tapered portion 7 of the tubular member 2). By making the cylindrical member 2 have such a shape toward the communication opening, it is possible to obtain the same effect as that of providing a filter, as shown in Examples described later, which is preferable.

  The cylindrical member of this example is not particularly limited in terms of dimensions and shape, but all are generally cylindrical. The shape may be a polygonal shape as long as it matches the rotor of the centrifuge used, but a cylinder is particularly preferable from the viewpoint of manufacturing, storage and the like.

  The cylindrical members 2, 3 and the cap 4 can be made of a resin such as acrylic, epoxy, polystyrene, synthetic quartz (SiO2) mainly composed of silicon oxide, ceramics, or a metal-based material. From the viewpoint of economic efficiency, and particularly from the problem of discardability after using a biological sample such as blood, a thermoplastic resin such as polypropylene or polystyrene resin is preferred. In addition, the inner surface of the separation structure (surface on which the sample can come into contact) is hydrophilic from the viewpoint of preventing non-specific adsorption of target cells or the like, or can be hydrophilized by a separate treatment. It is preferable that The treatment for hydrophilization is not particularly limited, and if it is a resin, for example, a method of activating the surface energy of the resin surface by corona discharge treatment to generate a polar group such as a carbonyl group and hydrophilizing, or, for example, A method of improving the hydrophilicity of the surface by irradiating the surface with electrons, ions, and radicals by oxygen plasma treatment and introducing -COOH or -CO can be exemplified. Moreover, for example, a method of applying a hydrophilic polymer such as polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, poly (hydroxyalkyl) methacrylate, polyacrylamide, or MPC polymer to make the surface hydrophilic can be exemplified.

  Next, the separation method of the present invention using the separation structure described above will be described. The present invention relates to biological samples such as urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, amniotic fluid, samples derived from organs and tissues such as cell aggregates, tumors, lymph nodes or arteries, cells It can be used to separate cells contained in a culture solution or the like. For organs and tissues, the separation method of the present invention may be carried out after preparing a cell suspension according to a normal treatment. In the present specification, the intended cell separation is synonymous with the concentration of the cells. That is, after the centrifugation operation, it is not possible to collect the target cells of the fraction that is maintained on the density gradient solution or the fraction that passes through the density gradient solution and moves below it. It is none other than the concentration of target cells by removing unintended components contained in the painting.

  A density gradient solution is a liquid substance that forms a density gradient by itself or by centrifugation. If a density (specific gravity) of a target cell is specified and an appropriate one is selected and used for the separation, good. Examples of selection indicators include nutrient components, pH, and isotonicity. Specifically, sucrose, glycerol, dextran, metrizamide, iodixanol, copolymer of sucrose and epichlorohydrin, colloidal silica particles with polyvinylpyrrolidone coating, sucrose polymer, diatrizoic acid, iohexol, nicodents Examples thereof include ionic or nonionic ones. Examples of commercially available density gradient solutions include trade names Ficoll, Ficoll-Paque or Percoll manufactured by GE Healthcare Biosciences, Inc., trade names Lymphoprep, Polymorphprep, or OptiPrep manufactured by Axis-Shield PoC AS.

  In the separation structure 1, the density gradient solution is injected from the bottom (bottom 5 of the cylindrical member 3) to the vicinity of the communication opening end (separation) 6. More specifically, when the separation structure 1 is allowed to stand, the liquid level height of the density gradient solution is higher than the communication port end of the upper cylindrical member 2 (on the cylindrical member 2 side). To do. That is, when a non-target component (cell or the like) in the sample solution is separated into the lower cylindrical member (tubular member 3), the target component (cell) maintained on the density gradient solution is cylindrical. Injection is performed so as to be as high as possible, preferably about 1 mm, while being maintained in the member 2.

  Thereafter, the sample solution is layered on the density gradient solution, the opening is sealed with the cap 4, and a centrifugal separation operation is performed. Centrifugation is generally performed at a low speed of about 1000 to 2000 × g, but the conditions maintained on the density gradient solution in consideration of the density of the target cells and the density gradient solution to be used. Select. For example, if the target cell is a tumor cell and centrifugation is performed as described above, the density of the density gradient solution is in the range of 1.060 to 1.095 g / mL depending on the type of tumor cell, physiological penetration. Examples include adjusting the pressure to a range of 200 to 450 mOsm / kg and adjusting the pH to a range of 6.8 to 7.8.

  By the centrifugal separation operation, the component having a density larger than that of the density gradient solution passes through the gradient layer of the density gradient solution and moves into the cylindrical member 3. On the other hand, target cells having a density smaller than that of the density gradient solution are maintained on the density gradient solution in the cylindrical member 2. Therefore, if the connected cylindrical members are maintained in the state shown in FIG. 1 while maintaining the sealing of the opening, the upper cylindrical member (cylindrical member 2) contains the target cells. The image can be collected. This fraction can be easily collected without requiring any special skill, for example, by dropping the cap 4 by releasing the cap 4 and dropping it downward. On the other hand, the fraction moved into the cylindrical member 3 can be discarded together with the cylindrical member, for example.

  As described above, the present invention has been described according to the embodiment shown in FIG. 1, but the separation structure of the present invention can also be composed of three or more cylindrical members. In this case, one of the cylindrical members is provided with a hermetically sealed opening, and one cylindrical member different from this forms a closed bottom, and other than these, communication openings are provided at both ends. It becomes the cylindrical member which has. If such a cylindrical member is used and the place of separation is changed according to the density of the target cell or the density gradient solution, the cell can be separated for each type (density) of the target cell. Is possible.

