Sample preparation system and sample preparation method

Abstract

The purpose of the present technology is to provide a sample preparation system for increasing the content ratio of target cells.　The present technology provides a sample preparation system including: a bioparticle capture module including a substrate on which a substance for capturing bioparticles is fixed; a reservoir for recovering bioparticles released from the substrate; and a hollow fiber membrane module through which the bioparticles in the reservoir pass. The present technology also provides a sample preparation method including: a capturing step for capturing bioparticles using a substrate on which a substance for capturing bioparticles is fixed; a recovering step for recovering bioparticles released from the substrate to a reservoir; and a hollow fiber membrane processing step for causing the bioparticles recovered to the reservoir to pass through a hollow fiber membrane module.

Description

[Technical Field]

[0001] The present technology relates to a sample preparation system and a sample preparation method, and in particular to a sample preparation system and a sample preparation method used to prepare a sample containing biological particles. [Background technology]

[0002] Bioparticle sorting and analysis are performed using various methods. For example, techniques for sorting and analyzing bioparticles in closed or open spaces have been developed. A representative technique for such sorting and analysis is flow cytometry (hereinafter also referred to as FCM). To sort or analyze bioparticles, cells are stained using fluorescently labeled antibodies. This cell staining makes it possible to analyze and / or sort specific cell populations. For example, T cells can be stained using an FITC-labeled CD3 antibody, and the stained T cells can be analyzed and / or sorted by, for example, FCM. FCM analyzes all cells one by one, allowing for very precise analysis and / or purification.

[0003] In bioparticle analysis, such as FCM, in which all cells are analyzed one by one, the analysis time correlates with the number of cells to be analyzed. Therefore, a sample containing a large number of cells to be analyzed but a small number of target cells can result in an unnecessary increase in the time required for the bioparticle analysis. Therefore, for example, a sample to be subjected to bioparticle analysis can be roughly purified to increase the proportion of target cells. By reducing the number of cells to be analyzed through crude purification, the analysis time can be shortened.

[0004] Several technologies related to this type of crude purification have been proposed. For example, Patent Document 1 listed below describes a "cell separation method for removing red blood cells from a cell suspension containing red blood cells and white blood cells and recovering white blood cells." The method includes the steps of: "(A) introducing a cell suspension into an inlet of a cell separation filter obtained by filling a container with a cell separation material and capturing white blood cells on the cell separation filter; (B) introducing a washing solution into the inlet of the cell separation filter to wash the cell separation filter; and (C) introducing a recovery solution into the outlet of the cell separation filter to recover the white blood cells captured on the cell separation filter; the washing solution and recovery solution contain a divalent cation chelating agent, and the ratio of the concentration (W / V) of the divalent cation chelating agent in the washing solution to the concentration (W / V) of the divalent cation chelating agent in the recovery solution is 1:200 to 200:1." Furthermore, Non-Patent Document 1 listed below describes a process for removing red blood cells from cord blood using a multilayer polyester nonwoven fiber as a filter. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2014 / 119529 [Non-patent literature]

[0006] [Non-Patent Document 1] Sato N, Fricke C, McGuckin C, Forraz N, Degoul O, Atzeni G, Sakurai H., Cord blood processing by a novel filtration system., Cell Prolif., 2015 Dec;48(6):671-81. Summary of the Invention [Problem to be solved by the invention]

[0007] It is desirable for a sample to be subjected to separation or analysis of biological particles to have a high content of target particles, which are the subject of separation or analysis. Therefore, the sample can be subjected to a process to increase the content of target particles before separation or analysis. This process requires selective and efficient recovery of target particles. Furthermore, this process also requires adjustment of the content of target particles, for example, by concentration, as needed. Therefore, the present technology aims to provide a sample preparation system for increasing the content of target particles. [Means for solving the problem]

[0008] The present inventors have discovered that the above problems can be solved by a specific sample preparation system. That is, this technology: a bioparticle capture module including a substrate on which a substance for capturing bioparticles is fixed; a reservoir in which bioparticles released from the substrate are collected; a hollow fiber membrane module through which the bioparticles in the reservoir flow; A sample preparation system is provided, comprising: The sample preparation system may be configured to provide a nucleic acid degrading substance to the bioparticle capture module. The substance that captures the bioparticles may be immobilized on the substrate via nucleic acids. The sample preparation system may be configured to allow the nucleic acid degrading substance to pass through the bioparticle capture module and reach the reservoir or the hollow fiber membrane module. The sample preparation system may be configured to supply a liquid containing biological particles to the biological particle capture module, and may be configured to supply a particle binding substance that binds to the biological particles to the reservoir. The particle-binding substance may be an antibody. The biological particle may be a cell. The sample preparation system may include a circulation channel that circulates bioparticles between the reservoir and the hollow fiber membrane module. The sample preparation system may further include a branch channel branching from the circulation channel and leading to the reservoir. The sample preparation system may further include an analytical device for analyzing the contents of the reservoir. The sample preparation system can control the flow of bioparticles through the hollow fiber membrane module based on the analysis results from the analysis device. The sample preparation system may include a pump provided on a flow path leading from the bioparticle capture module to the reservoir. The pump may be a tube pump. The sample preparation system may be configured such that the biological particles released from the substrate can be supplied to the reservoir by driving the pump. The sample preparation system may be configured such that a particle-binding substance that binds to biological particles can be supplied to the reservoir by driving the pump. A pump for circulating bioparticles may be provided on a flow path leading from the reservoir to the hollow fiber membrane module. The hollow fiber membrane module may have a discharge flow path through which the liquid separated from the bioparticles is discharged, and a discharge pump may be provided on the discharge flow path. The sample preparation system may be configured such that the sample containing the bioparticles is not in fluid communication with the external environment.

[0009] In addition, this technology: a capturing step of capturing bioparticles using a substrate to which a substance for capturing bioparticles is immobilized; a recovery step of recovering the bioparticles released from the substrate into a reservoir; a hollow fiber membrane processing step of passing the bioparticles collected in the reservoir through a hollow fiber membrane module; Also provided is a sample preparation method comprising: [Brief explanation of the drawings]

[0010] [Figure 1]1 is a schematic diagram showing an example of the configuration of a sample preparation system according to the present technology. FIG. [Figure 2] 1 is an example of a flow diagram of a sample preparation method using the sample preparation system of the present technology. [Figure 3] 1 is a schematic diagram showing an example of the configuration of a sample preparation system according to the present technology. FIG. [Figure 4] 1 is a schematic diagram for explaining the capture and release of target cells by a bioparticle capture module. FIG. [Figure 5] FIG. 10 shows the results of blood treatment using a substrate on which a bioparticle capture substance is immobilized. [Figure 6] 1 is a schematic diagram for explaining the capture and release of target cells by a bioparticle capture module. FIG. [Figure 7] FIG. 1 is a schematic diagram of an apparatus prepared to confirm the clogging prevention effect in a hollow fiber membrane treatment process. [Figure 8] FIG. 10 is a photograph of a cell aggregate formed in a reservoir. [Figure 9] FIG. 1 shows the results of analysis by a cell analyzer. [Figure 10] FIG. 1 shows the results of cell count measurement using a hemocytometer. [Figure 11] FIG. 1 shows a photograph of a cell aggregate. [Figure 12] FIG. 1 is a schematic diagram of the staining procedure. [Figure 13] 1 is a schematic diagram showing an example of the configuration of a sample preparation system according to the present technology. FIG. [Figure 14] 1 is an example of a flow diagram of a sample preparation method using the sample preparation system of the present technology. DETAILED DESCRIPTION OF THE INVENTION

[0011] Preferred embodiments for implementing the present technology will be described below. Note that the embodiments described below are representative embodiments of the present technology, and the scope of the present technology is not limited to these embodiments. Note that the present technology will be described in the following order. 1. First embodiment (sample preparation system) (1) Description of the First Embodiment (2) Example of a sample preparation system configuration according to this technology (3) Example of sample preparation method using the sample preparation system according to this technology (3-1) Priming process (3-2) Capture process (3-3) Recovery process (3-4) Hollow fiber membrane treatment process (3-5) Modifications (4) Example of processing using the bioparticle capture module (4-1) Example of the configuration and operation of the bioparticle capture module (4-2) Test Example (Blood Treatment Using a Substrate Immobilized with a Bioparticle Capture Substance) (4-3) Other Configuration and Operation Examples of the Bioparticle Capture Module (5) Evaluation of the effect of nucleic acid degrading substances on treatment using hollow fiber membrane modules (6) Evaluation of antibody staining using hollow fiber membrane modules 2. Second embodiment (sample preparation method)

[0012] 1. First embodiment (sample preparation system)

[0013] (1) Description of the First Embodiment

[0014] The sample preparation system of the present technology includes a bioparticle capture module including a substrate on which a substance for capturing bioparticles is fixed, a reservoir for collecting bioparticles released from the substrate, and a hollow fiber membrane module through which the bioparticles in the reservoir flow. The bioparticle capture module can selectively capture target bioparticles. Bioparticles released from the substrate of the bioparticle capture module are collected in the reservoir, and the bioparticles collected in the reservoir are passed through the hollow fiber membrane module. This allows for selective and efficient collection of target bioparticles and also enables adjustment of the concentration of the bioparticles.

[0015] (2) Example of a sample preparation system configuration according to this technology

[0016] An example of the configuration of a sample preparation system according to the present technology will be described below with reference to FIG.

[0017] The sample preparation system 1 shown in FIG. 1 includes a bioparticle capture module 10 , a reservoir 20 , and a hollow fiber membrane module 30 .

[0018] The biological particle capture module 10 has an inlet 11, a container 12, and an outlet 13. The container 12 is filled with a substrate to which a substance for capturing biological particles is fixed.

[0019] The biological particle capture module 10 may be configured so that a liquid containing biological particles can be supplied from an inlet 11 into a container 12. The liquid containing biological particles may contain target particles and non-target particles. 1, the bioparticle capture module 10 is connected to a container 40 (e.g., a bag) filled with a bioparticle-containing liquid L0 by flow paths 50 and 60. More specifically, the outlet of the container 40 and the inlet 11 are connected by the flow paths 50 and 60, and the bioparticle-containing liquid L0 in the container 40 can be supplied into the container 12.

[0020] The biological particle capture module 10 is also connected to a container 41 (e.g., a bag) filled with a cleaning liquid L1 (especially a cleaning buffer) by flow paths 51 and 60. More specifically, the outlet of the container 41 and the inlet 11 are connected by the flow path, and the cleaning liquid L1 in the container 41 can be supplied into the container 12.

[0021] The bioparticle capture module 10 is also connected to a container 42 (e.g., a bag) filled with a nucleic acid degrading substance-containing liquid L2 (particularly a nucleic acid degrading substance-containing buffer) by flow paths 52 and 60. More specifically, the outlet of the container 42 and the inlet 11 are connected by this flow path, and the nucleic acid degrading substance-containing liquid L2 in the container 42 can be supplied into the container 12.

