Cell bodies to be used for medical treatment

Abstract

To provide cell bodies to be used for medical treatment.SOLUTION: Cell bodies 9 to be used for medical treatment are prepared in medical treatment by a method including the steps of: preparing a sample holder including a holding space for holding a fluid medium 11; preparing a sample 7 including the one or more cell bodies in fluid medium in the holding space; preparing a functionalized wall surface portion 17 to be in contact with the sample in the holding space; bringing the sample into contact with the functionalized wall surface portion; pushing the one or more cell bodies of the sample in the holding space in a direction separating away from the functionalized wall surface portion; and removing at least some of the cell bodies of the sample from the holding space after the at least some of the cell bodies are separated away from the functionalized surface portion, where the removed cell bodies are used to be administered to a subject or another subject for medical treatment.SELECTED DRAWING: Figure 2A

Description

Technical Field

[0001] The present disclosure relates to cell bodies for use in medical treatment. The present disclosure also relates to methods and systems for studying biological cells.

Background Art

[0002] Biological cells have an outer membrane. This membrane is the boundary between the cell and its environment. The membrane constructs a platform for various processes in which biomolecules embedded in the membrane physically contact binding partners in the extracellular space.

[0003] Accordingly, numerous techniques have been developed for studying cells and intracellular processes through interaction with the cell membrane, and techniques for quantifying components on the cell surface have been devised to obtain information regarding a specific cell type (e.g., whether it is invasive breast cancer or not). Most of the techniques required physical interaction with the cells, but recently, a number of force-field techniques have been developed. However, they tend to require the adhesion of foreign substances to the cells and / or damage the cells. See, for example, the review by A.P. Liu, “Biophysical tools for cellular and subcellular actuation of cell signalling”, Biophys. J. 111:1112-1118 (2016).

[0004] However, there is a search for more powerful tools, particularly tools that give strong signals and are less likely to damage the cells if possible. In view of this, the present application provides a method and a system for manipulating and / or investigating cell bodies based on acoustic force.

[0005] Note that the use of acoustic forces to manipulate micron-sized particles and cells is known. For example, WO 2014 / 200341 describes examples of acoustic wave systems for use in studying biomolecules attached to microbeads. Further, cell manipulation by ultrasonic microbeams or “acoustic tweezers” is discussed in J. Lee et al., “Targeted cell immobilization by ultrasound microbeam”, Biotechnol. Bioeng. 108:1643 (2011); and D. Baresch et al., “Observation of a Single-Beam Gradient Force Acoustical Trap for Elastic Particles: Acoustical tweezers”, Phys. Rev. Lett. 116:024301 (2016). Additionally, a summary of current research in acoustofluidics can be found in V. Marx, “Biophysics: using sound to move cells”, Nature Methods, 12(1):41 (2015). Reviews are also published in H. Mulvana et al., “Ultrasound assisted particle and cell manipulation on-chip”, Adv. Drug Del. Rev. 65(11-12):1600 (2013); A. Lenshof et al., “Acoustofluidics 5: Building microfluidic acoustic resonators”, Lab on a Chip, 12:684 (2012); M. Evander, J. Nilsson, “Acoustofluidics 20: Applications in acoustic trapping”, Lab on a Chip, 12:4667 (2012).

[0006] However, all of these techniques had problems such as low signal-to-noise ratio, slow detection, small sample size, and / or interacting only locally and indirectly with the cell membrane of the probe cells. Therefore, development for biological research and / or for the measurement of samples of multiple specimens is still desired.

Summary of the Invention

[0007] In view of the above, the present application provides a method for manipulating and / or investigating cell bodies. Further, an operating system for investigating biological cell bodies is provided. Hereinafter, various embodiments and aspects will be described.

[0008] In accordance with the above considerations, the present application provides a method for manipulating and / or investigating cell bodies. The method includes the steps of preparing a sample holder including a holding space for holding a fluid medium, preparing a sample containing one or more cell bodies in the fluid medium within the holding space, and generating sound waves within the holding space to apply a force to the sample within the holding space. The method further includes the step of preparing a functionalized wall portion in the holding space for contacting the sample, and during at least a part of the step of applying the sound waves, the sample contacts the functionalized wall portion.

[0009] Therefore, the interaction between one or more cell bodies in the sample and the functionalized wall portion, for example, the relationship between the adhesion of the cell bodies to the functionalized wall portion and the sound wave (force) can be examined. The method provided in the present application can study the binding force of the cell bodies to the functionalized wall portion and can examine the entire contact surface between the cell bodies and the wall portion at once. This is related to the amount of binding and the binding force per binding. Further, a plurality of cell bodies can be studied simultaneously, and statistical distribution information can be provided by one or several measurements.

[0010] The cell body may be a part of a cell such as an organelle, nucleus and / or mitochondrion. However, the cell body may be a single cell or a multi-cell, such as a small aggregated cell population, a plant or animal biopsy, a dividing cell, a budding yeast cell, a colonial protist, etc. The cell body may be an animal embryo at an early stage of development (e.g., a mammalian morula, and in some cases a human embryo). In certain cases, different types of cell bodies can be studied together. For example, cell bodies obtained by mucosal swabs, blood samples or other probing techniques can be used.

[0011] The sample fluid is any fluid substance in which the cell body can move under the influence of an acoustic force within the time scale of the acoustic force study, and the cell body should be able to fall through the fluid onto the wall portion of the sample holder, and in particular, fall towards or from the wall portion functionalized according to the spatial orientation of the sample holder and the direction of the acoustic force. Note that the acoustic force study generally takes less than one second to several hours, and in some cases, several days, but the movement of the cell body within the range of less than one second to several seconds is preferred. The fluid should be capable of transmitting and maintaining ultrasonic waves for a long time. Suitable fluids are liquids and gels, such as water, aqueous fluids, biocompatible solvents, oils, gels, aerogels, hydrogels and body fluids, etc. However, in embodiments using optical studies, sufficient optical transparency for imaging may be desirable (see also below).

[0012] The sound wave is preferably a bulk acoustic wave, which can prevent localized forces and apply forces to the whole cell. The sound wave is preferably a standing acoustic wave, which gives a clear force profile in the transverse direction of the sample within the sample holder.

[0013] This method enables the study of various properties of one or more cell bodies. Specific examples are the quantification of the presence and / or abundance of biomolecules in the cell body, the surface adhesion force and / or adhesion kinetics of the cell body to the functionalized surface portion, and the differences in any of the above under the influence of active biological processes within the cell body.

[0014] Current cell adhesion assays and methods for the purpose of analyzing the biomolecular composition and abundance on the cell surface require a large number of cells, are very cumbersome, and rely on expensive equipment. For example, they require labeling and the risk of damaging the cells to be studied (e.g., cell lysis, antibody labeling). Furthermore, these techniques typically lack the ability to evaluate cell adhesion forces and cell adhesion kinetics, especially at the single-cell level. The method provided in the present application enables the parallel study of various characteristics of a plurality of individual cell bodies because the plurality of cell bodies in the sample can come into contact with and interact with the functionalized wall surface portion, thereby enhancing the accuracy of the research results and avoiding false positives or false negatives.

[0015] The method provided in the present application enables the study of cell bodies without adhering them to foreign substances such as microbeads, magnets, chromophores, antibodies, and various other labels generally required in current cell manipulation and research techniques. The cell bodies can be left essentially intact by this method, and after implementing the method, it is considered possible to administer the cell bodies to the test subject and / or return the cell bodies to the subject from whom they were provided for research. For example, one or more of T cells, white blood cells, red blood cells, and similar cell bodies are collected from a subject, studied by this method, and then can be further analyzed by various other methods (single-cell sequencing, fluorescence microscopy, cryo-electron microscopy) and / or administered to other subjects (e.g., blood donation) or returned to the original subject itself. Furthermore, even smaller cell bodies (e.g., those collected from plasma) can be studied before administration or return. Similarly, sperm and / or eggs can be studied before in vitro fertilization or in an artificial uterus, and fertilized eggs and / or early-stage embryos (e.g., morula or blastocyst stage) can be screened before implantation into a female subject for pregnancy.

[0016] A suitable interaction moiety may include an antibody for selective binding to a specific target such as a microbial cell or a cancer cell. For example, there are antibodies specific for several major nosocomial infections. Such an interaction moiety is generally effective for delivering a conjugate of the interaction moiety to a predetermined pathological site in a mammal. The pathological site may include a target moiety that constitutes a specific binding pair together with the interaction moiety. In the case of the present application, the interaction moiety can be attached to the wall of the functionalized wall moiety by direct attachment or by formation of the interaction moiety from or with a primer, and the binding pair may have a binding force sufficient to adhere the cell body to the functionalized wall moiety.