  Hereinafter, a separation structure that employs a configuration composed of three separable members will be described with reference to FIG. 11, but is not limited to a configuration composed of three separable members. As an effect of using a separation structure that employs a structure consisting of three separable members, it is possible to separate the target components (cells, etc.) for each type (density) of the target components (cells, etc.) The density distribution can be confirmed.

  In FIG. 11, first, a sample solution containing a target component (cell or the like) whose density is to be known is injected into a cylindrical member (upper side) 41, and the cylindrical member (center) 42 and the cylinder that are other cylindrical members are injected. The member (lower side) 43 is filled with a density gradient solution having a high density (for example, about 1.086 g / mL), and then centrifuged. The maximum density of the target component (cell or the like) can be found by finding a condition that the target component (cell or the like) can be recovered only by the cylindrical member (upper side) 41 by the above operation.

  Subsequently, the cylindrical member (center) 42 has a low density (for example, about 1.030 g / mL) density gradient solution and the cylindrical member (lower side) 43 has a target component (cell Etc.) and fill with a density gradient solution corresponding to a maximum density condition (eg, about 1.086 g / mL) and centrifuge. Similarly, the minimum density of the target component (cells, etc.) can be determined by finding a condition that allows the target component (cells, etc.) to be recovered only in the cylindrical member 42 (center). In this way, it is possible not only to simply separate the target components (cells etc.) and density gradient solution but also to know the properties (density) of the target components (cells etc.).

  In the present invention, by adding a substance that specifically binds to the target component (cells, etc.) or by adding a substance that specifically binds to a non-target component (cells, etc.) The components can be separated more efficiently. As shown in FIG. 2, a substance 22 that specifically binds to a non-target component (such as a cell) 21 (may be described as “non-target component 21”), and a substance for adjusting the density 23, the apparent density (specific gravity) of the non-target component 21 is increased by the combination of the two, and the target component (cell etc.) 24 (“target component 24” ”), The two can be separated efficiently. It should be noted that the apparent density can be reduced by combining a substance that specifically binds with a substance having a relatively low density such as porous silica particles. Thus, as a substance used for the purpose of adjusting the density, in addition to the porous silica particles, for example, polyvinyl compounds such as polyethylene, polypropylene, polyvinyl chloride, polyacrylonitrile, polyacrylate, polymethacrylate, and polycarbonate. Typical organic polymers, copolymers such as polystyrene latex, nylon and polyterephthalate, inorganic materials such as glass, silica and zirconia, and biopolymers such as cellulose, dextran, agarose, cellulose and sepharose Examples of the substance that specifically binds to a biopolymer such as an antibody, an antigen, a peptide, a polypeptide, a growth factor, a cytokine, and a lectin.

  Further, after separation with the separation structure of the present invention, the target component (cells and the like) can be more selectively recovered by further removing the non-target components (cells and the like). For example, by passing the solution after density gradient centrifugation through a filter, the non-target component can be passed through the filter, and the target component can be captured by the filter and concentrated. Conversely, the target component may be passed through a filter, and the non-target component may be captured by the filter. The through-hole formed in the filter can be appropriately adjusted depending on the intended use. For example, when capturing cancer cells (about 20 μm) in the filter, it is preferable that the opening has a circular shape, and the pore diameter is from 1 It is 20 μm, preferably 1 to 10 μm, particularly preferably 2 to 8 μm. If the pore size is smaller than 1 μm, the filter may be clogged, increasing the suction pressure near the filter and destroying the cells. On the other hand, when it becomes larger than 10 μm, small-diameter cancer cells may pass through the filter and be lost. Although there is no restriction | limiting in particular about the number and arrangement | positioning of a through-hole, In order to improve cell separation efficiency, providing many through-holes is preferable. When providing a large number of through holes, it is preferable that the distance between the through holes (the distance from the center of the opening of a certain through hole to the center point of the opening of another through hole) is equal. The distance between the through holes can be appropriately determined in consideration of the hole diameter of the through hole, but it can be exemplified as 20 μm or more. More specifically, for example, when the hole diameter is 8 to 10 μm, the distance between the through holes is particularly preferably about 50 μm.

  Specific examples of the filter in which the through hole described above is formed include a nickel substrate in which a through hole is formed using an electroforming technique, and a glass or quartz substrate in which a through hole is formed using a laser technique. With any technique, a through-hole as intended can be formed in a nickel substrate or the like.

  The filter is preferably hydrophilic. This is because adsorption of components contained in the sample is prevented and clogging or the like hardly occurs due to such properties. For this purpose, the filter may be manufactured from a material that is inherently hydrophilic, or its surface may be hydrophilized by any treatment. There is no particular limitation on the treatment for hydrophilizing the surface. Corona discharge treatment, plasma treatment, hydrophilic polymer coating, BSA (bovine serum albumin) or OVA (egg white) described in the method for hydrophilizing the separation structure of the present invention are not used. An example is a method of hydrophilizing the surface by dipping in a protein solution such as ovalbumin).