[0022] The nucleic acid degrading substance contained in the nucleic acid degrading substance-containing solution may be, for example, a nuclease, particularly a deoxyribonuclease, a ribonuclease, or both. The nuclease may be, for example, an endo-type, an exo-type, or both. Preferably, the nucleic acid degrading substance contains a deoxyribonuclease, particularly a deoxyribonuclease, but may not contain a ribonuclease. An example of a deoxyribonuclease is DNase I.

[0023] The present inventors have discovered that a liquid containing a nucleic acid-degrading substance, particularly a nuclease, more particularly a deoxyribonuclease, is suitable for preventing clogging in the sample preparation system of the present technology. Clogging can occur, for example, in various flow paths or in the hollow fiber membrane module described below. Clogging is particularly likely to occur in processes using the hollow fiber membrane module described below, particularly when washing or concentrating biological particles such as cells using the hollow fiber membrane module. In the present technology, the use of the nucleic acid-degrading substance can prevent clogging. The clogging is thought to be caused by DNA released from dead cells, etc. In this technology, by passing a liquid containing the nucleic acid degrading substance through various channels, a bioparticle capture module, a reservoir, and a hollow fiber membrane module, the DNA is degraded, which is thought to prevent clogging. As described above, the sample preparation system of the present technology may be configured to supply a nucleic acid degrading substance to the bioparticle capture module. More preferably, the sample preparation system may be configured to allow the nucleic acid degrading substance to pass through the bioparticle capture module and reach the reservoir and / or the hollow fiber membrane module.

[0024] As shown in FIG. 1, the flow paths 50 , 51 , and 52 are connected to the flow path 60 . Valves V0.1, V0.2, and V0.3 are provided on flow paths 50, 51, and 52, respectively. Opening or closing the valve V0.1 enables or disables the supply of the bioparticle-containing liquid L0 contained in the container 40 to the container 12. By opening or closing the valve V0.2, the supply of the cleaning liquid L1 contained in the container 41 to the container 12 is enabled or disabled. By opening or closing the valve V0.3, it becomes possible or impossible to supply the nucleic acid degrading substance-containing liquid L2 contained in the container 42 to the container 12. The flow paths 50, 51, 52, and 60 may be, for example, tubes. The material of the tubes may be selected appropriately by those skilled in the art. The cross-sectional dimensions of these flow paths may be set appropriately by those skilled in the art depending on the bioparticle-containing liquid to be passed through. The other flow paths shown in FIG. 1 may also be tubes, and the material and cross-sectional dimensions thereof may also be selected appropriately by those skilled in the art.

[0025] The container 12 of the bioparticle capture module 10 is filled with a substrate to which a substance for capturing bioparticles is fixed. The shape of the container 12 may be appropriately selected by a person skilled in the art, and may be, for example, a columnar, i.e., cylindrical, shape. A columnar shape is preferred to reduce the effect of water pressure on the container shape.

[0026] In this specification, a substance that captures bioparticles may refer to a substance used to capture bioparticles. The substance that captures bioparticles may be, for example, a substance that itself binds to bioparticles (also referred to as a "bioparticle-binding substance" in this specification), or a substance that captures bioparticles via another substance. In the latter case, the substance that captures bioparticles may not itself be a substance that binds to bioparticles, but the other substance may be a substance that binds to bioparticles.

[0027] As described above, the bioparticle-binding substance is a substance that itself binds to a bioparticle, and may be, for example, an antibody or antibody fragment, more particularly an antibody or antibody fragment that binds to an antigen present on the surface of a bioparticle, and even more particularly an antibody or antibody fragment that binds to a surface antigen on a cell. As described above, the substance that captures bioparticles via another substance does not have to be a substance that binds to bioparticles itself. The substance that captures bioparticles via another substance may be, for example, a substance that binds to a bioparticle-binding substance, particularly a protein that binds to an antibody or antibody fragment, and more particularly a protein that specifically binds to an antibody or antibody fragment. An example of such a protein is an antibody-binding protein. The antibody-binding protein may be, for example, any one or more of Protein A, Protein G, Protein L, and Protein A / G. The type of substance that captures the bioparticles may be appropriately selected by those skilled in the art depending on the type of bioparticles to be collected (particularly the type of substance present on the surface of the bioparticles).

[0028] As used herein, a bioparticle may be a biological particle, e.g., a particle that constitutes a living organism. A bioparticle may be a microparticle. The biological particles may be, for example, cells. Examples of cells include animal cells (such as blood cells) and plant cells. The cells may be, in particular, blood cells or tissue cells. Examples of blood cells include white blood cells (e.g., peripheral blood mononuclear cells), red blood cells, and platelets, and the blood cells particularly include white blood cells. Examples of white blood cells include monocytes (macrophages), lymphocytes, neutrophils, basophils, and eosinophils. The cells may be suspension cells, such as T cells and B cells. The tissue cells may be, for example, adherent cultured cells or adherent cells dissociated from tissue. The cells may also be tumor cells. The cells may be cultured or uncultured. The biological particles may be cell aggregates, such as spheroids and organoids. The biological particle may be a non-cellular biological component, such as an extracellular vesicle, in particular an exosome or a microvesicle. The biological particle may be a microorganism or a virus. Microorganisms may include bacteria such as E. coli and fungi such as yeast. Viruses may be, for example, DNA or RNA viruses, and may be enveloped or non-enveloped viruses. Bioparticles can also include biological macromolecules such as nucleic acids, proteins, and complexes thereof, which may be extracted from cells, for example, or contained in a blood sample or other liquid sample.

[0029] In this specification, the biological particle-containing liquid may be a liquid obtained from a living organism, such as a body fluid. The body fluid may be blood, lymph, tissue fluid (e.g., interstitial fluid, intercellular fluid, interstitial fluid, etc.), or body cavity fluid (e.g., serous cavity fluid, pleural fluid, peritoneal fluid, pericardial fluid, cerebrospinal fluid (spinal fluid), joint fluid (synovial fluid), etc.). The biological particle-containing liquid may also be a liquid obtained from these body fluids. In one embodiment of the present technology, the biological particle-containing liquid may be a blood sample, particularly a sample containing white blood cells. The biological particle-containing liquid may particularly be a blood sample that has been subjected to a red blood cell separation process. Note that the blood sample does not need to be one from which red blood cells have been completely removed, and may contain red blood cells. For example, the blood sample may be one in which the amount of red blood cells in blood collected from a living body has been reduced by the separation process.

[0030] The substrate may be formed from a material that allows the bioparticle-containing liquid to pass through, such as a porous material, a fibrous material, or a beaded material. Substrates formed from materials with such shapes or structures have a large surface area, can immobilize a large amount of substance that captures bioparticles, and can easily allow the bioparticle-containing liquid to pass through. The substrate material may be, for example, a proteinaceous material, an organic polymer-based material, or an inorganic material. Examples of the proteinaceous material include collagen or egg white. Examples of the organic polymer-based material include acrylamide, polystyrene, polycaprolactone, and lactic acid-based polymers. Examples of the inorganic material include titanium, particularly titanium fiber.

[0031] The substrate may be formed, for example, from a sponge-like material, for example, from a collagen sponge. The substrate may be formed, for example, from a cryogel material, for example, an acrylamide or egg white cryogel. The substrate may be formed, for example, from a scaffold material, such as polystyrene, polycaprolactone, a lactic acid-based polymer or a combination of a lactic acid-based polymer and hydroxyapatite, or a scaffold material formed from titanium fibers.

[0032] The biological particle capture module 10 may be configured to be able to discharge liquid from the outlet 13 .

[0033] 1 , the biological particle capture module 10 may be configured so that liquid discharged from the outlet 13 (particularly liquid containing biological particles that have been captured by a biological particle-capturing substance and then released from the substance) is supplied to the reservoir 20. To supply the liquid to the reservoir 20, the outlet 13 may be connected to the reservoir 20 via a flow path. More specifically, the outlet 13 is connected to a flow path 61, which is connected to a flow path 62, which is connected to a flow path 63, and the flow path 63 is connected to the reservoir 20. That is, the outlet 13 is connected to the reservoir 20 via the flow paths 61, 62, and 63.

[0034] 1, the bioparticle capture module 10 may be configured so that the waste liquid discharged from the outlet 13 is collected in a waste liquid collection container 45. To enable the collection of waste liquid, the flow path 61 branches into two flow paths 62 and 69, and of these two flow paths, the flow path 69 is used for collecting waste liquid. The flow path 69 is connected to the flow path 55, and the flow path 55 is connected to the waste liquid collection container 45. In other words, the outlet 13 is connected to the waste liquid collection container 45 via the flow paths 61, 69, and 55.

[0035] A valve V0.4 is provided on the flow path 69. By opening or closing the valve V0.4, collection of waste liquid into the waste liquid collection container 45 using the flow path 69 becomes possible or impossible.

[0036] The reservoir 20 is connected to a container 44 (particularly a bag) containing a particle-binding substance-containing liquid L4 via flow paths 54 and 63. More specifically, the outlet of the container 44 and the reservoir 20 are connected by tubes 54 and 63, allowing the particle-binding substance-containing liquid in the container 44 to be supplied into the reservoir 20. The particle-binding substance-containing liquid L4 may be a liquid used to bind the particle-binding substance to biological particles. The particle-binding substance may be, for example, an antibody, particularly a fluorescently labeled antibody. The type of particle-binding substance and the composition of the liquid can be appropriately selected by those skilled in the art depending on the sample to be prepared.

[0037] The reservoir 20 is connected to a container 43 (particularly a bag) containing a binding auxiliary liquid L3 via flow paths 53 and 63. More specifically, the outlet of the container 43 and the reservoir 20 are connected by the flow paths 53 and 63, and the binding auxiliary liquid L3 in the container 43 can be supplied into the reservoir 20. The binding auxiliary liquid may refer to a liquid used to bind a particle-binding substance (e.g., an antibody, particularly a fluorescently labeled antibody) to a biological particle. For example, the binding-assisting liquid may be a buffer used when staining bioparticles with antibodies, and may be a so-called staining buffer. The buffer may be used, for example, to dilute the antibody or to remove excess antibody after antibody labeling. The staining buffer may be, for example, PBS or physiological saline containing a protein (e.g., serum protein). The protein is added to prevent nonspecific binding of the antibody; for example, FBS or albumin (e.g., BSA) may be added to the physiological saline.