[0017] The interaction moiety may include an antibody, or an antibody fragment that binds to a cell surface antigen, or a ligand or ligand fragment that specifically binds to a cell surface receptor. Among this group, antibodies or antibody fragments that bind to cell surface antigens may be preferred because of their binding selectivity. For example, cancer cells usually have tumor-associated antigens on their surface. Their complementary antibodies bind very selectively to these tumor-associated antigens. However, ligands or ligand fragments are also suitable. It is known that various peptides bind to their cognate receptors with high affinity and would be ligands suitable for conjugation with the radioisotopes of the present invention. Receptors are cell membrane proteins that bind molecules such as growth factors, hormones, and neurotransmitters. Tumors arise from specific cell types that express a specific subset of such receptors. By exploiting the binding affinity between a receptor and a ligand, target-specific research and / or identification of cell bodies becomes possible.

[0018] Similarly, the immune response relies on a complex cascade of interactions between immune cells and their cell surfaces. For example, B cell activation relies on the binding of the B cell receptor expressed on the surface of B cells to an antigen exposed on the surface of an antigen-presenting cell (APC). This then triggers a cascade of intracellular and intercellular events leading to antibody secretion and pathogen attack by the complement system. Similarly, T cell activation occurs via the interaction of an antigen on the APC surface with the T cell receptor on the T cell surface. Furthermore, the recruitment of T cells to the site of inflammation / infection relies on the extravasation of T cells from the bloodstream into the tissue. Extravasation is initiated by a cytokine-regulated multi-step adhesion process to the vascular endothelium, followed by extravascular movement through the cell wall of the blood vessel. Immunodeficiency and autoimmune diseases represent imbalances in the immune response. The interaction between biomolecules on the cell surface and their binding partners in the extracellular environment is essential in all processes that may lead to abnormal immune responses, such as abnormal lymphocyte activation, cell adhesion, cell migration, and pathogen attack.

[0019] Representative examples of receptor-ligand pairs are shown below.

Table 1

[0020] This method may include at least one of introducing the sample fluid into the holding space and / or removing the sample fluid from the holding space, for example, may include flowing the sample fluid through the holding space. This can be particularly carried out during at least part of the step of applying the sound wave, but may also be carried out before and after that. The introduced sample fluid may contain one or more cell bodies.

[0021] For example, during at least part of the step of applying the sound wave, the sample may be introduced into the holding space and / or brought into contact with the functionalized wall portion. In addition or alternatively, the sample portion not adhered to the functionalized wall portion can be displaced by the fluid flow, and kinematic components, such as the lateral force component due to the flow, can also be included in the study.

[0022] Such embodiments can also be used to perform one or more of surface priming, sample introduction, sample recovery, exertion of force, transport of (a part of) the sample within the sample volume, introduction of one or more modulators of the sample, and / or priming for one or more functionalized wall portions. The modulator may include, for example, nutrients or harmful substances, or bioactive specimens for examining the effects on the properties of cells.

[0023] In one embodiment, one or more primers are provided on the functionalized wall portion. The primer may include one or more interaction portions. In particular, on the functionalized wall portion, there are provided one or more substances including one or more of antibodies, peptides, biological tissue factors, biological tissue portions, bacteria, antigens, proteins, ligands, cells, tissues, viruses, (synthetic) pharmaceutical compounds, lipid (double) layers, fibronectin, cellulose, nucleic acids, RNA, small molecules, allosteric modulators, (bacterial) biofilms, "organ-on-a-chip", etc., and / or specific atomic or molecular surface portions (such as gold surfaces) to which at least a part of the sample has a tendency to adhere preferentially over other surface portions as also shown elsewhere in this specification.

[0024] The method of the present application provides a wide variety of studies and / or interactions, which are mainly of physical or chemical or biological nature, although other intermediate or mixed types will be apparent.

[0025] One embodiment includes varying at least one of the frequency and amplitude of the sound wave in the holding space, preferably in a time-dependent manner.

[0026] For example, the force applied to the sample in the holding space can be adjusted. By varying the time-dependence of the force, one or more of the adhesion strength, adhesion kinetics, and (adhesion) kinetics can be examined.

[0027] One embodiment further includes the step of detecting and / or monitoring one or more characteristics of one or more cell bodies. The one or more characteristics are at least one or include at least one of cell integrity, adhesion of the cell body to at least a part of the functionalized wall portion, movement of one or more cell bodies, fluorescence emission, signs of viability of one or more cell bodies, interaction with sound waves, etc. The detecting and / or monitoring step may include optical detection, such as light intensity detection and / or optical imaging by one or more of photographing, video recording, microscopy. Additionally or alternatively, acoustic detection, such as surface acoustic detection, may be used.

[0028] The optical signal may depend on the amount of cell bodies adhered to the functionalized wall portion. Other optical signals depend on the position of one or more cell bodies within a one-dimensional, two-dimensional and / or three-dimensional volume and may in some cases also be time-dependent.

[0029] In one embodiment, interference tracking can be used to study one or more cell bodies. For example, a detection device is provided that includes a detector for generating (preferably digital) images of the focal plane of a detector and / or a light source. The detection device may be a camera or may include a camera. Next, an interference pattern, particularly a diffraction pattern, may be produced by out-of-focus cells. By processing such an interference pattern or a series of such interference patterns, the position and / or change in position of one or more cell bodies in a direction perpendicular to the focal plane can be calculated. Such a calculated series of positions can perhaps be combined with a time stamp and used to track the vertical movement of such cell bodies. Combining this with the lateral position of the cell body enables even higher-dimensional tracking. The illumination may preferably include plane wave front illumination and may be provided, for example, by a light emitting diode (LED).

[0030] This method may include at least one of cell sorting; tracking the movement of one or more cell bodies as a function of one or more of acoustic force, the flow of the sample fluid, and the composition of the sample fluid; monitoring one or more optical activities of the cell bodies, such as luminescence (phosphorescence, fluorescence, bioluminescence, photosynthesis, absorbance difference, etc.); changing the temperature and / or temperature profile of the sample holder; changing the illumination and / or illumination profile of the sample holder; and changing the composition of the sample fluid. Such techniques enable various studies of cellular (intracellular) processes that affect the membrane and / or the binding process itself. For example, cell sorting involves contacting at least a portion of the sample with a functionalized wall portion, generating a first acoustic wave in a holding space to apply a first force to the sample in the holding space, thereby desorbing a first amount of the cell bodies of the sample from the functionalized surface portion, displacing the first amount of the cell bodies, generating a second acoustic wave different from the first acoustic wave in the holding space to apply a second force different from the first force to the sample in the holding space, thereby desorbing a second amount of the cell bodies of the sample from the functionalized surface portion, and displacing the second amount of the cell bodies. Due to the different acoustic waves and forces, different types of cell bodies are selectively displaced, enabling the identification and / or separation of different types of cell bodies. The displacement may include removal from the holding space and removal from the sample holder. The optical effect may also be affected by cell interactions with the interaction portion of the surface. In one embodiment, the method includes quantifying the adhesion strength between one or more cell bodies and the functionalized surface portion.

[0031] The force (breaking force) by which the cell body is removed from the functionalized surface can be determined by measuring the speed at which the cell body separates from the surface portion. This speed can be measured by tracking the cell body over time. Assuming the cell body is spherical, the force applied to the cell body can be estimated by Stokes' law \(F_{acoustic}=F_{drag} = 6\pi\eta Rv\). Here, \(\eta\) is the kinematic viscosity of the medium (here, the sample fluid at the position of the cell body), \(R\) is the radius of the cell body, and \(v\) is the relative speed of the cell body with respect to the sample fluid in the direction of the force. This can also be used to calibrate the effective acoustic force with respect to the applied acoustic signal. One or more microbeads or other known particles may be included in the sample as a control for calibration.

[0032] In an embodiment of the method, an embodiment of the system includes a sample holder including a holding space for holding a sample containing one or more biological cell bodies in a fluid medium, and an acoustic wave generator connected to or connectable to the sample holder for generating acoustic waves in the holding space to apply a force to the sample. Typically, water or an aqueous buffer is used as the fluid. However, oils and (hydro)gels can also be used. In the case of a sample containing patient material, the fluid may be a body fluid. The sample holder includes a wall that provides a functionalized wall portion in contact with at least a part of the sample during use in the holding space.

[0033] The system, particularly the sample holder, can be designed such that the maximum acoustic amplitude is located at or near the functionalized wall, or such that a (high) force by which the cell body is particularly likely (expected) to be affected can be mainly detected at or near the functionalized wall.