  In addition, as a method for more selectively separating target cells, the target components are bound by antibody magnetic particles by reacting the cells collected after density gradient centrifugation with antibody magnetic particles, and then the target components are magnetically combined. Can be captured. Conversely, after binding the antibody magnetic particles to the non-target component, the non-target component may be removed by magnetic force. The antibody magnetic particles used at that time are preferably formed of an appropriate material that does not react with the target component. For example, when collecting cancer cells from blood, leukocytes, which are non-target components contained after density gradient centrifugation, are captured by antibody magnetic microparticles. The type of antibody of the antibody magnetic particle for removing the non-target component is selected from antibodies against surface markers that are expressed in the non-target component and not expressed in the target component (cells or the like). For example, when removing leukocytes from blood, CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD13, CD14, CD16, CD19, CD20, CD22, CD23, CD33, CD34, CD36, CD41, It is preferable to select from CD42, CD45, CD45RA, CD45RO, CD56, CD66b and the like.

  Further, when there are two or more non-target components, it is possible to concentrate using a binder that binds the non-target components. For example, when a very small amount of cancer cells are separated from blood or when a part of leukocyte components is separated, it is concentrated using a binding agent (for example, RosetteSep (manufactured by StemCell Technologies)) that can bind to red blood cells and white blood cells. can do. The binding agent only needs to be capable of binding to one or more white blood cells or red blood cells, or to bind to a cell surface antigen, and can aggregate white blood cells or bind white blood cells to red blood cells. Any antibody may be used. Particles (cell aggregates) that have been increased in density by the binder can be separated from the desired component (eg, cancer cells, etc.) during centrifugation.

  In the separation structure and the separation method of the present invention, after the centrifugation operation, when collecting the fraction containing the target cells and the like, the separation structure is separated by separating unnecessary fractions together with the separation structure. Only a very simple operation of opening the seal of the tubular member is performed. Therefore, inserting the tip of the pipette from the open end of the separation container, compared to collecting a very thin fraction on the density gradient solution, skill is not required, the target cells and the like can be stably recovered, There is an effect that the pipette tip is inserted into the density gradient solution, and there is a low possibility that the cells other than the target cells and the like are erroneously collected. Such an effect makes it possible to separate cells that do not depend on the skill of the operator, and it is also possible to automate the separation operation of the target cells and the like by machine operation.

  Moreover, the separation structure and the separation method of the present invention adopt a configuration comprising two or more separable members, so that unnecessary components are separated together with the structure, and each cell type (density) of the target cell is separated. Can be efficiently recovered.

It is a figure for demonstrating the isolation | separation structure of this invention. It is a figure for demonstrating the separation method of this invention. It is a figure for demonstrating the separation method of this invention. It is a figure which shows the isolation | separation structure used in Example 1 of this invention. It is a figure for demonstrating the measuring method of the cell implemented in Example 1, 2 and the comparative example of this invention. It is a figure which shows the cancer cell collection | recovery rate in Example 2 of this invention. It is a figure which shows the cancer cell collection | recovery rate in Example 4 of this invention. It is a figure which shows the cancer cell collection | recovery rate in Example 5 of this invention. It is a figure which shows the cancer cell collection | recovery rate in Example 6 of this invention. It is a figure which shows the cancer cell collection | recovery rate in Example 8 of this invention. It is a figure which shows the isolation | separation structure used in Example 10 of this invention. It is a figure for demonstrating the separation method of Example 10 of this invention. It is a figure which shows the cancer cell collection | recovery rate in Example 10 of this invention. It is a figure which shows the cancer cell collection | recovery rate in Example 11 of this invention.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to an Example.

Basic Principle Cancer cells were separated using the separation structure of the embodiment shown in FIG. The separation structure is
Specifically, the cylindrical member 2 is a cylindrical polypropylene member having an inner diameter of 16 mm, a length of 80 mm, and a capacity of 13 mL. The inner surface of the member is coated with bovine serum albumin (BSA),
Non-specific adsorption of cells and the like is prevented by making it hydrophilic. The inclination angle of the tapered portion 7 of the tubular member 2 is 30 °, and the communication opening with the tubular member 3 is Φ2 mm. Tubular member 3
Is a polypropylene member having an inner diameter of 10 mm, a length of 43 mm, and a capacity of 2 mL.

As schematically shown in FIG. 3, a density gradient solution 31 (Axis-Shield PoC AS, trade name Lymphoprep) having a density of 1.077 g / mL is applied to the cylindrical member 3 on the lower side of the separation structure. 2 mL was injected (the lower white portion in the figure is the portion filled with the density gradient solution). Specifically, the density gradient solution was injected such that the liquid level height was about 1 mm higher than the communication opening 6 of the upper cylindrical member 2 (therefore, the liquid level was located inside the upper cylindrical member). Subsequently, a mixed solution 32 of 3 mL of blood sample and 3 mL of physiological saline was layered on the density gradient solution (in the figure, the black coating portion is a portion of the mixed solution). The blood sample is a suspension in which about 30 human cancer cells are suspended in the blood of a healthy person obtained by obtaining informed consent. The cancer cells to be added are previously labeled with a fluorescent staining reagent (manufactured by Dojindo Laboratories, Inc., trade name Calcein AM). The cancer cells were statically cultured so that the cell density was about 2 × 10 ⁵ cells / cm ^2, and the cells were detached from the dish with 0.25% trypsin / 1 mM EDTA and adjusted by limiting dilution. Is.