[0038] As shown in FIG. 1, channels 53 , 54 and 62 are connected to channel 63 . Furthermore, valves V1.1, V1.2, and V1.4 are provided on the flow paths 53, 54, and 62, respectively. Opening or closing the valve V1.1 enables or disables the supply of the bonding aid liquid L3 contained in the container 43 to the reservoir 20. Opening or closing the valve V1.2 enables or disables the supply of the particle-binding substance-containing liquid L4 contained in the container 44 to the reservoir 20. Opening or closing valve V1.4 allows or prevents the liquid contained in container 12 from being delivered to reservoir 20.

[0039] A pump P1 is provided on the flow path 63. The pump P1 may be, for example, a tube pump, and more preferably a peristaltic pump. Such a pump is suitable for delivering liquid for capturing bioparticles by the bioparticle capture module and for discharging bioparticles released from the substrate. By driving the pump P1 and controlling the opening and closing of various valves, it is possible to supply various liquids to the bioparticle capture module 10, discharge liquids from the bioparticle capture module 10, and supply various liquids to the reservoir 20. Details of these liquid delivery operations will be described later. Thus, in the present technology, a pump may be provided in a flow path leading from the bioparticle capture module to the reservoir. The sample preparation system of the present technology may be configured to supply bioparticles released from the substrate to the reservoir by driving the pump. Furthermore, the sample preparation system of the present technology may be configured to supply a particle-binding substance that binds to bioparticles to the reservoir by driving the pump.

[0040] The hollow fiber membrane module 30 has an inlet 31, a container 32, an outlet 33, and a waste liquid outlet 34. The container 32 is filled with hollow fiber membranes.

[0041] The hollow fiber membranes included in the hollow fiber membrane module 30 may be selected by those skilled in the art depending on the size and type of bioparticles to be concentrated. The hollow fiber membranes may be formed, for example, from mPES (modified polyethersulfone), ME (mixed cellulose ester), PES (polyethersulfone), or PS (polysulfone). The pore size, expressed as the MWCO (molecular weight cut off), of the hollow fiber membranes may be, for example, from 1 kD to 1000 kD, particularly from 2 kD to 900 kD, and more particularly from 3 kD to 800 kD. The pore size of the hollow fiber membranes may be, for example, from 0.1 μm to 1.0 μm, particularly from 0.15 μm to 0.9 μm, and more particularly from 0.2 μm to 0.8 μm.

[0042] The hollow fiber membrane module 30 is configured to allow the bioparticle-containing liquid in the reservoir 20 to flow through it. For example, the hollow fiber membrane module 30 may be configured to allow the bioparticle concentration of the bioparticle-containing liquid in the reservoir 20 to be adjusted, for example, to increase or decrease the bioparticle concentration.

[0043] For example, as shown in Fig. 1, the hollow fiber membrane module 30 is connected to the reservoir 20 via flow paths 64 and 65. The flow paths 64 and 65 are also collectively referred to as a circulation flow path. The liquid in the reservoir 20 (particularly the liquid containing bioparticles) may be supplied to the hollow fiber membrane module 30 through the flow path 64, and the liquid containing bioparticles that has passed through the hollow fiber membrane module 30 may be supplied to the reservoir 20 through the flow path 65. This allows the bioparticles to circulate between the reservoir 20 and the hollow fiber membrane module 30. As such, the sample preparation system of the present technology may preferably include a circulation channel that circulates the bioparticles between the reservoir 20 and the hollow fiber membrane module 30. The circulation direction may be reversed. That is, the liquid in the reservoir 20 (particularly the liquid containing bioparticles) may be supplied to the hollow fiber membrane module 30 through the flow path 65, and the liquid containing bioparticles that has passed through the hollow fiber membrane module 30 may be supplied to the reservoir 20 through the flow path 64.

[0044] A pump P2 is provided on the flow path 64. The pump P2 may be, for example, a tube pump, more preferably a peristaltic pump. Such a pump is suitable for sending a liquid for treatment by the hollow fiber membrane module. In this way, in the present technology, a pump for circulating bioparticles may be provided on the flow path leading from the reservoir 20 to the hollow fiber membrane module 30. By controlling the operation of pumps P1, P2, and P3 and controlling the opening and closing of various valves, it becomes possible to supply various liquids to reservoir 20 and perform hollow fiber membrane treatment. Details of these liquid transfer operations will be described later.

[0045] Two valves V4 and V5 are provided on the flow path 65. Opening and closing the valves V4 and V5 enables or disables the flow of liquid between the reservoir 20 and the hollow fiber membrane module 30 (particularly the vessel 32). Although the number of valves may be one, it is preferable to provide two valves in order to perform an operation using a branch flow path 66, which will be described later.

[0046] A branch flow path 66 that branches off from between the valves V4 and V5 on the flow path 65 is provided on the flow path 65. The branch flow path 66 extends from the flow path 65 to the flow path 63 and is connected to the flow path 63 at a position between the pump P1 and the valve V2 on the flow path 63. In other words, the branch flow path 66 has one end located between the pump P1 and the valve V2 on the flow path 63 and the other end located between the valves V4 and V5 on the flow path 65. It can also be said that the branch channel 66 branches off from the circulation channel and leads to the reservoir 20 via the channel 63. Thus, the sample preparation system of the present technology preferably includes a branch channel that branches off from the circulation channel and leads to the reservoir. The branch channel 66 is used for dead volume recovery processing, as will be described later.

[0047] A valve V3 is provided on the branch channel 66. Opening and closing the valve V3 enables or disables the flow of liquid from the channel 65 to the channel 63, or the reverse flow.

[0048] A flow path 68 is connected to the hollow fiber membrane module 30, through which flows waste liquid (which can also be considered as permeate in hollow fiber membrane processing) resulting from the concentration of bioparticles by the module. The flow path 68 may lead to a waste liquid collection container. For example, as shown in FIG. 1 , the flow path 68 is connected to an outlet 34 through which the waste liquid is discharged, and the flow path 68 is further connected to a waste liquid collection container 45 via a flow path 55. As a result, the flow path 68 merges with a flow path 67 through which waste liquid from the bioparticle capture module flows, and is then connected to the flow path 55. In this way, the sample preparation system of the present technology may be configured so that the waste liquid from the bioparticle capture module and the waste liquid from the hollow fiber membrane module are collected in a single waste liquid collection container.

[0049] Pump P3 is provided on flow path 55. Pump P3 may be, for example, a tube pump, more preferably a peristaltic pump. Such a pump is suitable for recovering waste liquid from a bioparticle capture module or a hollow fiber membrane module. By driving pump P3 in combination with pumps P1 and / or P2 and further controlling the opening and closing of various valves, for example, waste liquid recovery becomes possible. Details of these liquid transfer operations will be described later. As described above, in the present technology, the hollow fiber membrane module has a discharge flow path (corresponding to flow paths 68 and 55) through which the liquid separated from the bioparticles is discharged, and a discharge pump may be provided on the discharge flow path.

[0050] A liquid recovery channel 67 may be connected to the reservoir 20. The liquid recovery channel 67 may be used to collect the bioparticle-containing liquid from the reservoir 20 before, during, or after treatment (particularly bioparticle concentration treatment) by the hollow fiber membrane module. In addition, the liquid recovery channel 67 may be used, for example, to collect biological particle-containing liquid from the reservoir 20 before, during, or after a process of binding the particle-binding substance contained in the container 44 to the biological particles.

[0051] The reservoir 20 may be equipped with an analyzer 90. The analyzer 90 analyzes the contents (particularly the bioparticle-containing liquid) in the reservoir 20. For example, the analyzer 90 may be configured as a concentration measuring device that measures the concentration of the bioparticle-containing liquid. The concentration measuring device can measure the concentration of the bioparticle-containing liquid, for example, by measuring the turbidity of the bioparticle-containing liquid or by measuring the fluorescence emitted from the bioparticle-containing liquid. Depending on the analysis results by the analyzer 90, the bioparticle-containing liquid in the reservoir 20 can be adjusted to a desired concentration by, for example, continuing or stopping the bioparticle concentration process using the hollow fiber membrane module 30.

[0052] The sample preparation system of the present technology may further include a control unit (not shown) that controls the operation of each element constituting the system. The control unit may, for example, control the operation of the pumps and / or valves described above. For example, the control unit may control the operation of the pumps and / or valves according to a predetermined program.

[0053] The control unit may also be configured to receive analysis results from the analysis device 90. The control unit may control the operation of the pumps and / or valves in response to the analysis results from the analysis device 90. For example, the control unit may control the operation of one or more of the pumps, particularly start or stop their operation, in response to receiving a predetermined analysis result. The control unit may also control the opening and closing of one or more of the valves in response to receiving a predetermined analysis result. This allows the sample preparation system of the present technology to control various steps included in the sample preparation method described below, for example, to control the start or end of the various steps, based on the analysis results from the analysis device.

[0054] For example, the sample preparation system of the present technology (particularly the control unit) is configured to be capable of controlling the priming step S101, capture step S102, recovery step S103, and hollow fiber membrane processing step S104 described below, and may be configured to be capable of controlling, for example, any one or more of these steps. Preferably, the sample preparation system of the present technology may be configured to control the flow of bioparticles through the hollow fiber membrane module based on the analysis results of the analyzer. More specifically, the sample preparation system of the present technology (particularly the control unit) may control the hollow fiber membrane processing step S104 described below and various processes included in this step, and particularly may control the start or end of this step or various processes included in this step. This makes it possible to automatically end the concentration adjustment process or concentration process, for example, when the bioparticle-containing liquid in the reservoir reaches a desired concentration.

[0055] The control unit may be configured as an information processing device (computer), and the functions of the control unit may be realized by, for example, a general-purpose computer.

[0056] Preferably, the sample preparation system of the present technology is configured so that the sample containing bioparticles is not in fluid communication with the external environment. For example, as can be seen from FIG. 1, the sample preparation system 1 shown in FIG. 1 is configured so that the space through which the bioparticle-containing liquid contained in the container 40 passes as the liquid passes through the bioparticle capture module 10 and is collected in the reservoir 20, and the space through which the liquid passes between the reservoir 20 and the hollow fiber membrane module 30, are not in contact with the external environment. In this way, the sample preparation system of the present technology can perform a series of processes from preparing a target sample from a bioparticle-containing liquid without contacting the external environment (e.g., external air or liquid), i.e., can perform the processes in a closed environment. This prevents contamination of the sample.

[0057] A sample prepared by the sample preparation system of the present technology may be used as a sample to be subjected to the sorting or analysis of biological particles. For example, a sample prepared by the sample preparation system of the present technology may be used as a sample to be subjected to an apparatus for sorting or analyzing biological particles in a closed space, or as a sample to be subjected to an apparatus for sorting or analyzing biological particles in an open space. An example of an apparatus for sorting or analyzing biological particles in a closed space is, for example, but is not limited to, the microparticle sorting apparatus described in Japanese Patent Application Publication No. 2020-76736. An example of an apparatus for sorting or analyzing biological particles in an open space is, for example, but is not limited to, the microparticle measuring apparatus described in Japanese Patent Application Publication No. 2020-51936. The sample preparation system of the present technology may be used to prepare a sample to be subjected to such an apparatus for sorting or analyzing biological particles.