[0034] The sample holder may be provided with a recess that forms a holding space. The sample holder may be of a single structure or composed of a plurality of objects. For example, it may include a member having at least a locally U-shaped cross-section and a cover member that covers and closes the U-shaped member to provide a closed holding space with a closed cross-section. The closed holding space may be closed in all directions, or may have one or more inlet and / or outlet ports and form a continuous flow path.

[0035] The acoustic wave generator may be permanently attached to the sample holder or may be integrated into a part of the sample holder in some cases. In another embodiment, the acoustic wave generator may be repeatedly attachable to the sample holder or may be connected via an acoustic transmission medium to form an acoustic cavity together with the sample holder. By way of example, ultrasonic waves can be generated by piezoelectric generators, electromechanical generators, optical generators (e.g., those subjecting a part to a series of laser pulses) and other techniques, and in some cases combinations thereof. For the generation of standing waves, the sample holder containing the sample fluid and, in some cases, the structures attached to the sample holder may form an ultrasonic cavity for a specific frequency in one or more directions. This sample holder can then be designed to prevent or promote mode mixing of acoustic modes in different and / or other directions.

[0036] The acoustic wave generator may be a bulk acoustic wave generator for generating bulk acoustic waves in the holding space and / or in the sample contained therein, or may include a bulk acoustic wave generator.

[0037] In one embodiment, one or more primers are applied to the functionalized wall surface portion. The primer may include one or more interaction portions and / or precursors thereof. In particular, antibodies, peptides, biological tissue factors, biological tissue portions, bacteria, antigens, proteins, ligands, cells, tissues, viruses, (synthetic) pharmaceutical compounds, lipid (double) layers, fibronectin, cellulose, nucleic acids, RNA, small molecules, allosteric modulators, (bacterial) biofilms, one or more substances including one or more of "biofunctional chips", and / or specific atomic or molecular surface portions (e.g., gold surfaces) to which at least a part of the sample tends to adhere preferentially over other surface portions, functionalized wall surface portions including nano-organized or micro-organized surface portions (e.g., micropillars, microridges, etc.) are provided.

[0038] By providing one or more primers on the functionalized surface portion, a predetermined interaction between the cells of the cell body and the functionalized surface portion may be affected or caused by the contact between the cell body and the portion. For example, the cell body may adhere to the wall. Also, one or more other biological processes within or on the cells of the cell body may be affected or caused, which can be investigated in this system.

[0039] In one embodiment, the functionalized wall surface portion includes a plurality of differently functionalized wall surface portions that contact the sample.

[0040] This contributes to the study of various predetermined interactions between the cell body and the functionalized surface portion.

[0041] The different portions can be formed using various techniques such as printing, and arranged in various patterns, in some cases patterns having repeating portions.

[0042] In one embodiment, the sample holder is connected to or connectable to a flow system for introducing fluid into and / or removing fluid from the holding space, e.g., for flowing fluid through the holding space. The fluid flow system may be incorporated into the operating system. The fluid flow system may comprise one or more of a reservoir, a pump, a valve, and a conduit for introducing and / or removing one or more fluids sequentially and / or simultaneously.

[0043] Thus, the fluid introduced into, removed from, or flowing through the holding space may include sample substances, such as one or more sample fluids, cell bodies, agents acting on the cell bodies, etc. For example, one or more portions of the sample may be recovered after the experiment, and / or different sample conditions may be provided, e.g., different dilution rates such as one or more different pH values, salt concentrations, different fluid compositions, and different cell bodies may be used sequentially or in parallel. Also, agents acting on the cell bodies, such as nutrients, chemicals, viruses, etc., may be introduced. The fluid may include one or more dissolved gases such as N2, CO2, O2, or entrained gases, especially in hydrogel and / or aerogel-based fluids, noble gases such as Ne, Ar, harmful gases such as CO, O3, NOx, other cyanide-containing gases, gases that may have biological effects such as ethylene. The gas may be a gas mixture of various components, may have a controlled composition, or may have an uncontrolled composition such as ambient air. Acoustic refractive index changes that can disrupt the desired acoustic force profile within the holding space, such as density changes in the fluid, especially bubbles on the order of the internal dimensions of the holding space that spread over a significant portion of the width and / or height of the holding space, should be prevented, at least during the study of the sample. However, microbubbles on the order of less than about one-tenth of the internal dimensions of the holding space, e.g., microbubbles less than 5 μm for a holding space with a height and / or width of about 50 μm, can be used as one or more of an image contrast, a force concentrator, a contrast agent, a force probe, etc. Note that large bubbles may be used to propel the sample fluid through the sample holder, and in such cases, they may be used to obtain beneficial effects.

[0044] Control of the amount and / or composition of fluid within the sample holder can also be used to control the sample volume.

[0045] The system can be configured to introduce fluid into and / or remove fluid from the holding space, for example, to flow fluid through the holding space, simultaneously with the operation of a sound wave generator for generating sound waves within the holding space to apply a force to the sample. For example, kinematic components, such as lateral force components resulting from flow, can be included in the study. Thus, the system may be included in a flow cytometry system or other flow systems and can be integrated with, for example, an online flush system.

[0046] The direction of flow can be perpendicular to the direction of the acoustic force or at least the major force direction component of the acoustic force.

[0047] In one embodiment, the sound wave generator is controllable to adjust at least one of the frequency and amplitude to generate an adjustable sound wave (preferably time-dependent), and in particular, the sound wave can be a standing wave.

[0048] By doing so, parameter research becomes possible. In certain cases, the amplitude can be adjusted over two or more digits. The acoustic frequency depends on the geometry of the sample holder but is generally in the range of 1 - 100 MHz. The open size of the sample holding space in any of the three orthogonal directions, for example, the height, width, and length of the holding space, can be 1 - 1000 μm, and as a result, the holding space can typically have a volume in the range of 0.1 μL - 100 μL and even up to 1 mL and can have a variety of geometries such as a "lab on a chip" sample holder. What is important is that the sample holder can effectively supply and sustain sound waves in the sample.

[0049] A single cell body or multiple cell bodies can be manipulated and / or measured simultaneously. A large number of cell bodies may form a surface bulk, such as a bacterial colony. In one embodiment, contrary to surface bulk research, multiple individual cell bodies can be manipulated and / or measured separately but in parallel.

[0050] The acoustic signal can vary over several orders of magnitude and may, for example, depending on the nature and robustness of the sample and the process in the sample, last less than 1 second to several tens of minutes or even more.

[0051] The appropriate frequency of the acoustic wave is determined by the dimensions of at least a part of the sample holder (which may or may not be combined with the acoustic generator), for example the acoustic cavity, and the frequency can be the resonant frequency of the acoustic wave in that part and is probably related to the standing wave in that part. The optimal frequency can be determined in advance and / or during operation. The resonant frequency is thought to be able to exert an acoustic force several orders of magnitude higher than that of a non-resonant acoustic wave.

[0052] Time-dependent adjustment enables research based on a predetermined frequency and / or amplitude pattern, such as sweeps like pulses, ramps, iterative modulations and / or frequency chirps. Different frequency spectra can also be applied. Using such techniques, particles can be manipulated selectively or collectively, for example by pushing or pulling.

[0053] The system may comprise multiple acoustic generators, which are configured to generate acoustic waves that are different from each other with respect to at least one of frequency, amplitude, and the time-dependence of each frequency and / or amplitude, and / or to adjust the frequency and / or amplitude of each acoustic wave (preferably time-dependently) so that at least one of them is controllable to generate an adjustable acoustic wave. Thus, one or more additional acoustic generators can be provided to generate a complex force field, and in certain embodiments, multiple acoustic generators are connected to the sample holder to generate acoustic waves from multiple vertical directions into the holding space and they are controllable separately.

[0054] In one embodiment, the operating system includes a detector for detecting the response of one or more cell bodies to sound waves (or the force applied by sound waves). By doing so, the effect of the manipulation of one or more cell bodies can be examined.

[0055] The detector may include an optical detector. The optical detector may include a photodiode, an array of photodiodes, a camera and / or a microscope. However, in some experiments, such as bulk measurements, a photocell without image resolution may be sufficient. A digital photo and / or a film camera may be appropriate and preferably have an adjustable image frame rate. The detector may be wavelength selective and include one or more color filters. In some cases, a plurality of detectors with wavelength selectivity for different wavelengths are provided.

[0056] In certain embodiments, the detector includes a confocal microscope and / or a super-resolution microscope, a (near-field) scanning optical microscope ("SOM" / "NSOM"), a structured illumination microscope ("SIM"), and a multi-color excitation light source.