  After sample injection, the opening of the separation structure was sealed with a cap 4 (made of polypropylene), and centrifuged at 1100 × g for 10 minutes at room temperature. As shown in the left of FIG. 4, the cells were maintained at the density gradient solution / sample interface (the top of the Lymphoprep solution) 33 by the centrifugation operation. After separating the cylindrical members 2 and 3 constituting the separation structure without removing the cap at the separation portion, the upper cylindrical member 2 is opened by removing the cap and opening the seal as shown in the right of FIG. A part of the density gradient solution and the cells maintained thereon are discharged from the communication opening 6 and collected by a 50 mL tube installed below, and the inner wall of the upper cylindrical member is washed, and the cells attached to the wall are also removed. Collected simultaneously.

  The collected cell suspension is made up to 30 mL with 0.4% sodium citrate / PBS solution, centrifuged at 300 × g for 10 minutes at room temperature, and the liquid at the top of the pellet is removed with a pipette. Of cells were resuspended in 30 mL of 300 mM mannitol solution and centrifuged at 300 × g for 5 minutes at room temperature. This centrifugation operation is for removing cell debris and platelets and concentrating the target cells.

  For the measurement of the collected cells, a method of applying to a slide or observing the cells trapped in a well under a microscope or a flow cytometry method can be used. In Example 1, a method was adopted in which the separated cells were captured and measured in holding holes (about 1 million) having a hole diameter of 30 μm and a depth of 30 μm provided on the substrate by dielectrophoretic force.

Using the apparatus shown in FIG. 5, it was confirmed that the separated cells were the intended human breast cancer cells. The apparatus shown in FIG. 5 has a power source 34 and electrode substrates 35 and 36, and applies a voltage between the substrates to cause a dielectrophoretic force 38 to act on the cells 37, and introduces and captures them in the holding holes 39. (See Japanese Patent No. 4910716). The separated cell suspension was subjected to this apparatus, and the cells captured in the holding holes were observed with a fluorescence microscope 40. As a result, about 27 cancer cells are captured together with about 4 million normal leukocytes in the holding hole (recovery rate is about 90%), and the structure is constituted by using the separation structure of the present invention. The target cells can be separated with a high recovery rate by a simple operation of separating the cylindrical member and removing the cap that seals the opening.

  The same operation as in Example 1 was performed to separate cancer cells. In detail, in the separation structure, the cylindrical member 2 is a cylindrical polypropylene member having an inner diameter of 18 mm, a length of 70 mm, and a capacity of 15 mL. The inner surface of the member is coated with bovine serum albumin (BSA) to make it hydrophilic, thereby preventing nonspecific adsorption of cells and the like. The inclination angle of the tapered portion 7 of the tubular member 2 is 30 °, and the communication opening with the tubular member 3 is Φ2 mm. The cylindrical member 3 is a polypropylene member having an inner diameter of 10 mm, a length of 54 mm, and a capacity of 2 mL.

As schematically shown in FIG. 3, a density gradient solution 31 (Axis-Shield PoC AS, trade name Lymphoprep) having a density of 1.077 g / mL is applied to the cylindrical member 3 on the lower side of the separation structure. 2 mL was injected (the lower white portion in the figure is the portion filled with the density gradient solution). Specifically, the liquid level height of the density gradient solution is about 1 mm higher than the communication opening end (separation part) 6 of the upper cylindrical member 2 (therefore, the liquid level is located inside the upper cylindrical member). ) Injected. Subsequently, a 3 mL blood sample and 3 mL physiological saline mixed solution (blood sample and physiological saline mixed solution (portion of layered mixed solution)) 32 was overlaid on the density gradient solution (black in the figure). The coated part is the part of the mixed liquid that is overlaid). The blood sample is a suspension in which about 30 human cancer cells are suspended in the blood of a healthy person obtained by obtaining informed consent. The cancer cells to be added are previously labeled with a fluorescent staining reagent (manufactured by Dojindo Laboratories, Inc., trade name Calcein AM). The cancer cells were statically cultured so that the cell density was about 2 × 10 ⁵ cells / cm ^2, and the cells were detached from the dish with 0.25% trypsin / 1 mM EDTA and adjusted by limiting dilution. Is.

  After sample injection, the opening of the separation structure was sealed with a cap 4 (made of polypropylene), and centrifuged at 1100 × g for 10 minutes at room temperature. As shown in the left of FIG. 4, the cells were maintained at the density gradient solution / sample interface (the top of the Lymphoprep solution) 33 by the centrifugation operation. After separating the cylindrical members 2 and 3 constituting the separation structure without removing the cap at the separation portion, the upper cylindrical member 2 is opened by removing the cap and opening the seal as shown in the right of FIG. A part of the density gradient solution and the cells maintained thereabove are discharged from the open end (separation part) 6 and collected in a 50 mL tube 50 (hereinafter sometimes referred to as “tube 50”) installed below. At the same time, the inner wall of the upper cylindrical member was washed, and the cells attached to the wall were also collected at the same time.

  The collected cell suspension is made up to 30 mL with 0.4% sodium citrate / PBS solution, centrifuged at 300 × g for 10 minutes at room temperature, and the liquid at the top of the pellet is removed with a pipette. Of cells were resuspended in 30 mL of 300 mM mannitol solution and centrifuged at 300 × g for 5 minutes at room temperature. This centrifugation operation is for removing cell debris and platelets and concentrating the target cells.