[0058] (3) Example of sample preparation method using the sample preparation system according to this technology

[0059] An example of a sample preparation method using a sample preparation system according to the present technology will be described below with reference to Figures 2 and 3. In this example, a process is performed in which target cells are selectively collected into a reservoir from a cell-containing solution containing target cells and non-target cells using a bioparticle capture module, and a process is performed in which the concentration of target cells in the reservoir is increased using a hollow fiber membrane module. In this example, a process is further performed in which the target cells are stained with fluorescently labeled antibodies. The cell-containing fluid may be, for example, a blood sample, particularly a sample containing white blood cells. The cell-containing fluid may particularly be a blood sample that has been subjected to a red blood cell separation process. Note that the blood sample does not need to be one from which red blood cells have been completely removed, and may contain red blood cells. For example, the blood sample may be a blood sample in which the amount of red blood cells in blood collected from a living body has been reduced by the separation process.

[0060] Figure 2 shows an example of the flow of the sample preparation method. As shown in Figure 2, the sample preparation method includes, for example, a priming step S101, a capture step S102, a recovery step S103, and a hollow fiber membrane treatment step S104. These steps will be described below with reference to Figure 1.

[0061] Figure 3 is a diagram of the configuration example shown in Figure 1, with the types of liquids used in this example written in each container. As shown in Figure 3, container 41 contains a washing buffer. Container 42 contains a DNase I-containing buffer as a liquid containing a nucleic acid degrading substance. Container 43 contains a staining buffer as a binding auxiliary liquid. Container 44 contains a fluorescently labeled antibody-containing liquid as a liquid containing a particle-binding substance.

[0062] The container 12 of the bioparticle capture module 10 is filled with a substrate on which an antibody that specifically binds to a target cell is immobilized. The antibody corresponds to the substance that captures the bioparticle in this technology. The antibody is immobilized on the substrate via DNA; that is, the DNA is bound to the substrate, and the antibody is bound to the DNA. Therefore, the antibody is released from the substrate by cleaving or decomposing the DNA.

[0063] (3-1) Priming process

[0064] In the priming step S101, the flow path through which the cell-containing liquid passes is filled with, for example, a washing buffer or a staining buffer. In the priming step S101, the bioparticle capture module 10, the reservoir 20, and the hollow fiber membrane module 30 can also be filled with the washing buffer or the staining buffer. This prevents the flow path from being blocked by nonspecific binding between the flow path and the cells.

[0065] For example, priming with a wash buffer can be performed by controlling the valves and pumps as follows. The following valve and pump control may be performed by the user or by the sample preparation system of the present technology itself (particularly the control unit). Furthermore, the valves and pumps in each step (and the various processes included in each step) after the priming step may also be controlled by the user or by the sample preparation system of the present technology itself (particularly the control unit). The sample preparation system of the present technology itself, particularly the control unit, controls the valves and pumps, thereby enabling automatic sample preparation.

[0066] For example, the pump P1 is driven with the valves V0.2, V1.4, and V2 open, thereby priming the flow path 60, the bioparticle capture module 10, the flow paths 61, 62, and 63, and the reservoir 20 with the washing buffer.

[0067] Next, with valves V0.2 and V0.4 open, pump P3 is driven, thereby priming channels 69 and 55 with wash buffer.

[0068] Next, with valves V4 and V5 open and valves V2 and V3 closed, pumps P2 and P3 are driven, thereby priming the flow paths 64, 65, 68, and 55 and the hollow fiber membrane module 30 with the washing buffer.

[0069] Next, with valves V2, V3, and V5 open and valve V4 closed, pump P2 is driven, thereby priming flow path 66 with wash buffer.

[0070] By changing the way the pumps and valves are controlled, for example, channels 64, 65, and 66 can also be primed with a staining buffer.

[0071] (3-2) Capture process

[0072] (3-2-1) Capture process

[0073] In the capture step S102, the cell-containing liquid in the container 40 is supplied to the bioparticle capture module 10. As described above, an antibody that specifically binds to the target cells is immobilized in the container 12 of the bioparticle capture module 10. Therefore, the target cells bind to the antibody, and as a result, the target cells are captured in the container 12.

[0074] For example, pump P3 is driven with valves V0.1 and V0.4 open to supply the cell-containing liquid to the bioparticle capture module 10. During this drive, other valves may be closed, or other pumps may not be driven.

[0075] As a result of this actuation, the cell-containing liquid passes through channels 50 and 60 and enters container 12 from inlet 11 of bioparticle capture module 10. As a result, target cells are captured by antibodies in container 12 as described above, while non-target cells pass through container 12 and exit outlet 13, and are then collected in waste container 45 through channels 61, 69, and 55.

[0076] (3-2-2) Excess cell removal process

[0077] In the above operation (3-2-1), while some of the non-target cells are collected in the waste liquid container 55, there may also be non-target cells that are not bound to the antibody but remain in the container 12 of the bioparticle capture module 10. Therefore, in order to remove such non-target cells from the container 12, an excess cell removal process using a washing buffer may be performed. This removal process may be performed by passing the washing buffer in the container 41 through the bioparticle capture module 10.

[0078] For the removal process, for example, pump P3 is driven with valves V0.2 and V0.4 open. During this drive, other valves may be closed, or other pumps may not be driven.

[0079] By this actuation, the washing buffer passes through the flow paths 51 and 60 and enters the container 12 from the inlet 11 of the bioparticle capture module 10, then passes through the container 12 and exits from the outlet 13. As a result, the non-target cells exit from the outlet 13 of the container 12 and are collected in the waste container 55 through the flow paths 61, 69, and 55.

[0080] (3-3) Recovery process

[0081] In the recovery step S103, the target cells in the container 12 of the bioparticle capture module 10 are released from the substance that captures the bioparticles and then recovered into the reservoir 20. To perform the release, a DNase I-containing buffer in the container 42 is supplied to the bioparticle capture module 10. The DNase I decomposes the DNA that immobilizes the antibody to the substrate, and the target cells bound to the antibody are released from the substrate. The target cells released from the substrate then exit the outlet 13 and are recovered into the reservoir 20.

[0082] To perform the recovery, for example, pump P1 is driven with valves V0.3, V1.4, and V2 open, while other valves may be closed or other pumps may not be driven.

[0083] As a result of this actuation, the DNase I-containing buffer passes through channels 52 and 60 and enters container 12 from inlet 11 of bioparticle capture module 10. DNase I degrades the DNA that immobilizes the antibody on the substrate, liberating the target cells from the substrate. The liberated cells exit outlet 13, pass through channels 61, 62, and 63, and are collected in reservoir 20. Like the liberated cells, the DNase I-containing buffer is also collected in reservoir 20. Therefore, the action of DNase I is exerted in the following hollow fiber membrane treatment process as well.

[0084] (3-4) Hollow fiber membrane treatment process

[0085] (3-4-1) Density adjustment process

[0086] In the hollow fiber membrane treatment step S104, the target cells in the reservoir 20 are passed through the hollow fiber membrane module 30. This allows the concentration of the target cells to be adjusted, for example, the concentration of the target cells can be increased.

[0087] To adjust the concentration, for example, pumps P2 and P3 are driven with valves V4 and V5 open. During this driving, other valves may be closed, or other pumps may not be driven.

[0088] By this actuation, the liquid containing the target cells in the reservoir 20 passes through the flow path 64 and enters the container 32 from the inlet 31 of the hollow fiber membrane module 30, and the liquid containing the target cells that passes through the container 32 exits from the outlet 33 and returns to the reservoir 20 through the flow path 65. As a result, the target cells are concentrated by the hollow fiber membrane in the container 32.

[0089] The waste liquid (permeate from the hollow fiber membrane treatment) resulting from the concentration is discharged from the waste liquid outlet 34 and collected in the waste liquid container 45 through the flow paths 68 and 55 .

[0090] (3-4-2) Solvent exchange treatment

[0091] In the hollow fiber membrane processing step S104, a binding process may be performed to bind an antibody (e.g., a fluorescently labeled antibody) to the target cells. To perform this binding process, it is preferable to exchange the solvent in which the target cells are held for a solvent suitable for this binding process. Therefore, for this binding, the solvent of the target cell-containing liquid is exchanged for a staining buffer, which is a binding-assisting liquid. For this solvent exchange, for example, diafiltration using a hollow fiber membrane module may be performed.

[0092] For the solvent exchange, for example, pump P1 is driven with valves V1.1 and V2 open, thereby supplying staining buffer into reservoir 20 and increasing the proportion of staining buffer in the target cell-containing solution. Simultaneously with pump P1 operating with valves V1.1 and V2 open, pumps P2 and P3 are operated with valves V4 and V5 open. By operating pumps P2 and P3, the target cell-containing liquid in reservoir 20 passes through flow path 64 and enters container 32 from inlet 31 of hollow fiber membrane module 30. The target cell-containing liquid that passes through container 32 exits outlet 33 and returns to reservoir 20 through flow path 65. This concentrates the target cells using the hollow fiber membranes in container 32. The waste liquid resulting from the concentration exits waste outlet 34 and passes through flow paths 68 and 55 to be collected in waste container 55. By driving pump P1 with valves V1.1 and V2 open and by driving pumps P2 and P3 with valves V4 and V5 open, the solvent in the target cell-containing solution is exchanged for the staining buffer. In the above explanation, the operation of pump P1 with valves V1.1 and V2 open and the operation of pumps P2 and P3 with valves V4 and V5 open are performed simultaneously in parallel, but these two operation operations may be performed sequentially, or may be performed sequentially and repeatedly.

[0093] (3-4-3) Dead volume collection process

[0094] Hollow fiber membrane processing has the problem of so-called dead volume, as described below. Specifically, in hollow fiber membrane processing, a process such as concentration or diafiltration is performed while circulating a liquid containing a hollow fiber membrane column. Therefore, at the stage where the process is completed, sample remains in the flow paths and hollow fiber membrane modules other than the collection container, and the amount of this remaining sample is the dead volume. For example, in the sample preparation system shown in FIG. 3, at the stage where the solvent exchange process described above in (3-4-2) is completed, cell-containing liquid remains in the flow path 64, hollow fiber membrane module 30, and flow path 65, and the amount of this remaining cell-containing liquid is the dead volume.