[0057] Typically, bright-field illumination (e.g., LED illumination) can be used. However, dark-field imaging can also be used. Even if the microscope itself is not a super-resolution microscope, software can be used to examine a sample (a part of it) with a precision below the diffraction limit. However, the detector does not necessarily have to be a microscope. In the case of only measuring the presence of cells on the surface, a simple detector, such as total internal reflection fluorescence microscopy ("TIRF microscopy"), may be sufficient. Other options include surface plasmon resonance detection and / or lensless imaging (e.g., imaging without a lens by placing a camera chip directly below the sample).

[0058] Furthermore, acoustic detection can be used. Surface acoustic waves enable the detection of objects on the surface based on the effects on the sound waves, such as differences in amplitude, phase, and / or differences in the propagation direction due to absorption and / or reflection of acoustic energy. For example, bulk acoustic waves can be used to acoustically manipulate cell bodies, and surface acoustic waves along each surface can be used to detect the amount of cell bodies, especially those bound to the surface. A suitable acoustic detector comprises a piezoelectric actuator that functions as a sensor element.

[0059] Contact detection and / or other forms of detection can be used in combination with acoustic force research, but optical detection is preferred because it can be performed with little or no effect or interaction on biological cell bodies. Furthermore, numerous established optical techniques and systems can be utilized. For optical detection, the system may comprise a light source. The light source can be multi-color or single-color, and may induce or exclude optical interaction with the sample (a part thereof), and may reduce or prevent optical (chromatic) aberration for optical detection. Optical detection relying on any of optical interference, refraction, and diffraction can obtain beneficial effects from a light source that provides illumination with a plane wavefront or other controlled wavefront at the position of the sample.

[0060] In an embodiment, the system comprises one or more of a light source, a memory, a tracking system for tracking one or more cell bodies, and a controller for performing microscopy calculations and / or analysis related to microscopy techniques. The tracking system can be configured to perform two-dimensional ("2D") and / or three-dimensional ("3D") tracking. In one embodiment, 3D tracking can be used to determine the velocity of cell bodies passing through the surrounding medium, and can also be used to determine the acoustic force of the system on the cell bodies and / or the interaction between the cell bodies and the acoustic force of the system. The tracking system and / or the controller may be connected to an acoustic wave generator to control the operation of the system.

[0061] The system may comprise a sensor and a controller connected to or connectable to an acoustic wave generator for controlling the operation of the acoustic wave generator in response to signals from the sensor. Accordingly, a feedback system may be provided.

[0062] However, the system and method are already extremely effective and beneficial in simply quantifying the amount of cell bodies adhered to the functionalized surface as an absolute number and / or relative fraction without investigating in detail the interaction or behavior of the individual components.

[0063] In one embodiment, the system includes one or more of an optical tweezer system, a magnetic tweezer system, an electrostatic tweezer system, and a contact probe for manipulating one or more cell bodies, and one or more cell bodies may be appropriately prepared, and a preparation system therefor may be provided.

[0064] In one embodiment, the system includes a light source for exciting and / or inspecting one or more optically active moieties (e.g., chromophores) in a sample for utilizing fluorescence detection. The light source may include a laser, and the light source is preferably wavelength tuneable.

[0065] The system may be provided as a stand-alone system, as a worktop device, and further as a hand-held device.

[0066] In one embodiment, the system further comprises a detector for generating a digital image of the focal plane, and the system includes a computing device for calculating the position of one or more cell bodies in a direction perpendicular to the focal plane by arithmetic processing of an interference pattern caused by one or more out-of-focus cell bodies. This enables interference detection, determination, and tracking of cell bodies in a direction along the imaging direction that is perpendicular to the direction of the sound wave and / or to the functionalized surface. In one embodiment, it may include a thermal element for adjusting the temperature and / or temperature profile of the sample holder. The thermal element may include a simple electric heater wire, or may include complex elements such as one or more Peltier elements capable of precise thermal control and both heating and cooling.

[0067] In another aspect, a sample holder for the system and / or method described herein is provided. The sample holder includes a holding space for holding a sample containing one or more cell bodies in a fluid medium, and an acoustic wave generator connected to the sample holder for generating acoustic waves that apply a force to the sample. The sample holder includes a wall that provides a functionalized wall portion that contacts at least a portion of the sample during use in the holding space.

[0068] The sample holder may be formed together with a connection portion for connecting to a corresponding opposing connector of a device including an attached ultrasonic generator and a detection system (e.g., one or more light sources and optical detectors) for providing the above-described system and / or implementing the above-described method.

[0069] Thus, provided are a method and a system capable of detecting the presence and / or quantifying the abundance of biomolecules on the surface of a cell body. Since the adhesion rate and adhesion strength can be quantified in parallel for hundreds to thousands of cell bodies at the single-cell level because individual cell bodies may interact differently with the functionalized surface, this method and system can perform force spectroscopy on cell bodies in a highly parallelized manner at the single-cell level, combining aspects of acoustic force spectroscopy, microfluidics, surface functionalization, and live (super-resolution) video tracking of multiple cell bodies, and in some cases single-cell live super-resolution video tracking. When a resonant bulk acoustic wave is generated in a microfluidic cavity, a force can be directly and instantaneously applied to the cell body.

[0070] In a typical design, the microfluidic chamber that forms the holding space itself does not form an acoustic cavity. Instead, a standing wave is formed in the entire sample holder (e.g., a chip including a piezo element, a top glass, a fluid layer, a bottom glass layer). If the standing wave is generated only on the microfluidic layer, the force at the boundary becomes very small and is insufficient to pull the cell body away from the surface, potentially compromising detectability. When combined with live (single-cell) video tracking, this technology can accurately detect cell adhesion events and quantify the adhesion forces transmitted by the interaction between the (bio)molecules on the surface of the cell body and the functionalized wall portion of the microfluidic sample volume. Note that motion detection may not require precise position detection at a given time point, and determining the position of the sample portion at a specific time point may not require detection and / or tracking of the motion of the sample portion at that time.

[0071] The above aspects will be further described in more detail below, together with additional details and advantages, with reference to the drawings showing numerous embodiments by way of example.

Brief Description of the Drawings

[0072]

Figure 1

Figure 2

Figure 2A

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Mode for Carrying Out the Invention

[0073] The drawings are schematic diagrams and are not necessarily to scale, and details that are not necessary for understanding the present invention may be omitted. Terms such as "upper", "lower", "down", "up", etc. relate to the embodiments of the orientation shown in the drawings unless otherwise specified. Further, components that are at least substantially the same or components that perform at least substantially the same function are given the same reference numerals, and alphabetical suffixes are added when it is useful to distinguish them individually.

[0074] FIG. 1 is a schematic diagram of an embodiment of an operating system 1 according to the present invention, FIG. 2 is a cross-sectional view of a sample holder, and FIG. 2A is a detailed view of a portion indicated by "IIA" of the sample holder of FIG. 2.

[0075] System 1 includes a sample holder 3 that includes a holding space 5 for holding a sample 7 containing one or more biological cell bodies 9 in a fluid medium 11. The fluid is preferably a liquid or a gel. System 1 further includes an acoustic wave generator 13 (e.g., a piezoelectric element) connected to the sample holder 3 for generating acoustic waves in the holding space 5 that apply a force to the sample 7 and the cell bodies 9 in the sample 7. The acoustic wave generator 13 is connected to a controller 14 and a power source (these are integrated in this figure) as optional components.

[0076] The sample holder 3 includes a wall 15 that provides a functionalized wall portion 17 that contacts a portion of the sample 7 during use to the holding space 5.

[0077] The operating system 1 shown in the figure includes a microscope 19 having an adjustable objective lens 21 and a camera 23 connected to a computer 25 having a controller and a memory. The computer 25 may be programmed to track one or more cell bodies based on signals from the camera 23 and / or to perform microscopy calculations and / or to perform analysis related to super-resolution microscopy and / or video tracking (which may be sub-pixel video tracking). The computer 19 or another controller (not shown) can be connected to other parts of the system 1 (not shown) to control at least a portion of the microscope 19 and / or other detectors (not shown). In particular, the computer 25 can be connected to one or more of the acoustic wave generator 13, its power source, and its controller 14 shown in FIG. 1.

[0078] This system further includes a light source 27. The light source 27 illuminates the sample 7 using an appropriate optical system (not shown) to provide a desired illumination intensity and intensity pattern (e.g., plane wave illumination, Köhler illumination, etc., which are known per se). In the system shown in the figure, the light 31 emitted from the light source 27 is directed through the acoustic wave generator 13 towards the sample holder 3 (and the sample 7 therein), and the sample light 33 from the sample 7 passes through the objective lens 21, through the optional eyepiece lens 22 and / or additional optical system, and reaches the camera 23. The objective lens 21 and the camera 23 may be integrated. In one embodiment, two or more optical detection tools (e.g., with different magnifications) may be used simultaneously, for example, for detecting the sample light 33 using a beam splitter.