  For the measurement of the collected cells, a method of applying to a slide or observing the cells trapped in a well under a microscope or a flow cytometry method can be used. In Example 1, a method was adopted in which the separated cells were captured and measured in holding holes (about 1 million) having a hole diameter of 30 μm and a depth of 30 μm provided on the substrate by dielectrophoretic force.

Using the apparatus shown in FIG. 5, it was confirmed that the separated cells were the target cancer cells. The apparatus shown in FIG. 5 has a power source 34 and electrode substrates 35 and 36, and applies a voltage between the substrates to cause a dielectrophoretic force 38 to act on the cells 37, and introduces and captures them in the holding holes 39. (See Japanese Patent No. 4910716). The separated cell suspension was subjected to this apparatus, and the cells captured in the holding holes were observed with a fluorescence microscope 40. The recovery rates of two types of breast cancer cells (SKBR3, MDA-MB-231), two types of non-small cell lung cancer (PC9, A549), and two types of small cell lung cancer (H69, SBC-1) are shown in FIG. It was 74.6 to 92.4%. In addition, about 4 million normal leukocytes were mixed with cancer cells. By using the separation structure of the present invention, various cell types can be separated with a high recovery rate by a simple operation of separating the cylindrical member constituting the structure and removing the cap that seals the opening.

  As a sample, 3 mL of a suspension obtained by mixing about 30 human breast cancer cells (SKBR3) in blood of a healthy person was subjected to a centrifugation operation in the same manner as in Example 1 to remove the cells from the density gradient solution and the sample. Maintained at the interface. After completion of the centrifugation operation, the cap 2 is removed, the cylindrical members of the separation structure are turned over without being separated, and a part of the density gradient solution and the cells maintained thereon are allowed to flow out from the opening, and the tube 50 In addition, the inner wall of the upper cylindrical member was washed, and the cells attached to the wall were also collected at the same time. At this time, the pellet mainly containing red blood cells moved to the lower structure by the tapered portion toward the communication opening of the upper cylindrical member of the separation structure flows back to the upper cylindrical member. There was no.

Using the apparatus shown in FIG. 5, as in Example 1, it was confirmed that the separated cells were target human breast cancer cells. As a result, about 26 cancer cells are captured along with about 4 million normal leukocytes in the holding hole (recovery rate: about 85%), and the cap that seals the opening by using the separation structure of the present invention. The target cells can be separated with a high recovery rate by a simple operation of removing the structure and falling the structure.

Basic Principle of Aggregation Method Similar to Example 1, a sample prepared by mixing about 30 cancer cells in 3 mL of healthy human blood onto a density gradient solution, 3 mL of physiological saline, and 75 μL of a binder (trademark RosetteSep, StemCell) (Technologies Inc) was overlaid, centrifuged and collected.

Using the apparatus shown in FIG. 5, as in Example 1, it was confirmed that the separated cells were the target cancer cells. The recovery rates of two types of breast cancer cells (SKBR3, MDA-MB-231), two types of non-small cell lung cancer (PC9, A549), and two types of small cell lung cancer (H69, SBC-1) are shown in FIG. 76.8 to 91.4%. In addition, there are about 300,000 normal leukocytes mixed with cancer cells, and the density is increased by binding non-target cells (red blood cells, white blood cells) to each other with a binding agent. By increasing the density difference and performing separation using the separation structure of the present invention, the target cells can be separated with high recovery rate and selectivity.

Optimization of Centrifugal Conditions in Density Difference Separation In the same manner as in Example 3, a sample prepared by mixing approximately 30 human breast cancer cells (SKBR3) with healthy blood, a physiological saline, and a mixed solution of a binder are density gradient solutions. The mixture was layered on top, centrifuged and collected. In addition, the centrifugation conditions in density difference separation were implemented at room temperature for 1 to 3000 × g for 3 to 10 minutes. After centrifuging, the cylindrical members 2 and 3 constituting the separation structure are separated by the separation part without removing the cap, and then the cap is removed to open the seal, thereby opening the communication opening end of the upper cylindrical member 2. (Separation part) A part of the density gradient solution and the cells maintained thereon are drained from the separator 6 and collected by the tube 50 disposed below, and the inner wall of the upper cylindrical member is washed to adhere to the wall. Was also collected at the same time.

  The collected cell suspension was diluted to 30 mL with 0.4% sodium citrate / PBS solution, and centrifuged at 300 × g for 10 minutes at room temperature. The liquid at the top of the pellet was removed with a pipette, the cells in the pellet were resuspended in 30 mL of 300 mM mannitol solution, and then centrifuged at 300 × g for 5 minutes at room temperature.

Using the apparatus shown in FIG. 5, as in Example 1, it was confirmed that the separated cells were the intended breast cancer cells. FIG. 8 shows the results of centrifugation conditions and cancer cell recovery. Centrifugation conditions in density difference separation reached 2000 × g and a maximum of 88.1% in 5 minutes, and when the centrifugal acceleration was further increased, the load on the cells increased and the recovery rate decreased.