[0095] The problem of dead volume is not apparent when a large amount of the target substance is collected in the collection container by hollow fiber membrane processing. However, in bioparticle analysis, such as flow cytometry, the amount of sample used is often relatively small, for example, about 5 mL. This amount is smaller than the expected collection amount in general hollow fiber membrane processing, for example, by about one order of magnitude. Therefore, in bioparticle analysis, it is desirable to minimize the dead volume in hollow fiber membrane processing. Furthermore, in bioparticle analysis, rare cells, such as certain immune cells, may be targeted for analysis. Therefore, in order to ensure sufficient cell numbers, it is desirable to minimize the dead volume in hollow fiber membrane processing.

[0096] The inventors have found that the problem of dead volume can be solved by using a branch flow path 66 that branches off from the circulation flow path that circulates bioparticles between the reservoir 20 and the hollow fiber membrane module 30 and leads to the reservoir 20.

[0097] For example, the dead volume after the solvent exchange process in (3-4-2) above can be recovered in the reservoir 20 by carrying out the following operation.

[0098] First, pump P1 is driven with valves V1.1, V3, and V4 open. During this drive, other valves may be closed, or other pumps may not be driven. As a result, the target cell-containing solution in flow channel 64 from the junction between branch flow channel 66 and flow channel 65 (the junction between valves V4 and V5) to the end of reservoir 20 is collected into reservoir 20.

[0099] Next, with pump P1 open and valves V1.1, V3, and V5 open, pump P2 is driven in the reverse direction (i.e., so that the liquid proceeds in the order of flow path 65, hollow fiber membrane module 30, and flow path 64). As a result, the target cell-containing liquid in the portion of flow path 65 from the junction of branch flow path 66 and flow path 65 (the junction between valves V4 and V5) to the outlet 33 of the hollow fiber membrane module 30, the target cell-containing liquid in the hollow fiber membrane module 30, and the target cell-containing liquid in flow path 64 (i.e., the target cell-containing liquid in the flow path from the inlet 31 of the hollow fiber membrane module 30 to the inside of reservoir 20) are recovered into reservoir 20.

[0100] When driving the pump P2 in the reverse direction, the pump P3 can preferably be driven in the reverse direction (i.e., so that the liquid flows from the flow path 68 into the container 32 of the hollow fiber membrane module 30). This driving is performed to encourage the cells to detach from the hollow fiber membranes. This driving may be performed, for example, at a low speed. This driving may also be performed, for example, for a short time (e.g., 0.1 to 5 seconds, particularly 0.5 to 3 seconds). For example, this short-time driving may be performed one or more times, for example, 1 to 10 times, particularly 2 to 5 times. Driving the pump P3 in this manner makes it easier for the cells to detach from the hollow fiber membranes, and the number of cells recovered in the reservoir 20 can be increased.

[0101] (3-4-4) Binding treatment for binding antibodies to cells

[0102] As described above, in the hollow fiber membrane processing step S104, a binding treatment may be performed to bind the antibody to the cells. For this binding treatment, the antibody-containing liquid in the container 44 is introduced into the reservoir 20. Then, incubation is performed to allow the antibody to bind to the cells. The incubation conditions (e.g., time and temperature) may be appropriately selected by those skilled in the art.

[0103] To introduce the antibody-containing liquid into reservoir 20, for example, pump P1 is driven with valves V1, 2, and V2 open. During this drive, other valves may be closed, or other pumps may not be driven. This drive causes the antibody-containing solution in container 44 to pass through channels 54 and 63 and be supplied into reservoir 20. Then, the incubation is performed. For the incubation, the temperature of reservoir 20 may be maintained at, for example, about 4°C.

[0104] (3-4-5) Cell concentration treatment

[0105] After the cell labeling process described in (3-4-4) above, it is preferable to remove excess antibody. This removal can be performed, for example, by a process similar to the solvent exchange process described in (3-4-2) above. However, antibody removal can be more efficiently performed by increasing the cell concentration in the reservoir, i.e., concentrating the cells, before the cell labeling process. Therefore, a concentration process can be performed as described in (3-4-1) above.

[0106] For the concentration process, for example, pumps P2 and P3 are driven with valves V4 and V5 open, while other valves may be closed or other pumps may not be driven.

[0107] As a result of this actuation, the liquid containing antibody-labeled target cells in reservoir 20 passes through flow path 64 and enters container 32 from inlet 31 of hollow fiber membrane module 30. The liquid containing target cells that passes through container 32 then exits outlet 33 and returns to reservoir 20 through flow path 65. This causes the antibody-labeled target cells to be concentrated by the hollow fiber membrane in container 32. For example, since the sample used in bioparticle analysis such as flow cytometry is approximately 5 ml, when a sample prepared by a sample preparation system according to the present technology is to be used in such bioparticle analysis, the sum of the amount of liquid in the reservoir after the concentration process and the amount of dead volume remaining in the flow path can be adjusted to about that amount.

[0108] (3-4-6) Excess antibody removal treatment

[0109] As described in (3-4-5) above, it is preferable to remove excess antibody after the cell labeling process. Therefore, the liquid containing the target cells after the concentration process described in (3-4-5) above is subjected to a solvent exchange process using a staining buffer. The solvent exchange process may be performed, for example, as described in (3-4-2) above.

[0110] Specifically, as described in (3-4-2) above, the operation of pump P1 with valves V1.1 and V2 open and the operation of pumps P2 and P3 with valves V4 and V5 open can be performed sequentially or simultaneously.

[0111] This actuation causes diafiltration of the target cell-containing solution with the staining buffer, and excess antibody is removed from the target cell-containing solution.

[0112] (3-4-7) Density adjustment processing

[0113] After antibody removal in (3-4-6) above, the target cell concentration in the target cell-containing solution may be adjusted. The concentration adjustment may be, for example, a dilution treatment to decrease the concentration or a concentration treatment to increase the concentration. The target cell concentration may be appropriately selected depending on the bioparticle analysis to which the prepared sample will be subjected.

[0114] For example, to perform a dilution process, pump P1 is driven with valves V1.1 and V2 open, which allows the staining buffer to be supplied into reservoir 20 through channels 53 and 63, thereby decreasing the concentration of target cells. During the dilution process, valves V4 and V5 may be opened and pump P2 may be driven. This allows the cells in the target cell-containing liquid to be uniformly dispersed. This also prevents the pores of the hollow fibers from becoming clogged with cells.

[0115] Furthermore, to perform the concentration process, as described in (3-4-5) above, pumps P2 and P3 are driven with valves V4 and V5 open. During this driving, other valves may be closed, or other pumps may not be driven.

[0116] In adjusting the concentration, concentration measurement may be performed using an analysis device 90 that analyzes the liquid in the reservoir 20. The analysis device 90 may be, for example, an analysis device that performs turbidity measurement or fluorescence measurement, and the concentration of the target cells can be determined from the measured turbidity or fluorescence. Depending on the measured concentration, it can be determined whether to start, continue, or end the concentration adjustment process (dilution process or concentration process).

[0117] (3-4-8) Dead volume collection process

[0118] After the concentration adjustment process described in (3-4-7) above, the dead volume collection process can be performed again. This allows the dead volume remaining in the flow path and the hollow fiber membrane module after the concentration adjustment process to be collected. The dead volume process can be performed as described in (3-4-3) above.

[0119] A liquid sample containing target cells is prepared by the process described above. The prepared sample may be collected in a container outside reservoir 20, for example, via flow path 67, and the container may be used to subject the sample to subsequent bioparticle analysis processing. Alternatively, reservoir 20 itself may be used as a container for subjecting the sample to subsequent bioparticle analysis processing.

[0120] (3-5) Modifications

[0121] In the above-described process, the bioparticle capture module process is carried out first, and then the hollow fiber membrane module process is carried out. In the present technology, for example, a hollow fiber membrane processing step (hereinafter also referred to as a "first hollow fiber membrane processing step") may be performed first, followed by a bioparticle capture step. A hollow fiber membrane processing step (hereinafter referred to as a "second hollow fiber membrane processing step") may then be performed. These steps may be performed by the sample preparation system 1 described above with reference to Figures 1 and 3, but may also be performed by, for example, the sample preparation system 3 shown in Figure 13. Below, the sample preparation system 3 will be described with reference to Figure 13, and then an example of sample preparation processing using the sample preparation system 3 will be described with reference to the flow chart in Figure 14.

[0122] The sample preparation system 3 shown in FIG. 13 has a configuration in which the following changes have been made to the sample preparation system 1. In sample preparation system 1, container 40 for supplying cell-containing liquid is connected to flow path 60 via flow path 50, but in sample preparation system 3, it is connected to flow path 63 via flow path 56. In addition, valve V6 is provided on flow path 56. Opening and closing valve V6 enables or disables the supply of cell-containing liquid (sample) contained in container 40 to reservoir 20. In sample preparation system 1, flow path 60 was connected to container 40, but in sample preparation system 3, flow path 60 is not connected to container 40 but is connected to flow path 65, particularly to a portion of flow path 65 between valves V4 and V5. In addition, valve V8 is provided in flow path 60 between the connection point to flow path 65 and the connection point to flow path 51. Container 43 contains a staining buffer in sample preparation system 1, but in sample preparation system 3 contains a staining buffer containing DNaseI. In addition to the container 40 being connected to the flow path 63 as described above, a container 46 containing a staining buffer that does not contain DNase I is also connected to the flow path 63 via a flow path 57. A valve V7 is provided on the flow path 57. Opening and closing the valve V7 enables or disables the supply of the DNase I-free staining buffer in the container 40 to the reservoir 20.

[0123] 14 shows an example of a flow diagram of the sample preparation process by the sample preparation system 3. The process may be performed, for example, as follows.

[0124] (3-5-1) Priming process

[0125] In the priming step S201, as described in (3-1) above, the flow path through which the cell-containing liquid passes is filled with, for example, a washing buffer or a staining buffer. In the priming step S201, the bioparticle capturing module 10, the reservoir 20, and the hollow fiber membrane module 30 may also be filled with the washing buffer or the staining buffer. The valves and pumps in the priming step may be controlled, for example, as described in (3-1) above.

[0126] (3-5-2) Supply process

[0127] In supply step S202, the cell-containing solution in container 40 and the DNase I-containing staining buffer in container 43 are supplied to reservoir 20. To supply these, for example, pump P1 is first driven with valves V6 and V2 open, and then pump P1 is driven with valves 1.1 and V2 open. By driving the former, the cell-containing solution is introduced into reservoir 20, and then by driving the latter, the staining buffer is supplied to reservoir 20.

[0128] (3-5-3) First hollow fiber membrane treatment process

[0129] In the first hollow fiber membrane processing step S203, the concentration of the cell-containing solution in the reservoir 20 can be adjusted using the hollow fiber membrane module 30. After this adjustment, the cells in the reservoir 20 are labeled with the antibody in the container 44. To perform this labeling, the hollow fiber membrane processing step S203 may perform, for example, the "(3-4-1) concentration adjustment process," "(3-4-2) solvent exchange process," "(3-4-3) dead volume recovery process," "(3-4-4) binding process for binding the antibody to the cells," and "(3-4-5) cell concentration process" described in (3-4) above. These processes may be as explained in (3-4) above, and the explanation also applies to this modified example.