[0079] In another embodiment, not shown but detailed in WO 2014 / 200341, the system includes a partially reflective reflector, and the light emitted from the light source is guided from the objective lens to the sample through the reflector. The light from the sample is reflected back to the objective lens, passes through the partially reflective reflector, and heads towards the camera through the intervening optical system of the optional components. Other embodiments will be apparent to the reader.

[0080] The sample light 33 may include the light 31 affected (e.g., scattered and / or absorbed) by the sample and / or light emitted by one or more parts of the sample 7 itself (e.g., chromophores adhered to the cell body 9).

[0081] Some of the optical elements in the system 1 may be one or more of partially reflective, dichroic (having a wavelength-specific reflectivity, e.g., high reflectivity for one wavelength and high transmittance for other wavelengths), polarization selective, and other configurations suitable for the illustrated configuration. To configure the system 1 for a specific type of microscopy, additional optical elements, such as lenses, prisms, polarizers, diaphragms, reflectors, etc., may be provided.

[0082] The sample holder 3 can also be formed from a single piece of material (e.g., glass, injection-molded polymer, etc. (not shown)) having a flow path inside, or separate layers made of a suitable material can be formed, for example, by welding, glass bonding, adhesive bonding, taping, clamping, etc., so that at least during the experimental period, a holding space 5 for accommodating the fluid sample 7 is formed by fixing them more or less permanently. As shown in FIGS. 1 and 2, the sample holder 3 may include a member 3A having a recess with at least a locally U-shaped cross section, and a cover member 3B that covers and closes the U-shaped member (recess) to provide a closed holding space 5 with a closed cross section.

[0083] As shown in FIG. 2, the sample holder 3 is connected to a fluid flow system 35 of an optional component, for example, to flow fluid through the holding space (see the arrow in FIG. 2), in order to introduce fluid into the holding space 5 of the sample holder 3 and / or remove fluid from the holding space 5. The fluid flow system 35 may be included in an operation and / or control system. The fluid flow system 35 may include one or more of a reservoir 37, a pump, a valve, and a conduit 39 to sequentially and / or simultaneously introduce and / or remove one or more fluids. The sample holder 3 and the fluid flow system 35 may include connectors that can be disposed at appropriate positions of the sample holder 3 in order to couple / separate without damaging at least one of the members 3, 35 and preferably to repeatedly couple / separate so that one or both of the members 3, 35 can be reused later.

[0084] FIG. 2A is a schematic view of two cell bodies 9 in the sample holder 3 of FIG. 2. On the wall 15 of the sample holder 3, there are a portion (left side of FIG. 2A) where a functionalized wall portion 17 is provided and a portion (right side of FIG. 2A) where it is not provided.

[0085] FIGS. 3A and 3C are microscopic images of the experimental situation in the line-of-sight direction perpendicular to the functionalized wall portion of the sample holder (not recognizable in FIGS. 3A and 3C). Five cell bodies 9A, 9B in the sample fluid 7 in the sample holder can be visually recognized.

[0086] Figures 3B and 3D are schematic diagrams of the respective situations of FIGS. 3A and 3B in the line-of-sight direction along the functionalized wall portion 17 of the sample holder (i.e., perpendicular to FIGS. 3A and 3C, similar to FIG. 2A).

[0087] In either case, the functionalized wall portion 17 is provided with one or more primers. The primer may contain one or more interaction portions.

[0088] FIGS. 3B and 3D each show a cross-sectional view of the functionalized wall portion 17 of the wall 15 of the holding space 5 of the sample holder 3 according to an embodiment of the system 1. The wall portion 17 is functionalized with an interaction portion 41 (e.g., an antibody), which is one type of antibody in this example. Cell bodies 9A, 9B containing different target portions 43A, 43B (antigens 43A, 43B) of the sample are brought into contact with the functionalized wall portion 17, and the cell bodies 9A, 9B are engaged (caused to engage) with the interaction portion 41.

[0089] FIGS. 3A - 3B show a stationary state in which no sound wave is applied to the holding space. FIGS. 3C - 3D show a situation where a sound wave is applied to the holding space and a force F is applied to the cell bodies 9A, 9B in the sample (see the arrows in FIGS. 2A and 3D). The sound wave is a bulk sound wave having a propagation direction perpendicular to the wall 15. The sound wave may be a traveling wave or preferably a standing wave. For example, the force F is in the propagation direction perpendicular to the wall 5 (see the arrow) and towards the node of the sound field indicated by the dashed line N in FIG. 2A. Note that the position of the node (which may be a node line or a node plane) in the sample holder and / or the intensity of the acoustic force can be adjusted by appropriately selecting one or more of the acoustic frequency, acoustic power, geometric shape of the sample holder, and composition (such as viscosity) of the liquid medium.

[0090] In the sample of FIG. 2A, the cell body 9 adhered to the functionalized wall portion 17 remains adhered (left side), and the cell body 9 located beside the functionalized wall portion 17 is lifted from the wall 15 (right side).

[0091] In the samples of FIGS. 3A to 3B, a binding pair is formed in which a certain interaction coincides with and strongly binds to the target portions 41 and 43A, and each cell body 9A strongly binds to the wall 15 (the functionalized wall portion 17). Other interactions do not coincide with the target portions 41 and 43A, forming a weakly bound binding pair or no binding at all, and under the influence of the force F, each cell body 9B is released from the wall 15 (the functionalized wall portion 17). In the microscopic images of FIGS. 3A and 3C, this can be visually recognized by the cell bodies 9A, 9B at the focal position of the microscope or the cell bodies 9A, 9B that move away from the focus of the microscope.

[0092] Thus, by observing the response of the cell bodies when a force is applied to the sample by sound waves, the binding interaction can be detected. For example, this can distinguish different cell bodies 9A, 9B (characteristics). In an embodiment where a fluid flow is applied to the sample parallel to the wall 15, the detached cell bodies 9B may move (be washed away), and the cell bodies 9A remaining adhered to the wall 15 can stay in a predetermined position. In this way, the cell bodies 9A, 9B can be separated.

[0093] Note that instead of not treating any film or process as shown in FIG. 2(A), a non-stick coating such as polytetrafluoroethylene (PTFE, "Teflon" (registered trademark)) may be provided on the wall surface portion adjacent to the non-functionalized wall surface portion, or different functionalized wall surface portions may be provided. For example, a portion containing an antibody compatible with the antigen of the cell body detached from the upstream functionalized wall surface portion may be provided so that the cell body binds to the adjacent functionalized wall surface portion.

[0094] Figures 4A to 4C show two cell bodies 9C, 9D having the same target portion 43 but different amounts in sample 5. The cell bodies 9C, 9D interact with the functionalized wall portion 17 (upper interaction portion 41), and are subjected to the force F of the sound wave on the sample (cell body in the sample) due to the change in the amplitude of the sound wave. In Figure 4A, the force F is 0 (compared with Figure 3B), and both cell bodies 9C, 9D are adhered to the wall portion 17. In Figure 4B, the force F is 1 unit, which is sufficient to break the bond between the interaction portions 41, 43 of one cell body 9D and desorb the cell body 9D. In Figure 4C, the force increases to 3 units, which is also sufficient to break the bond between the interaction portions 41, 43 of the other cell body 9C and the wall portion 17 and desorb the cell body 9C. The appropriate binding force between a single cell and the surface is on the order of piconewtons (pN), and the force applicable with sound waves, particularly standing bulk sound waves, has been achieved in the range of about 0.1 pN to several thousand pN with microbeads.

[0095] As a result, by observing the responses of the cell bodies 9C, 9D to the change in the force of the sound wave on the sample, the strength of the binding interaction can be detected, and the quantification of the target portion on the cell body surface becomes possible.

[0096] Figure 4D shows a statistical analysis (histogram) of the measured binding forces, i.e., the force F_rupt required to break the bond, of a plurality of normal healthy cells with respect to the wall portion functionalized with an antibody as the interaction portion. In some cases, diseased cells form more antigens (target portions) on their membranes than healthy cells, resulting in a stronger binding force to the functionalized wall surface portion (antibody). Compare with Figures 4A to 4C. This can be detected by adhesion force statistics (simulation-based curve). Similarly, a decrease in the target portion (e.g., due to overexpression of a gene) can be detected by a decrease in the binding strength of diseased cells compared to healthy cells.

[0097] In addition to changing the amplitude of the sound wave, the frequency of the sound wave may be changed to affect and examine the interaction portion - target portion binding parameter. The results can be obtained in the same manner as shown in Figures 4A to 4D.