Optimization of the centrifugation conditions in the collection operation In the same manner as in Example 3, a sample obtained by mixing approximately 30 human breast cancer cells (SKBR3) with healthy blood, physiological saline, and a mixed solution of a binder were mixed with a density gradient solution. Layered up, centrifuged and collected. In addition, the centrifugation conditions in density difference separation were implemented at 2000 xg for 5 minutes at room temperature. After centrifuging, the cylindrical members 2 and 3 constituting the separation structure are separated by the separation part without removing the cap, and then the cap is removed to open the seal, thereby opening the communication opening end of the upper cylindrical member 2. (Separation part) A part of the density gradient solution and the cells maintained thereon are drained from the separator 6 and collected by the tube 50 disposed below, and the inner wall of the upper cylindrical member is washed to adhere to the wall. Was also collected at the same time.

  The collected cell suspension was diluted to 30 mL with a 0.4% sodium citrate / PBS solution, and centrifuged at 300 to 900 × g for 5 to 10 minutes at room temperature. The liquid at the top of the pellet was removed with a pipette, the cells in the pellet were resuspended in 30 mL of 300 mM mannitol solution, and then centrifuged at 300 × g for 5 minutes at room temperature.

Using the apparatus shown in FIG. 5, as in Example 1, it was confirmed that the separated cells were the intended breast cancer cells. FIG. 9 shows the results of centrifugation conditions and cancer cell recovery. Centrifugation conditions at the time of collection reach a maximum of 89.2% at 300 × g for 10 minutes, and if the centrifugation time is less than 10 minutes at a centrifugal acceleration of 300 × g, the cells do not settle sufficiently. The recovery rate decreased due to cell death over time. When the centrifugal acceleration was set to 900 × g or more, a remarkable gravity load on the cells was observed, and the recovery rate was greatly reduced.

Optimization of taper-shaped inclination angle (density gradient solution 1.077 g / mL)
In the same manner as in Example 3, a sample obtained by mixing about 30 human breast cancer cells (SKBR3) with healthy human blood, a physiological saline solution, and a binder mixture were layered on the density gradient solution, centrifuged, A recovery operation was performed. Centrifugation in density difference separation was performed at 2000 × g for 5 minutes, and centrifugation in recovery was performed at 300 × g for 10 minutes at room temperature. In the separation structure used in Example 6, the cylindrical member 2 is a cylindrical polypropylene member having an inner diameter of Φ18 mm, a length of 70 mm, and a capacity of 15 mL. The inner surface of the member is coated with bovine serum albumin (BSA) to make it hydrophilic, thereby preventing nonspecific adsorption of cells and the like. The inclination angle of the tapered portion 7 of the cylindrical member 2 is 30, 50, and 70 °, and the communication opening with the cylindrical member 3 is Φ2 mm. The cylindrical member 3 is a polypropylene member having an inner diameter of 10 mm, a length of 54 mm, and a capacity of 2 mL.

Using the apparatus shown in FIG. 5, as in Example 1, it was confirmed that the separated cells were the intended breast cancer cells. The inclination angle of the tapered portion 7 of the cylindrical member 2 reaches a maximum of 86.5% at 30 °, and as the inclination angle increases, the interface between the density gradient solution and the sample on the cylindrical member 2 side (the top of the Lymphoprep solution) Since the cancer cells maintained in this state are easily moved to the cylindrical member 3 side, the recovery rate was reduced to 75% at the inclination angle 50 ° of the tapered portion 7 of the cylindrical member 2 and 73% at 70 °.

Optimization of density gradient solution In the same manner as in Example 3, a mixture of approximately 30 human breast cancer cells (SKBR3) mixed with healthy human blood, a mixture of physiological saline, and binder onto the density gradient solution. Layered, centrifuged and collected. Centrifugation in density difference separation was performed at 2000 × g for 5 minutes, and centrifugation in recovery was performed at 300 × g for 10 minutes at room temperature. In Example 7, 2 mL of density gradient solution having densities of 1.077, 1.082, 1.084, and 1,091 g / mL was injected into the cylindrical member 3 of the separation structure 1.

Using the apparatus shown in FIG. 5, as in Example 1, it was confirmed that the separated cells were the intended breast cancer cells. FIG. 10 shows the density gradient solution density and cancer cell recovery rate results. As the density of the density gradient solution increased, the recovery rate of cancer cells improved, and the recovery rate was 96.9% at a density of 1.091 g / mL.

Optimization of communication opening diameter (specification that can hold liquid by sealing the cap)
In the same manner as in Example 3, a sample obtained by mixing about 30 human breast cancer cells (SKBR3) with healthy human blood, a physiological saline solution, and a binder mixture were layered on the density gradient solution, centrifuged, A recovery operation was performed. In the separation structure used in Example 9, the cylindrical member 2 is a cylindrical polypropylene member having an inner diameter of 18 mm, a length of 70 mm, and a capacity of 15 mL. The inner surface of the member is coated with bovine serum albumin (BSA) to make it hydrophilic, thereby preventing nonspecific adsorption of cells and the like. The inclination angle of the tapered portion 7 of the cylindrical member 2 is 70 °, and the communication opening with the cylindrical member 3 is Φ2, 4, 6 mm. The cylindrical member 3 is a polypropylene member having an inner diameter of 10 mm, a length of 54 mm, and a capacity of 2 mL.

  After centrifuging, when the cylindrical members 2 and 3 constituting the separation structure were separated by the separation part without removing the cap, the liquid held in the cylindrical member 2 did not flow out when the communication opening was Φ2, 4 mm. However, a slight outflow was observed at Φ6 mm.