[0130] After the cell concentration process, a process for removing excess antibody from reservoir 20 may be performed. The excess antibody process may be performed by exchanging the solvent in reservoir 20. Specifically, pump P1 may be driven with valves V7 and V2 open, and pumps P2 and P3 may be driven with valves V4 and V5 open, either simultaneously or sequentially. This replaces the liquid in reservoir 20 with the DNase I-free staining buffer in container 46. In other words, the liquid containing cells no longer contains DNase I.

[0131] (3-5-4) Capture process

[0132] (3-5-4-1) Capture process

[0133] In the capture step S204, the cell-containing liquid in the reservoir 20 is supplied to the bioparticle capture module 10. As described above in (3-2-1), an antibody that specifically binds to the target cells is immobilized in the container 12 of the bioparticle capture module 10. Therefore, the target cells bind to the antibody, and thereby the target cells are captured in the container 12.

[0134] For example, pump P3 is driven with valves V4, V8, and V0.4 open to supply the cell-containing liquid to the bioparticle capture module 10. During this drive, other valves may be closed, or other pumps may not be driven.

[0135] This actuation causes the cell-containing liquid in reservoir 20 to pass through flow path 60 (more specifically, from reservoir 20, through the portion of flow path 60 where valve V4 is located and then the portion of flow path 60 where valve V8 is located) and enter container 12 from inlet 11 of bioparticle capture module 10. As a result, the target cells are captured by the antibody in container 12 as described above, while non-target cells pass through container 12 and exit from outlet 13, and are then collected in waste container 45 through flow paths 61, 69, and 55.

[0136] (3-5-4-2) Excess cell removal process

[0137] In the operation (3-5-4-1) above, the excess cell removal process described in (3-2-2) above may be performed. The valves and pumps for the removal process may be operated as described in (3-2-2) above.

[0138] (3-5-5) Recovery process

[0139] In the recovery step S205, as described in (3-3) above, the target cells in the container 12 of the bioparticle capture module 10 are released from the substance that captures the bioparticles and then recovered into the reservoir 20. To perform the release, a DNase I-containing buffer in the container 42 is supplied to the bioparticle capture module 10. The DNase I decomposes the DNA that immobilizes the antibody to the substrate, and the target cells bound to the antibody are released from the substrate. The target cells released from the substrate then exit the outlet 13 and are recovered into the reservoir 20.

[0140] To perform the recovery, for example, pump P1 is driven with valves V0.3, V1.4, and V2 open, while other valves may be closed or other pumps may not be driven.

[0141] As a result of this driving, the DNase I-containing buffer passes through channels 52 and 60 and enters container 12 from inlet 11 of bioparticle capture module 10. DNase I degrades the DNA that immobilizes the antibody on the substrate, liberating the target cells from the substrate. The liberated cells exit outlet 13, pass through channels 61, 62, and 63, and are collected in reservoir 20. Like the liberated cells, the DNase I-containing buffer is also collected in reservoir 20. Therefore, the action of DNase I is exerted in the next process as well.

[0142] (3-5-6) Second hollow fiber membrane treatment process

[0143] In the second hollow fiber membrane processing step S206, a washing process for cells in the cell-containing solution containing the target cells collected in the reservoir and a cell concentration process for concentrating the target cells may be performed. The cell-containing solution obtained by the second hollow fiber membrane processing step may be treated as a sample prepared by the system of the present technology. The washing process may be performed, for example, by carrying out the same operation as the solvent exchange process described in (3-4-2) above. The cell concentration process may be performed, for example, as described in (3-4-5) above. It should be noted that the target cell-containing liquid collected in the reservoir 20 in (3-5-5) above may be treated as a sample prepared by the system of the present technology without carrying out the second hollow fiber membrane treatment step.

[0144] (4) Example of processing using the bioparticle capture module

[0145] As described above, the bioparticle capture module includes a substrate on which a substance for capturing bioparticles is immobilized. The substance for capturing bioparticles captures the target bioparticles. Cells other than the target cells are not captured and can be discarded, for example, by washing. This allows the target bioparticles to be selectively supplied to the reservoir. Specific examples of the operation of the bioparticle capture module are described below.

[0146] (4-1) Example of the configuration and operation of the bioparticle capture module

[0147] The capture and release of target cells by the bioparticle capture module will be described with reference to Figure 4. As shown in Figure 4A, the bioparticle capture module includes a substrate S on which a substance for capturing bioparticles is immobilized. The substrate S may be a fibrous substrate as shown in Figure 4A, but it does not have to be fibrous. For example, the substrate S may be a porous substrate or a beaded substrate.

[0148] A substance that captures bioparticles is immobilized on the substrate S. Preferably, the substance that captures bioparticles is immobilized on the substrate S via nucleic acids. This allows the target cells to be released by the nucleic acid degrading substance described above.

[0149] The substrate S can be packed in the bioparticle capture module so as to have gaps of, for example, 100 μm to 1000 μm, particularly 200 μm to 800 μm, 300 μm to 700 μm, and more particularly about 500 μm. The size of the gaps can be set so that, for example, bioparticles (particularly cells) can easily pass through.

[0150] As described above, the substance that captures the bioparticles is preferably a substance that binds to the target bioparticles, particularly an antibody. The type of antibody may be selected depending on the target bioparticles, particularly depending on the surface antigens of the target bioparticles. Thus, in a preferred embodiment of the present technology, the antibody may be immobilized on the substrate via a nucleic acid.

[0151] 4B, a liquid containing a biological particle is supplied to the biological particle capturing module, and the liquid contains target biological particles Pt and non-target biological particles Pn.

[0152] As shown in Figure 4C, by passing the bioparticle-containing liquid through the bioparticle capture module, the target particles Pt are captured by the bioparticle capture substance fixed to the substrate S. On the other hand, the non-target particles Pn pass through the bioparticle capture module without being captured by the bioparticle capture substance. Note that, as shown in Figure 4C, it is possible that not all of the non-target particles pass through the bioparticle capture module, and some remain in the bioparticle capture module.

[0153] In order for the bioparticles to bind to the substance that captures them, the bioparticles must come into contact with the substrate S. The frequency of this contact may be controlled, for example, by the flow rate. If necessary, after supplying the bioparticle-containing liquid into the bioparticle capture module, the bioparticle-containing liquid may be maintained in the module for a predetermined time, i.e., incubation for the binding may be performed. The incubation time may be selected as appropriate, but may be, for example, 10 minutes to overnight, 10 minutes to 12 hours, 10 minutes to 5 hours, 10 minutes to 3 hours, 20 minutes to 2 hours, or 30 minutes to 60 minutes.

[0154] As shown in Figure 4D, the target particles Pt are released from the substrate S. For this release, a nucleic acid degrading substance may be used, as described above. The nucleic acid degrading substance releases the bioparticle-capturing substance from the substrate, and the bioparticle-capturing substance is then collected in a reservoir.

[0155] (4-2) Test Example (Blood Treatment Using a Substrate Immobilized with a Substance Capturing Bioparticles)

[0156] An antibody fixation solution containing 62.5 μg / mL anti-porcine CD3 antibody (Mouse Anti-Porcine CD3ε, clone PPT3, Southern Biotechnology Associates, Inc.), 7.5 μg / mL N-Hydroxysuccinimide (NHS) (Nacalai Tesque), and 5.0 μg / mL 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl (EDC) (Nacalai Tesque) was prepared in a solution of 0.1 M MES (pH 6.0), 0.5 M NaCl, and the antibody fixation solution was allowed to stand for 15 minutes. After this standing, a four-fold volume of 5xPBS(-) was added to the antibody fixation solution, and a collagen sponge (Collagen Sponge Mighty, sterile φ5 x 3 mm, Koken Co., Ltd.) was then added. After 1 hour, the collagen sponge was removed and transferred to D-PBS(-). Through these steps, the anti-porcine CD3 antibody was fixed to the collagen sponge. Three pieces of the fixed collagen sponge were loaded into a 1 mL syringe. A photograph of the filled syringe is shown in Figure 5 A. Medium was added to the syringe to wash the inside of the syringe.

[0157] After this washing, 1 mL of porcine whole blood was added to the syringe, as shown in Figure 5B. As a result, it was visually confirmed that red blood cells naturally passed through the collagen sponge and fell downward, as shown in Figure 5C. Some whole blood remained in the syringe, but by pressing the plunger, the remaining whole blood could also be forced to pass through the collagen sponge. These results confirmed that red blood cells passed through the collagen sponge without being trapped.

[0158] The fraction captured by the collagen sponge was then collected by applying water pressure to the syringe. The collected fraction was analyzed by flow cytometry. The whole blood was also analyzed by flow cytometry. The analysis results for the whole blood are shown in Figure 5D, and the analysis results for the fraction are shown in Figure 5E. As shown in Figure 5D, for the whole blood, almost no cells were observed within the leukocyte gate. On the other hand, for the fraction, 40% of the detected cells were observed within the leukocyte gate. These results confirmed that the collagen sponge with the antibody immobilized on it can increase the percentage of leukocytes.

[0159] (4-3) Other Configuration and Operation Examples of the Bioparticle Capture Module

[0160] In the present technology, as described above, the substance that captures bioparticles may be a substance that captures bioparticles via another substance, for example, a substance that captures bioparticles via a bioparticle-binding substance. An example of the configuration and operation of a bioparticle capture module including a substrate to which the substance that captures bioparticles via another substance is fixed will be described below with reference to FIG.

[0161] As shown in Fig. 6A, the bioparticle capture module includes a substrate S on which a substance for capturing bioparticles is immobilized. The substrate S may be a fibrous substrate as shown in Fig. 6A, but it does not have to be fibrous. For example, the substrate S may be a porous substrate or a beaded substrate.

[0162] A substance that captures bioparticles is immobilized on the substrate S. Preferably, the substance is immobilized on the substrate S via nucleic acids. This allows the target cells to be released by the nucleic acid degrading substance described above.

[0163] The substrate S can be packed in the bioparticle capture module so as to have gaps of, for example, 100 μm to 1000 μm, particularly 200 μm to 800 μm, 300 μm to 700 μm, and more particularly about 500 μm. The size of the gaps can be set so that, for example, bioparticles (particularly cells) can easily pass through.

[0164] As described above, the substance that captures bioparticles via another substance may be a substance that binds to a bioparticle-binding substance, such as an antibody-binding protein. The antibody-binding protein may be, for example, any one or more of Protein A, Protein G, Protein L, and Protein A / G. Thus, in a preferred embodiment of the present technology, the antibody-binding protein may be immobilized on a substrate via a nucleic acid.