[0098] FIG. 5 shows a schematic diagram and a graph versus time t similar to FIGS. 3A - 4D, where the force F applied to sample 5 is varied with a series of off - on pulses (different panels of the schematic diagram as indicated by the arrows), and by measuring a signal S indicating a change in one or more characteristics (such as visibility, motility, etc.) of cell body 9 to be examined, it shows how the adhesion kinetics can be investigated. This may provide additional information about the cell body under study. For example, it may show the probability that a cell adheres to a specific antibody (indicated as "v") or does not adhere (indicated as "x"). The acoustic force can also be varied differently (e.g., gradually). For example, in an experiment, the difference between when the acoustic force is suddenly stopped and the cell body is dropped onto the surface and when the acoustic force is gradually decreased and the cell body is slowly approached to the functionalized surface can be examined.

[0099] FIGS. 6A - 6C show the experimental results in biological cells imaged by confocal fluorescence microscopy. FIGS. 6A and 6B are images of a part of the sample at different times, and FIG. 6C shows a series of images from the details shown in FIGS. 6A and 6B at different times, as will be described later. In this example, the focal plane of the microscope was positioned to coincide with the acoustic node, i.e., the position where the cells are pushed away. At the start of the experiment, i.e., at t = 0 seconds (FIG. 6A; t = 0s in FIG. 6C), no acoustic signal was applied, and all cells in the sample settled to the bottom of the holding space and the cells were not visible. Subsequently, the acoustic force was switched on, and the cells were pushed from the surface to the acoustic node point, and a large number (a cluster) of cells became visible at t = 1 second. See t = 1s in FIGS. 6B and 6C. During the application of the acoustic force, the cells remained trapped at the acoustic node and remained visible (FIG. 6C, t = 2 seconds). Two seconds after applying the acoustic force, when the acoustic force was stopped, the cells slowly settled again and dropped out of the field of view within a few seconds (FIG. 6C, t = 3 seconds - 7 seconds). The fact that the brightness of different cells in FIGS. 6B and 6C is different (and the colors are also different in the original images) suggests that different types of cells were present in this sample, but this aspect was not examined in this experiment.

[0100] It will be apparent to the reader that other imaging methods can be used simultaneously with the acoustic manipulation of the cell body.

[0101] Figures 7A - 9C show the use of the technology provided in the present application in the selection of T cells based on avidity (the strength of binding to a binding partner). The experiment was conducted as follows. A tumor cell line derived from a melanoma patient was cultured inside a sample holder such that the culture adhered to the wall portion to form a functionalized wall surface portion. Next, T cells (fluorescently stained red) genetically engineered to express a T cell receptor against an antigen present in the tumor cell line or non - genetically engineered T cells (fluorescently stained green) without specificity for the tumor cell line were injected, and the chip was incubated at 37 °C for 30 minutes to cause T cell - tumor cell binding. Figure 7A shows an image of the T cells within the sample holder. In this figure, specific (red - stained) cells appear as light gray, and non - specific cells (green - stained) appear as dark gray. For comparison and clarity, the signals of each cell are shown separately in Figure 7B (red - stained / specific) and Figure 7C (green - stained / non - specific). Next, to select and isolate specifically bound T cells, acoustic waves were generated within the sample holder to apply an acoustic force. T cells that were not bound to the tumor cells or were weakly bound floated towards the acoustic node (located approximately 20 μm above the tumor cells) due to the acoustic force (compare Figures 3A - 3D). Due to mode mixing between the axial and transverse normal modes of the acoustic cavity, within the sample holder, acoustic nodes are formed not only axially but also transversely. As a result, unbound floating T cells tended to aggregate along lines formed by the combination of axial and transverse nodes. In Figure 8A, these appear as multiple separated horizontal bands, but as can be best seen by comparing Figure 8A with Figures 8B and 8C, the bound cells remain substantially in place. Note that Figures 8A, 8B, and 8C show all cells, different specific and non - specific cells for comparison and clarity, respectively (see Figures 7A - 7C). The transverse acoustic node structure that causes aggregation of unbound cells into multiple lines can facilitate the detection of unbound cells and enhance the visibility of the cells (especially with respect to bound cells). However, it is clear that such structuring is not essential for the detection of the unbound state itself or the ability to select cells based on avidity.For example, the distinction between the bound state and the unbound state can also be made based on tracking the axial and / or lateral movement of cells without a lateral node structure.

[0102] In addition, a complex acoustic force field having a plurality of nodes can also be generated by a plurality of sound wave generators connected to the sample holder for generating sound waves from a plurality of vertical directions in the holding space. These sound wave generators may be controllable separately or may be an unsteady force field, for example, inducing the movement of cells (populations) at moving acoustic nodes.

[0103] In the next step, the sample space is gently flushed. For example, by flowing a sample fluid into the sample space, unbound cells are removed, causing a relative enrichment of the specific T cell population in the sample space. Similar to FIGS. 7A to 7C and 8A to 8C, FIGS. 9A to 9C show the remaining cells (FIG. 9A shows all cells, and FIGS. 9B to 9C show different specific and non-specific cells respectively). FIG. 10 shows the fractional incidence rates (relative proportions of the total cell number) of specific and non-specific cells in the field of view for the three steps shown in FIGS. 7A, 8A, and 9A.

[0104] Increasing the level of the applied acoustic force and repeating the above protocol (incubation, acoustic wave application, flushing, optionally additional incubation, acoustic wave application, flushing, repeating as necessary), T cells can be screened and recovered based on tumor cell avidity. This selection process is shown in comic form in FIG. 11 (see FIGS. 4A-4C (and their descriptions)). FIG. 11 shows, from left to right, the introduction and incubation of T cells (shown as hemispheres) having a target moiety (shown as the "legs" of the cells); application of weak acoustic waves (shown by the thin symbol "∧") and floating of unbound cells (bond breakage); flushing and recovery of unbound cells in the vial; application of moderately strong acoustic waves (shown by the medium-thick symbol "∧") and floating of weakly bound cells (bond breakage); flushing and recovery of the detached weakly bound cells in the vial; application of strong acoustic waves (shown by the thick symbol "∧") and floating of strongly bound cells (bond breakage); and flushing and recovery of the detached strongly bound cells in the vial.

[0105] In a therapeutic setting, one or more of the T cell fractions isolated in this manner can be selected for treatment of a patient from whom the target tumor cells studied as above were obtained. The same principle of cell screening and / or selection for treatment and / or research based on this avidity can be applied to basic research in immunology and / or cell biology, research on other forms of cell applications based on avidity, and research on their use in immunotherapy and the like (including, for example, immunosuppressive therapy for patients who have received a donor organ transplant).

[0106] The present disclosure is not limited to the above-described embodiments, and numerous modifications can be made within the scope of the claims as described above.