  As in Example 1, using the apparatus shown in FIG. 5, it was confirmed that the separated cells were the intended breast cancer cells. When the communication opening was Φ2, 4 mm, the recovery rate was 86, respectively. 2% and 85.5%, while in Φ6mm, the recovery rate was significantly reduced to 72.2%.

Example of Use of Tripartite Structure Cancer cells were separated using the separation structure of the embodiment shown in FIG. Specifically, the separation structure includes a cap 49 and cylindrical members 41, 42, and 43. The cylindrical member 41 is a cylindrical polypropylene member having an inner diameter of 20 mm, a length of 60 mm, and a capacity of 11 mL. The inclination angle of the tapered portion 44 of the cylindrical member 41 is 70 °, and the communication opening with the cylindrical member 42 is Φ2 mm. The cylindrical member 42 is a cylindrical polypropylene member having an inner diameter of 14 mm, a length of 30 mm, and a capacity of 3 mL. The inclination angle of the tapered portion 45 of the cylindrical member 42 is 30 °, and the communication opening with the cylindrical member 43 is Φ2 mm. The cylindrical member 43 is a polypropylene member having an inner diameter of 14 mm, a length of 41 mm, and a capacity of 2.5 mL. The inner surface of the member is coated with bovine serum albumin (BSA) to make it hydrophilic, thereby preventing nonspecific adsorption of cells and the like.

  2.5 mL of a density gradient solution (a portion filled with the lower cylindrical member) 46 having a density of 1.077 to 1.086 g / mL was injected into the lower cylindrical member 43 of the separation structure (see FIG. The middle and lower white parts are filled with the density gradient solution). Subsequently, a density gradient solution (central cylindrical member at the center) having a density of 1.030 to 1.086 g / mL is formed in the intermediate cylindrical member (center) 42 (sometimes referred to as the cylindrical member 42) of the separation structure. (Part that may be described as “density gradient solution 47”) was injected 3 mL (the middle hatched portion in the figure is the portion filled with the density gradient solution). Finally, on the density gradient solution 47, a mixture of 3 mL of sample obtained by mixing about 30 human breast cancer cells (SKBR3) with healthy human blood, 3 mL of physiological saline, and 75 μL of a binder (sample, physiological saline) Water (binder) 48 (which may be referred to as a mixed solution 48) was overlaid (in the figure, the black coating portion is the portion of the mixed solution overlaid).

  After sample injection, the opening of the separation structure was sealed with a cap 49 (made of polypropylene), and centrifuged at 2000 × g for 5 minutes at room temperature. As shown in FIG. 12, after centrifuging, the cylindrical members 42 and 43 constituting the separation structure are separated by the separation part without removing the cap, and then the tubular members 41 and 42 are separated by the separation part. Thus, only a part of the density gradient solution and the cells in the cylindrical member 42 are allowed to flow out from the communication opening of the cylindrical member 42 and are collected by the tube 50 disposed below, and the inner wall of the cylindrical member 42 is washed, At the same time, cells attached to the cells were collected. Finally, by removing the cap and opening the seal, a part of the density gradient solution and the cells in the cylindrical member 41 are allowed to flow out from the communication opening of the cylindrical member 41, and the tube installed below in the same manner as described above. At 50, the inner wall of the cylindrical member 41 was washed, and the cells attached to the wall were also collected at the same time.

  Each collected cell suspension is diluted to 30 mL with 0.4% sodium citrate / PBS solution, centrifuged at 300 × g for 10 minutes at room temperature, and the liquid at the top of the pellet is removed with a pipette. The cells inside were resuspended in 30 mL of 300 mM mannitol solution and centrifuged at 300 × g for 5 minutes at room temperature. This centrifugation operation is for removing cell debris and platelets and concentrating the target cells.

Using the apparatus shown in FIG. 5, as in Example 1, it was confirmed that the separated cells were the intended breast cancer cells. FIG. 13 shows the density gradient solution density injected into the cylindrical members 42 and 43 and the results of the cancer cell recovery rate from each cylindrical member. By using the separation structure of the present invention, each cell type (density) can be obtained for each target cell type (density) by a simple operation of separating each cylindrical member constituting the structure and removing a cap that seals the opening. Can be separated.

Secondary separation by magnetic beads of fractions after density centrifugation Using 3 mL of suspension obtained by mixing approximately 30 cancer cells in healthy human blood as a sample, the centrifugation operation was performed in the same manner as in Example 1. The cells were then maintained at the density gradient solution-sample interface. After completion of the centrifugation operation, the cylindrical members 2 and 3 constituting the separation structure are separated at the separation part without removing the cap, and then the cap is removed as shown in the right of FIG. 4 to open the seal. Then, a part of the density gradient solution and the cells maintained thereon are discharged from the communication opening end (separation part) 6 of the upper cylindrical member 2 and collected by a tube installed below, and the upper cylindrical member The inner wall was washed, and the cells attached to the wall were collected at the same time.

  The collected cell suspension was diluted to 30 mL with a 0.4% sodium citrate / PBS solution, centrifuged at 300 × g for 10 minutes at room temperature, and the liquid at the top of the pellet was removed with a pipette. The pellet was resuspended in 500 μL of PBS solution, and 25 μL of antibody magnetic particles (Dynabeads CD45, Invitrogen) for removing leukocytes were added, followed by antigen-antibody reaction at 4 ° C. for 20 minutes with stirring. By placing the container after the reaction on a magnet, the leukocytes bound to the antibody magnetic particles were captured and removed from the container wall, and at the same time, a solution containing cancer cells was collected. The collected cell suspension was diluted to 30 mL of a 300 mM mannitol solution and centrifuged at 300 × g for 5 minutes at room temperature.