[0165] As shown in Figure 6B, an antibody-containing liquid is supplied to the bioparticle capture module. As the antibody-containing liquid passes through the module, the antibody AB, which is a bioparticle-binding substance, binds to the antibody-binding protein. As a result, as shown in Figure 6C, the antibody AB is immobilized on the substrate S. The antibody AB may be an antibody that specifically binds to a target particle.

[0166] 6D, a liquid containing a biological particle is supplied to the biological particle capturing module, and the liquid contains target biological particles Pt and non-target biological particles Pn.

[0167] As shown in FIG. 6E, by passing the bioparticle-containing liquid through the bioparticle capturing module, the target particles Pt are captured by the antibodies AB immobilized on the substrate S. On the other hand, the non-target particles Pn are not captured by the antibodies AB and pass through the bioparticle capturing module. As shown in Figure 6E, some of the unintended particles may not pass through the biological particle capture module, and some may remain in the biological particle capture module. Note that, in order to expel as many unintended particles as possible from the module, a washing step may be performed, for example, by flowing a washing buffer through the module.

[0168] After FIG. 6E, as shown in FIG. 6F, a process for binding an antibody (especially a fluorescently labeled antibody) to the surface antigen of the target bioparticle Pt may be performed. For this process, an antibody-containing liquid is passed through the module. This process may be performed, for example, as an antibody staining process. In this way, in the present technology, the antibody staining process may be performed within the bioparticle capture module.

[0169] After the treatment described with reference to FIG. 6E or 6F, the target particle Pt is released from the substrate S. For this release, a nucleic acid degrading substance may be used, as described above. The antibody-binding protein is released from the substrate by the nucleic acid degrading substance. As a result of this release, the antibody AB bound to the antibody-binding protein and the target bioparticle Pt bound to the antibody AB are released from the substrate and collected in a reservoir.

[0170] (5) Evaluation of the effect of nucleic acid degrading substances on treatment using hollow fiber membrane modules

[0171] In the sample preparation method described in (3) above, in the recovery step S103, the target cells are liberated from the substrate using DNase I, a nucleic acid degrading substance. The nucleic acid degrading substance is recovered together with the target cells into the reservoir 20. Then, in the hollow fiber membrane treatment step S104, the nucleic acid degrading substance flows through the hollow fiber membrane module 30 together with the target cells. In this technology, by flowing the nucleic acid degrading substance through the hollow fiber membrane module 30 in this manner, clogging of the hollow fiber membrane module 30 can be prevented.

[0172] To confirm the effect of preventing clogging in the hollow fiber membrane treatment process using the nucleic acid degrading substance, an apparatus 2 having the configuration shown in Figure 7 was prepared. The apparatus is equipped with a reservoir 20 and a hollow fiber membrane module 30, and is capable of concentrating the cell-containing liquid in the reservoir 20. A container 43 containing a staining buffer is connected to the reservoir via a flow path. The reservoir 20 and the hollow fiber membrane module 30 are as described above in (3).

[0173] A cell-containing solution containing DNase I was placed in reservoir 20 of device 2, and a set of processes similar to the cell concentration process described above in (3-4-1), the solvent exchange process described above in (3-4-2), the dead volume recovery process described above in (3-4-3), and the binding process for binding antibodies to cells described above in (3-4-4) were carried out using hollow fiber membrane module 30. Note that in this binding process, the antibodies were not introduced from a flow path connected to reservoir 20, but were added directly into reservoir 20 by hand. More specifically, the following operations were carried out.

[0174] First, the components in the device 2 that come into contact with the liquid (flow path, reservoir, and hollow fiber membrane module) were primed. Next, 1x10 8 12 mL of mononuclear cell suspension was added. The cell concentration of the suspension was doubled using the hollow fiber membrane module 30 of the device 2, and the concentrated suspension was then subjected to solvent exchange with four times the volume of DNase buffer. After the solvent exchange, DNase was added to the reservoir and circulated for approximately 10 minutes. After the solvent exchange, a dead volume recovery process was performed. The recovery volume from this recovery process was 20 mL. Three types of cell staining antibodies were added to the reservoir 20, and a binding process (4°C, 30-60 minutes) was performed to bind the antibodies to the cells. The suspension after the binding process was concentrated approximately four times, and a solvent exchange process was performed with five times the volume of staining buffer. The dead volume recovery process was then performed to recover the cells. The recovery volume from this recovery process was 5.5 mL. A small amount (approximately 2 mL) of staining buffer was then circulated, and the cells were recovered in the same manner.

[0175] In addition, the same set of treatments was carried out except that the cell-containing solution did not contain DNase I. Specifically, the treatments were as follows.

[0176] First, the components in the device 2 that come into contact with the liquid (flow path, reservoir, and hollow fiber membrane module) were primed. Next, 1x108 12 mononuclear cell suspensions 1 mL was added. Using the hollow fiber membrane module 30 of the device 2, the cell concentration of the suspension was concentrated two-fold, and the concentrated suspension was then subjected to solvent exchange with four times the volume of staining buffer. After the solvent exchange, a dead volume recovery process was performed. The recovery volume from this recovery process was 20 mL. Three types of cell staining antibodies were added to the reservoir 20, and a binding process (4°C, 30 to 60 minutes) was performed to bind the antibodies to the cells. The suspension after the binding process was concentrated approximately four-fold, and a solvent exchange process was performed with five times the volume of staining buffer. The dead volume recovery process was then performed to recover the cells. The recovery volume from this recovery process was 5.5 mL. Furthermore, a small amount (approximately 2 mL) of staining buffer was circulated, and the cells were recovered in the same manner.

[0177] When a cell-containing liquid containing DNase I was used and the above-mentioned set of treatments was performed, no clogging occurred in the hollow fiber membrane module 30 or in the circulation flow path connecting the hollow fiber membrane module 30 and the reservoir 20. On the other hand, when a cell-containing solution not containing DNase I was used, cell aggregates were observed in the reservoir 20 after the solvent exchange treatment, as shown in Figure 8. In addition, clogging occurred in the hollow fiber membrane module 30. The above results indicate that the use of a nucleic acid degrading substance, such as DNase I, can prevent cell aggregation and prevent clogging in the hollow fiber membrane module. Therefore, the use of a nucleic acid degrading substance is preferred in the present technology. For example, the sample preparation system of the present technology may be configured so that the nucleic acid degrading substance can pass through the bioparticle capture module and reach the reservoir or the hollow fiber membrane module.

[0178] (6) Evaluation of antibody staining using hollow fiber membrane modules

[0179] In the sample preparation method described in (3) above, a binding process for binding antibodies to cells is carried out in the hollow fiber membrane processing step S104. This binding process is also called an antibody staining process. An evaluation was carried out as follows to confirm whether the cells were antibody-stained by the antibody staining process using the hollow fiber membrane module.

[0180] First, the apparatus described in (5) above with reference to Figure 7 was prepared. Using this apparatus, PBMCs (peripheral blood mononuclear cells) were subjected to the solvent exchange process described in (3-4-2) above and the antibody binding process described in (3-4-4) above. The PBMCs were also subjected to antibody staining using the same antibodies. The materials used in these processes and the specific operating procedures are as follows. A schematic diagram of the operating procedures is also shown in Figure 12.

[0181] (6-1) Experimental materials Hollow fiber membrane module: C02-E65U-07-N (Repligen, non-sterile, pore size 0.65 μm, membrane material mPES) Pressure sensor for measuring the pressure of the hollow fiber membrane module: ACPM-799-01N (Repligen, polysulfone) Tubing that constitutes the flow path: Pharmapure #14 Frozen PBMC: 1 x 10 cells per tube 8 Five vials Antibodies: PE-labeled anti-CD3 antibody, FITC-labeled anti-CD4 antibody, and VioBlue-labeled anti-CD8 antibody

[0182] (6-2) Preparation and manual staining of PBMCs Step 1-1. The frozen PBMCs in each vial were heated in a 37°C water bath for about 2 minutes. Step 1-2: When only a small amount of ice remained in each vial, 5 mL of TM buffer containing 10% human albumin was added to each vial and pipetted, and the contents of each vial were then transferred to a 50 mL tube. Step 1-3. 1 mL of the TM buffer was added to an empty vial and pipetted into each vial in step 2. Step 1-4: The TM buffer was added to the 50 mL tube so that the total volume was 13 mL, and the mixture was passed through a 40 μm cell strainer. Step 1-5: 1 mL of the permeate from the cell strainer was collected, and more than 100 μL of this 1 mL was stained with PI. The white blood cell count and cell count were measured using a cell analyzer (SP-6800, Sony Corporation) and a multiparameter automated hemocytometer (pocH-100I, Sysmex Corporation). The white blood cell count was 93 × 10 2 The cell count was 9 x 10 6 There were 100 pieces. Step 1-6: The remaining 12 mL of the permeate through the cell strainer was subjected to treatment using the device containing the hollow fiber membrane module. This treatment will be described below in <Dyeing treatment using a hollow fiber membrane module>. Step 1-7: The remaining 1 mL of the solution obtained in step 1-5 was centrifuged at 200 g for 10 minutes at room temperature. Step 1-8: After the centrifugation in step 1-7, the supernatant was removed, leaving the pellet, followed by tapping. Step 1-9: 200 μL of wash buffer (WB) containing 200 U / mL of DNase I was added to the pellet, and the mixture was mixed by inversion. Step 1-10: Repeat Step 1-9 above with a total volume of 1.4 mL (44 x 10 white blood cells). 2 cells / μL, number of cells 6×10 6 Staining buffer was added so that the total volume of the stained cells was 1000 μg / ml. Step 1-11. Four 100 μL samples were taken from the 1.4 mL sample obtained in Step 1-10. Two of the four 100 μL samples were stained with three antibodies (PE-labeled anti-CD3 antibody, FITC-labeled anti-CD4 antibody, and VioBlue-labeled anti-CD8 antibody) at room temperature or 4°C for 1 hour, and then immediately washed manually. They were then stained with PI (propidium iodide) and then analyzed using the cell analyzer. Step 1-12: Of the four 100 μL samples prepared in Step 1-11 above, the remaining two were stained with the antibody at room temperature or 4°C for 1 hour. After staining, the samples were manually washed after the treatment described below using the device including the hollow fiber membrane module. Subsequently, the samples were stained with PI (propidium iodide) and then analyzed using the cell analyzer.