[0107] The components and aspects described with respect to or in relation to particular embodiments of the method or system can be combined as appropriate with the components and aspects of other embodiments of the system or method, unless otherwise explicitly stated. The first aspect of the present invention is as follows. [Item A1] A method for manipulating and / or studying cell bodies, comprising: preparing a sample holder including a holding space for holding a fluid medium; preparing a sample containing one or more cell bodies in the fluid medium within the holding space; and comprising: the method includes preparing a functionalized wall surface portion in the holding space to be contacted with the sample; during at least a part of the step of applying sound waves, the sample is in contact with the functionalized wall surface portion; the method includes generating sound waves within the holding space to apply a force to one or more cell bodies of the sample within the holding space in a direction away from the functionalized wall surface portion. [Item A2] The method according to Item A1, including at least one of introducing a sample fluid into the holding space and / or removing the sample fluid from the holding space, for example, flowing the sample fluid through the holding space during at least a part of the step of applying sound waves, and in particular, the introduced sample fluid may contain one or more cell bodies. [Item A3] One or more primers are provided on the functionalized wall surface portion, and the primers are one or more interaction portions, in particular antibodies, peptides, biological tissue factors, biological tissue portions, bacteria, antigens, proteins, ligands, cells, tissues, viruses, (synthetic) pharmaceutical compounds, lipid (double) layers, fibronectin, cellulose, nucleic acids, RNA, small molecules, allosteric modulators, (bacterial) biofilms, biofunctional chips, and one or more of specific atomic or molecular surface portions (such as a gold surface) where at least a part of the sample has a tendency to adhere preferentially over other surface portions. The method according to Item A1 or Item A2. [Item A4] The method according to any one of Items A1 to A3, including changing at least one of the frequency and amplitude of the sound waves within the holding space, preferably in a time-dependent manner. [Item A5] further comprising the step of detecting and / or monitoring one or more characteristics of the one or more cell bodies, wherein the one or more characteristics are or include at least one of cell integrity, adhesion of the cell body to at least a part of the functionalized wall portion, movement of the one or more cell bodies, fluorescence emission, signs of viability of the one or more cell bodies; wherein the detecting and / or monitoring step comprises at least one of optical detection by means of one or more of photography, videography, microscopy, such as light intensity detection and / or optical imaging, and acoustic detection, such as surface acoustic detection The method according to any one of claims A1 to A4. [Claim A6] Cell sorting; tracking the movement of one or more cell bodies as a function of one or more of acoustic forces, flow of the sample fluid, and composition of the sample fluid; monitoring the optical activity of one or more cell bodies; changing the temperature and / or temperature profile of the sample holder; changing the illumination and / or illumination profile of the sample holder; changing the composition of the sample fluid The method according to any one of claims A1 to A5, comprising at least one of the above. [Claim A7] The method according to any one of claims A1 to A6, comprising the step of quantifying the adhesion strength between one or more cell bodies and the functionalized surface portion. [Claim A8] An operating system for studying cell bodies, the system comprising a sample holder including a holding space for holding a sample containing one or more cell bodies in a fluid medium, and an acoustic wave generator connected to or connectable to the sample holder for generating acoustic waves in the holding space to apply a force to the sample and the sample holder comprises a wall providing a functionalized wall portion in the holding space that is brought into contact with at least a part of the sample during use, An operating system configured such that when a sample is contained in the holding space, a force is applied to one or more cell bodies of the sample in the holding space in a direction away from the functionalized wall portion. [Item A9] The operating system according to Item A8, wherein one or more primers are provided on the functionalized wall portion, and the primer is one or more interaction portions, particularly an antibody, a peptide, a biological tissue factor, a biological tissue portion, a bacterium, an antigen, a protein, a ligand, a cell, a tissue, a virus, a (synthetic) drug compound, a lipid (double) layer, fibronectin, cellulose, a nucleic acid, RNA, a small molecule, an allosteric modulator, a (bacterial) biofilm, a biofunctional chip, and one or more specific atomic or molecular surface portions (for example, a gold surface) where at least a part of the sample has a tendency to adhere preferentially over other surface portions. [Item A10] The operating system according to Item A8 or Item A9, wherein the functionalized wall portion includes a plurality of wall portions that are differently functionalized and are contacted with the sample. [Item A11] The operating system according to any one of Items A8 to A10, wherein the sample holder is connected to or connectable to a flow system for introducing fluid and / or gas into the holding space and / or removing fluid and / or gas from the holding space, for example, for flowing fluid through the holding space, and particularly the fluid and / or gas includes a sample substance, for example, a sample fluid and / or one or more cell bodies. [Item A12] The operating system according to any one of Items A8 to A11, wherein the sound wave generator is controllable to adjust at least one of the frequency and the amplitude in order to generate an adjustable sound wave, preferably a time-dependent one, in the holding space, and particularly the sound wave is a standing wave. [Item A13] The operating system according to any one of items A8 to A12, wherein the system includes a detector for detecting the response of one or more cell bodies to sound waves, and the detector can include one or more of an acoustic detector, such as a piezoelectric element, and an optical detector, such as a photodiode, an array of photodiodes, a camera and / or a microscope. [Item A14] wherein the system a light source; a memory for storing data serving as an indicator of the operation of the system and / or signals from the detector; a tracking system for tracking one or more cell bodies, and a controller connected to or connectable to the detector for performing analysis related to microscopic examination calculation and / or microscopy; a sensor, and a controller connected to or connectable to the sound wave generator for controlling the operation of the sound wave generator in response to a signal from the sensor; a thermal element, such as a Peltier element, for adjusting the temperature and / or temperature profile of the sample holder The operating system according to any one of items A8 to A13, comprising one or more of the above. [Item A15] The operating system according to any one of items A8 to A14, further comprising a detector for generating a digital image of the focal plane, and the system comprising a computing device for calculating the position of one or more cell bodies in a direction perpendicular to the focal plane by arithmetic processing of an interference pattern caused by one or more out-of-focus cell bodies. [Item A16] A sample holder for use in the method according to any one of items 1 to 7 and / or the operating system according to any one of items 8 to 15, a holding space for holding a sample containing one or more cell bodies in a fluid medium, and a sound wave generator connected to the sample holder for generating sound waves in the holding space to apply a force to the sample A sample holder that is provided with a wall that provides a holding space having a functionalized wall surface portion that is brought into contact with at least a part of the sample during use. A second aspect of the present invention is as follows. [Item B1] A method for manipulating and / or studying cell bodies, comprising the step of providing a sample holder that includes a holding space for holding a fluid medium, the step of providing a sample containing one or more cell bodies in the fluid medium within the holding space and the method includes providing, in the holding space, a functionalized wall surface portion that is brought into contact with the sample, the functionalized wall surface portion includes cells where at least a part of the sample has a tendency to adhere, where the cells and / or the cell bodies include at least one of tumor cells, immune cells, T cells, and B cells, and the cell bodies are lacking microbeads and / or magnets, during at least a part of the step of applying a force, the sample is in contact with the functionalized wall surface portion, the method applies a force to one or more cell bodies of the sample within the holding space in a direction away from the functionalized wall surface portion, and promotes one or more cell bodies of the sample within the holding space in a direction away from the functionalized wall surface portion by the force, and where the method further includes determining the relationship between the force and the adhesion strength of one or more cell bodies to the functionalized wall surface portion. [Item B2] The method according to item B1, including at least one of introducing a sample fluid into the holding space and / or removing a sample fluid from the holding space. [Item B3] The method according to item B2, wherein the introduced sample fluid contains one or more cell bodies. [Item B4] Tracking and / or recovering at least a part of the cell bodies based on the relationship between the force of the one or more cell bodies against the functionalized wall portion and the adhesion strength and / or the adhesion force, the method according to any one of items B1 to B3. [Item B5] The above functionalized wall portion is further provided with one or more primers, and the primers include antibodies, peptides, biological tissue factors, bacteria, antigens, proteins, ligands, tissues, viruses, drug compounds, lipid (double) layers, fibronectin, cellulose, nucleic acids, RNA, small molecules, allosteric modulators, biofilms, biofunctional chips, and at least one of the atomic or molecular surface portions having a tendency to preferentially adhere to the other surface portion of the holding space than at least a part of the above sample, the method according to any one of items B1 to B4. [Item B6] Further including the step of detecting and / or monitoring one or more characteristics of the above one or more cell bodies, The one or more characteristics are at least one or include the integrity of the cells, the adhesion of the cell bodies to at least a part of the above functionalized wall portion, the movement of the one or more cell bodies, fluorescence emission, and signs of the viability of the one or more cell bodies, The detection and / or monitoring step is Optical detection by one or more of photography, video recording, and microscopy, such as light intensity detection and / or optical imaging, and Acoustic detection, such as surface acoustic detection The method according to any one of items B1 to B5, including at least one of them. [Item B7] Cell sorting; Tracking the movement of one or more cell bodies as a function of one or more of the acoustic force, the flow of the sample fluid, and the composition of the sample fluid; Monitoring the optical activity of one or more cell bodies; Changing the temperature and / or temperature profile of the sample holder; Changing the illumination and / or illumination profile of the sample holder; Changing the composition of the sample fluid The method according to any one of items B1 to B6, comprising at least one of [Item B8] The method according to any one of items B1 to B7, comprising the step of quantifying the adhesion strength between one or more cell bodies and a functionalized surface portion. [Item B9] Tracking the movement of one or more cell bodies as a function of the flow of the sample fluid and the composition of the sample fluid; and Changing the composition of the sample fluid The method according to item B7, comprising at least one of [Item B10] The method according to any one of items B1 to B9, wherein at least a part of the cell bodies is provided with a chromophore and / or a fluorescent stain. [Item B11] After the sample has contacted the functionalized wall surface portion, removing at least a part of the cell bodies from the holding space, and further analyzing at least a part of the cell bodies removed from the holding space by one or more other methods selected from single cell sequencing, fluorescence microscopy, and cryo-electron microscopy. The method according to any one of items B1 to B10. [Item B12] The method according to any one of items B1 to B11, wherein the sample containing at least cell bodies and / or the cells of the functionalized wall surface portion are obtained from a subject. [Item B13] After the sample containing the cell bodies has contacted the functionalized wall surface portion, removing at least a part of the cell bodies from the holding space, wherein the removing is performed within the sample holder. The method according to item B12. [Item B14] The method according to item B13, wherein the sample holder is connected to or connectable to a flow system. [Item B15] A cell body used in the treatment of a subject, wherein the cell body is obtained by the method described in item B13, and the cell body is used for administration to the subject from whom the sample was obtained and / or another subject. [Item B16] A cell body used in the treatment of a subject, wherein in the treatment, the cell body is prepared by the method described in item B13, and the cell body is used for administration to the subject from whom the sample was obtained. [Item B17] A cell body used in the treatment of a subject, wherein in the treatment, the cell body is prepared by the method described in item B13, and the cell body is used for administration to a subject different from the subject from whom the sample was obtained. The third aspect of the present invention is as follows. [Item C1] A cell body for use in a medical treatment, wherein in the medical treatment, the cell body Preparing a sample holder including a holding space for holding a fluid medium; Preparing a sample containing one or more cell bodies in the fluid medium in the holding space; Preparing a functionalized wall surface portion in the holding space to be contacted with the sample, wherein the functionalized wall surface portion includes cells to which at least a part of the sample is intended to adhere, the cells include tumor cells, the cell bodies include one or more of immune cells, T cells, and B cells, the cell bodies are free of microbeads and / or magnets, and the sample containing the cell bodies and / or the cells of the functionalized wall surface portion is obtained from a subject. Contacting the sample with the functionalized wall surface portion; Applying a force to one or more cell bodies of the sample in the holding space in a direction away from the functionalized wall surface portion, and pushing one or more cell bodies of the sample in the holding space in a direction away from the functionalized wall surface portion by the force. After the sample containing the cell bodies contacts the functionalized wall portion, the force is applied, and at least some of the cell bodies are separated from the functionalized surface portion, removing at least a portion of the cell bodies of the sample from the holding space prepared by a method comprising wherein the removed cell bodies are used for administration to the subject or another subject for the medical treatment the cell bodies [Item C2] Cell bodies for use in the medical treatment of a subject, wherein in the medical treatment, the cell bodies providing a sample holder comprising a holding space for holding a fluid medium providing a sample containing one or more cell bodies in the fluid medium within the holding space providing a functionalized wall portion in the holding space to be contacted with the sample, wherein the functionalized wall portion comprises cells to which at least a portion of the sample is intended to adhere, the cells comprising tumor cells, the cell bodies comprising one or more of immune cells, T cells, and B cells, the cell bodies lacking microbeads and / or magnets, wherein the sample containing the cell bodies is obtained from the subject and / or the cells of the functionalized wall portion are obtained from the subject contacting the sample with the functionalized wall portion applying a force to one or more cell bodies of the sample within the holding space in a direction away from the functionalized wall portion, and pushing one or more cell bodies of the sample within the holding space in a direction away from the functionalized wall portion by the force After the sample containing the cell bodies contacts the functionalized wall portion, the force is applied, and at least some of the cell bodies are separated from the functionalized surface portion, removing at least a portion of the cell bodies of the sample from the holding space prepared by a method comprising wherein the removed cell bodies are used for administration to the subject for the medical treatment The cell body. [Item C3] The cell body according to Item C1 or Item C2, wherein preparing the cell body includes a flushing step of flushing the holding space after the contacting step and before applying the force, thereby removing unbound cell bodies. [Item C4] The cell body according to any one of Items C1 to C3, wherein in preparing the cell body, the sample containing the cell body is obtained from the subject. [Item C5] The cell body according to any one of Items C1 to C4, wherein in preparing the cell body, the sample containing the cells of the functionalized wall surface portion is obtained from the subject. [Item C6] The cell body according to any one of Items C1 to C5, wherein in preparing the cell body, in the removing step, at least a part of the cell body is collected based on the relationship between the force of one or more cell bodies on the functionalized wall surface portion and the adhesion strength and / or adhesion rate. [Item C7] The cell body according to any one of Items C1 to C6, wherein in preparing the cell body, strongly bound cell bodies are collected in the removing step. [Item C8] Preparing the cell body applies a first force to one or more cell bodies of the sample in the holding space in a direction away from the functionalized wall surface portion, thereby separating a first amount of cell bodies of the sample from the functionalized surface portion, and removing the first amount of cell bodies of the sample from the holding space, and applies a second increased force to one or more cell bodies of the sample in the holding space in a direction away from the functionalized wall surface portion, thereby separating a second amount of cell bodies of the sample from the functionalized surface portion, and moving and removing the second amount of cell bodies of the sample from the holding space including Here, the second amount of the removed sample is used for administration to the subject for the medical treatment. The cell body according to any one of items C1 to C7. [Item C9] The cell body according to any one of items C1 to C8, wherein in preparing the cell body, the fluid composition of the sample changes. [Item C10] The cell body according to claim 9, wherein in preparing the cell body, the change is due to one or more of different pH values, different dilutions including salt concentrations, and different fluid compositions. [Item C11] The cell body according to any one of items C1 to C10, wherein in preparing the cell body, the functionalized wall portion contains tumor cells. [Item C12] The cell body according to item C11, wherein in preparing the cell body, the cell body contains T cells genetically engineered to express a T cell receptor for an antigen present in the tumor cells.