Using the apparatus shown in FIG. 5, as in Example 1, it was confirmed that the separated cells were the target cancer cells. The recovery rates of two types of breast cancer cells (SKBR3, MDA-MB-231), two types of non-small cell lung cancer (PC9, A549), and two types of small cell lung cancer (H69, SBC-1) are shown in FIG. 37 to 87.3%. The number of normal leukocytes mixed with cancer cells is about 2 million, and the objective is high recovery and selectivity by separation using the separation structure of the present invention and removal of leukocytes using antibody magnetic particles. Cells can be separated.
Comparative Example 1
Samples used in Example 1 using a commercially available blood separation kit by density gradient centrifugation (Axis-Shield PoC AS, trade name Lymphoprep Tube, density gradient solution (Lymphoprep) density 1.077 g / mL) Of human breast cancer cells (SKBR3) from The sample was layered on the density gradient solution in the separation container included in the kit, and then centrifuged at 1100 × g for 10 minutes at room temperature. After centrifugation, cells maintained at the interface between the density gradient solution and the sample (the top of the Lymphoprep solution) are tumbled out of the separation container and collected in the tube 50, and the inner wall of the container and the filter in the separation container are washed. The cells attached to them were collected at the same time.

  The collected cell suspension is made up to 30 mL with 0.4% sodium citrate / PBS solution, centrifuged at 300 × g for 10 minutes at room temperature, and the liquid at the top of the pellet is removed with a pipette. Of cells were resuspended in 30 mL of 300 mM mannitol solution and centrifuged at 300 × g for 5 minutes at room temperature. This centrifugation operation is for removing cell debris and platelets and concentrating the target cells.

  After the centrifugation operation, the liquid at the top of the pellet was removed with a pipette, and the cells in the pellet were resuspended in 1 mL of a 300 mM mannitol solution. Using the apparatus shown in FIG. 5, as in Example 1, it was confirmed that the separated cells were target human breast cancer cells. As a result, about 4.4 million normal leukocytes were captured in the holding holes, and about 25 cancer cells (recovery rate: about 83%). The same operation was performed again. As a result, about 4 million normal leukocytes were captured in the holding hole, and about 14 cancer cells (recovery rate: about 45%). Cell recovery varied greatly. From this result, it can be seen that cancer cells can be stably recovered at a high recovery rate by the present invention in which a part of the density gradient solution and cells maintained thereon are flowed out from the communication opening of the upper cylindrical member.

Comparative Example 2
After centrifugation at 1100 × g, the same procedure as in Example 1 was performed except that the cells maintained at the interface between the density gradient solution and the sample (the top of the density gradient solution) were aspirated and collected in a tube. Carried out. As a result, approximately 4.1 million normal white blood cells were captured in the holding hole, but only about 21 cancer cells (recovery rate: about 71%), and the operation of aspirating cancer cells with a pipette after centrifugation As a result, the recovery rate of cancer cells decreased. From this result, it can be seen that cancer cells can be stably recovered at a high recovery rate by the present invention in which a part of the density gradient solution and cells maintained thereon are flowed out from the communication opening of the upper cylindrical member.

1: Separation structure 2: Cylindrical member (upper side)
3: Cylindrical member (lower side)
4: Cap 5: Bottom 6: Open end of communication (separation part)
7: Tapered portion 21 of cylindrical member: Unintended component (cells, etc.)
22: A substance that specifically binds to a non-target component 23: A substance for adjusting the density 24: A target component (cell, etc.)
31: Density gradient solution (part filled with density gradient solution)
32: Mixed solution of blood sample and physiological saline (layered mixed solution part)
33: Interface between density gradient solution and sample 34: AC power source 35: Upper electrode substrate 36: Lower electrode substrate 37: Cell 38: Dielectrophoretic force 39: Holding hole 40: Fluorescence microscope 41: Cylindrical member (upper side)
42: cylindrical member (center)
43: Cylindrical member (lower side)
44: Tapered shape (upper cylindrical member)
45: Tapered shape (center tubular member)
46: Density gradient solution (part filled with lower cylindrical member)
47: Density gradient solution (part filled with the central cylindrical member)
48: Mixed solution (sample, physiological saline, binder)
49: Cap 50: 50 mL tube

Claims (2)

One end is closed to form the bottom, and the other end is composed of a cylindrical structure that is open and a cap that seals the opening. The structure is composed of two or more cylindrical members that can be separated at the separation part. der, of the tubular member, the tubular member at the bottom side has an opening and a bottom, the other tubular member has the separation unit and the opening,
The separation structure according to claim 1, wherein the separation portion has a tapered shape with an inclination angle of 30 to 70 °, has a communication opening of φ2 to 4 mm at a tip portion thereof, and is not provided with a filter . A method for separating a target component from a liquid sample, wherein the separation structure according to claim 1 is used to fill a density gradient solution from the bottom of the structure to the vicinity of the separation portion, and stack the samples. A step of centrifuging in a state in which the opening is sealed, and a step of separating the structure while centrifuging and maintaining the sealed state of the opening, and recovering a fraction containing target cells by releasing the sealing. A separation method comprising:

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