[0183] (6-3) Dyeing process using hollow fiber membrane module Step 2-1. 12 mL of the solvent from the permeate described in 6. of (6-2) above was exchanged with the staining buffer using the device including the hollow fiber membrane module. The volume of the sample after solvent exchange was 20 mL. The 20 mL sample was passed through a 40 μm cell strainer. The 20 mL of permeate from the cell strainer was divided into 1 mL and 19 mL portions. To the 19 mL sample, 237.5 μL of each of the three antibodies was added. The antibody addition was performed in a 50 mL tube. Step 2-2: After adding the antibody in step 2-1 above, the mixture was incubated in a cool, dark place for 1 hour. Step 2-3: The sample incubated in step 2-2 above was subjected to a concentration treatment using the hollow fiber membrane module using the device. As a result of this concentration treatment, the cell concentration of the cell suspension was concentrated four times. Step 2-4: After step 2-3, the device was used to perform a washing process to remove excess antibody with staining buffer. This washing process was a solvent exchange process using staining buffer in an amount five times the volume of the concentrated cell suspension. Step 2-5: After step 2-4 above, a recovery process was also performed on the circulation flow path between the reservoir 20 and the hollow fiber membrane module 30 and the dead volume within the hollow fiber membrane module 30. After recovery by this recovery process, the staining buffer was circulated, and the cells were recovered in the same manner. Step 2-6: The cell count was measured for the combined total of the sample in the reservoir obtained by the washing process in step 2-4 and the collected sample obtained by the collection process in step 2-5 using a multi-parameter automated hemocytometer (pocH-100I, Sysmex Corporation). The sample was also stained with PI (propidium iodide) and then analyzed using the cell analyzer.

[0184] Figure 9 shows the results of cell analyzer analysis of the cells stained manually as described in (6-2) above, and the results of cell analyzer analysis in step 2-6 of (6-3) above. As shown in Figure 9, for all antibodies, the percentage of positive cells for each antibody among the cells stained using the hollow fiber membrane module was higher than that of the manual staining. This indicates that the staining process using the hollow fiber membrane module has better staining efficiency than the manual process. Furthermore, although the staining process using the hollow fiber membrane module involves concentration and washing processes, it was also confirmed that appropriate staining was possible.

[0185] The results of measuring the cell count using the hemocytometer for the cells stained using the hollow fiber membrane module described in (6-3) above are shown in Figure 10. As shown in Figure 10, at the stage of step 1-4, the liquid volume was 13 mL and the white blood cell count was 93 x 10 2 After the washing process in step 2-4, the liquid volume was 5.3 mL, and the white blood cell count was 127 × 10 2 The white blood cell recovery rate was 80.5%. The recovery rate is the ratio of the number of white blood cells after the concentration and washing processes to the number of white blood cells before these processes.

[0186] Furthermore, when the recovered amount obtained by the recovery treatment in step 2-5 was added to the recovered amount after the washing treatment in step 2-4, the recovery rate was further improved to 85.4%. Therefore, it can be seen that the recovery rate can be further improved by adding the staining buffer and washing. When the dyeing process was performed manually without using the hollow fiber membrane module, the recovery rate was 75%. Therefore, it can be seen that the use of the hollow fiber membrane module can produce results that are comparable to those obtained by manual dyeing.

[0187] In the treatment using the hollow fiber membrane module described above in (6-3), DNase I was added to the staining buffer used in step 2-1, as described above. In step 2-1, no cell aggregates were observed in the sample after cell strainer and solvent exchange. The same treatment as that using the hollow fiber membrane module described in (6-3) above was performed, except that DNase I was not added. As a result of this treatment, as shown in the photographs A and B in Figure 11, it was confirmed that cell clumps remained on the cell strainer in step 2-1. Furthermore, as shown in the photographs C and D in Figure 11, cell clumps were also confirmed in the sample after solvent exchange in step 2-1. These results demonstrate that the nucleic acid degrading substance can prevent the formation of cell aggregates during treatment with a hollow fiber membrane module, thereby suppressing clogging, for example. It also demonstrates that suppressing the formation of aggregates can increase the cell recovery rate.

[0188] 2. Second embodiment (sample preparation method)

[0189] The present technology also provides a sample preparation method including: a capture step of capturing bioparticles using a substrate to which a substance that captures bioparticles is immobilized, a recovery step of recovering the bioparticles released from the substrate into a reservoir, and a hollow fiber membrane processing step of passing the bioparticles recovered in the reservoir through a hollow fiber membrane module. The sample preparation method may be performed using, for example, the sample preparation system according to the present technology described in 1 above, but may also be performed using other systems.

[0190] The capture step, recovery step, and hollow fiber membrane treatment step are as explained in (3-2), (3-3), and (3-4) of 1. above, respectively, and these explanations also apply to this embodiment.

[0191] Furthermore, the sample preparation method of the present technology may further include a priming step as described in 1.(3-1) above.

[0192] The present technology can also be configured as follows. [1] a bioparticle capture module including a substrate on which a substance for capturing bioparticles is fixed; a reservoir in which bioparticles released from the substrate are collected; a hollow fiber membrane module through which the bioparticles in the reservoir flow; A sample preparation system comprising: [2] The sample preparation system described in [1], wherein the sample preparation system is configured to be able to supply a nucleic acid degrading substance to the biological particle capture module. [3] The sample preparation system according to [1] or [2], wherein the substance that captures the biological particles is immobilized on the substrate via a nucleic acid. [4] The sample preparation system described in [2], wherein the sample preparation system is configured so that the nucleic acid degrading substance can pass through the bioparticle capture module and reach the reservoir or the hollow fiber membrane module. [5] The sample preparation system is configured to supply a sample containing bioparticles to the bioparticle capture module, and to supply a particle-binding substance that binds to the bioparticles to the reservoir. The sample preparation system is described in any one of [1] to [4]. [6] The sample preparation system described in [5], wherein the particle-binding substance is an antibody. [7] The sample preparation system described in [5], wherein the biological particles are cells. [8] The sample preparation system according to any one of [1] to [7], which is provided with a circulation channel for circulating bioparticles between the reservoir and the hollow fiber membrane module. [9] The sample preparation system described in [8], further comprising a branched channel branching from the circulation channel and leading to the reservoir.

[10] The sample preparation system according to any one of [1] to [9], further comprising an analytical device for analyzing the contents of the reservoir.

[11] The sample preparation system according to

[10] , wherein the flow operation of bioparticles through the hollow fiber membrane module is controlled based on the analysis results of the analysis device.

[12] The sample preparation system according to any one of [1] to

[11] , wherein a pump is provided on a flow path leading from the bioparticle capture module to the reservoir.

[13] The sample preparation system described in

[12] , wherein the pump is a tube pump.

[14] The sample preparation system according to

[12] or

[13] , which is configured so that bioparticles released from the substrate can be supplied to the reservoir by driving the pump.

[15] A sample preparation system described in any one of

[12] to

[14] , which is configured so that a particle-binding substance that binds to biological particles can be supplied to the reservoir by driving the pump.

[16] The sample preparation system according to any one of [1] to

[15] , wherein a pump for circulating bioparticles is provided on a flow path leading from the reservoir to the hollow fiber membrane module.

[17] The sample preparation system according to any one of [1] to

[16] , wherein the hollow fiber membrane module has a discharge flow path through which the liquid separated from the bioparticles is discharged, and a discharge pump is provided on the discharge flow path.

[18] A sample preparation system according to any one of [1] to

[17] , which is configured so that the sample containing the biological particles is not in fluid communication with the external environment.

[19] a capturing step of capturing bioparticles using a substrate to which a substance for capturing bioparticles is immobilized; a recovery step of recovering the bioparticles released from the substrate into a reservoir; a hollow fiber membrane processing step of passing the bioparticles collected in the reservoir through a hollow fiber membrane module; A sample preparation method comprising: [Explanation of symbols]

[0193] 1. Sample Preparation System 10 Bioparticle Capture Module 20 reservoir 30 Hollow fiber membrane module

Claims

1. a bioparticle capture module including a substrate on which a substance for capturing bioparticles is fixed, and an outlet capable of discharging bioparticles released from the substrate; a reservoir connected to the outlet of the bioparticle capture module via a flow path, to which the bioparticles released from the substrate are supplied; a hollow fiber membrane module connected to the reservoir via a flow path and supplied with bioparticles in the reservoir; Equipped with the bioparticle capture module is connected to a container containing a nucleic acid degrading substance via a flow path, and the nucleic acid degrading substance is supplied from the container to the bioparticle capture module; the substance that captures the bioparticle is immobilized on the substrate via a nucleic acid, and The release of the bioparticles from the substrate is carried out by cleaving or decomposing the nucleic acid using the nucleic acid degrading substance. Sample preparation system.

2. The bioparticle capture module is connected to the reservoir or the hollow fiber membrane module via a flow path, The sample preparation system according to claim 1 , wherein the nucleic acid degrading substance can pass through the bioparticle capture module and reach the reservoir or the hollow fiber membrane module via the flow path.

3. The bioparticle capture module is connected to a container filled with a bioparticle-containing liquid via a flow path, and the bioparticle-containing liquid can be supplied from the container to the bioparticle capture module, and The sample preparation system of claim 1, wherein the reservoir is connected via a flow path to a container containing a particle-binding substance that binds to the biological particles, and the particle-binding substance can be supplied from the container to the reservoir.

4. The sample preparation system of claim 3 , wherein the particle-binding substance is an antibody.

5. The sample preparation system of claim 1 , wherein the biological particles are cells.

6. The sample preparation system according to claim 1 , further comprising a circulation channel for circulating bioparticles between the reservoir and the hollow fiber membrane module.

7. The sample preparation system of claim 6 , further comprising a branch channel branching off from the circulation channel and leading to the reservoir.

8. The sample preparation system of claim 1 , further comprising an analytical device for analyzing the contents of the reservoir.

9. Further comprising a control unit that receives an analysis result from the analysis device, The sample preparation system according to claim 8 , wherein the control unit automatically terminates the concentration adjustment process or the concentration process when the bioparticle-containing liquid in the reservoir reaches a desired concentration according to the analysis result.

10. The sample preparation system of claim 1 , further comprising a pump provided on a flow path leading from the bioparticle capture module to the reservoir.

11. The sample preparation system of claim 10 , wherein the pump is a tube pump.

12. The sample preparation system according to claim 10 , wherein the pump is configured to supply biological particles released from the substrate to the reservoir by driving the pump.

13. The sample preparation system according to claim 10, wherein the pump is configured to supply a particle-binding substance that binds to biological particles to the reservoir by driving the pump.

14. The sample preparation system according to claim 1 , further comprising a pump for circulating bioparticles provided on a flow path leading from the reservoir to the hollow fiber membrane module.

15. 2. The sample preparation system according to claim 1, wherein the hollow fiber membrane module has a discharge flow path through which the liquid separated from the bioparticles is discharged, and a discharge pump is provided on the discharge flow path.

16. A sample preparation system as described in claim 1, wherein the space through which a sample containing bioparticles passes before passing through the bioparticle capture module and being supplied to the reservoir, and the space between the reservoir and the hollow fiber membrane module through which the sample containing the bioparticles passes, do not come into contact with the external environment.

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