Claims

1. 1. A cell body for use in a medical treatment, the cell body comprising: providing a sample holder including a holding space for holding a fluid medium; providing a sample comprising one or more cell bodies in a fluid medium within the holding space; providing a functionalized wall portion in the holding space to be contacted with the sample, wherein the functionalized wall portion comprises cells to which at least a portion of the sample is intended to adhere, the cells comprising tumor cells, the cell bodies comprising one or more of immune cells, T cells and B cells, the cell bodies being devoid of microbeads and / or magnets, wherein the sample comprising the cell bodies and / or the cells of the functionalized wall portion has been obtained from a subject; contacting the sample with the functionalized wall portion; applying a force to one or more cell bodies of the sample within the holding space in a direction away from the functionalized wall portion, and using the force to push one or more cell bodies of the sample within the holding space in a direction away from the functionalized wall portion; removing at least some of the cell bodies of the sample from the holding space after the sample containing the cell bodies contacts the functionalized wall portion, the force is applied, and at least some of the cell bodies detach from the functionalized surface portion. The method includes the steps of: wherein the removed cell bodies are used for administration to the subject or another subject for the medical treatment. The cell body.

2. 1. A cell body for use in a medical treatment of a subject, the medical treatment comprising: providing a sample holder including a holding space for holding a fluid medium; providing a sample comprising one or more cell bodies in a fluid medium within the holding space; providing a functionalized wall portion in the holding space to be contacted with the sample, wherein the functionalized wall portion comprises cells to which at least a portion of the sample is intended to adhere, the cells comprising tumor cells, the cell bodies comprising one or more of immune cells, T cells and B cells, the cell bodies being devoid of microbeads and / or magnets, wherein the sample comprising the cell bodies has been obtained from the subject and / or the cells of the functionalized wall portion have been obtained from the subject; contacting the sample with the functionalized wall portion; applying a force to one or more cell bodies of the sample within the holding space in a direction away from the functionalized wall portion, and using the force to push one or more cell bodies of the sample within the holding space in a direction away from the functionalized wall portion; removing at least some of the cell bodies of the sample from the holding space after the sample containing the cell bodies contacts the functionalized wall portion, the force is applied, and at least some of the cell bodies detach from the functionalized surface portion. The method includes the steps of: wherein the removed cell bodies are used for administration to the subject for the medical treatment. The cell body.

3. The cell body of claim 1 or 2, wherein preparing the cell body includes a flushing step of flushing the holding space after the contacting step and before applying the force, thereby removing unbound cell bodies.

4. The cell body according to any one of claims 1 to 3, wherein in preparing the cell body, the sample containing the cell body is obtained from the subject.

5. The cell body according to any one of claims 1 to 4, wherein in preparing the cell body, the sample containing the cells of the functionalized wall portion is obtained from the subject.

6. A cell body according to any one of claims 1 to 5, wherein in preparing the cell body, in the removing step, at least a portion of the cell body is collected based on a relationship between the force of one or more cell bodies against the functionalized wall portion and adhesion strength and / or adhesion rate.

7. The cell body according to any one of claims 1 to 6, wherein in preparing the cell body, tightly bound cell bodies are collected in the removing step.

8. Preparing the cell body, applying a first force to one or more cell bodies of the sample within the holding space in a direction away from the functionalized wall portion, thereby separating a first amount of cell bodies of the sample from the functionalized wall portion and removing the first amount of cell bodies of the sample from the holding space; and applying a second increased force to one or more cell bodies of the sample within the retention space in a direction away from the functionalized wall portion, thereby dissociating a second quantity of cell bodies of the sample from the functionalized wall portion, and displacing and removing the second quantity of cell bodies of the sample from the retention space. Including, wherein the second amount of the removed sample is used to be administered to the subject for the medical procedure. A cell body according to any one of claims 1 to 7.

9. The cell body according to any one of claims 1 to 8, wherein in preparing the cell body, the fluid composition of the sample is changed.

10. 10. The cell body of claim 9, wherein in preparing the cell body, the changes are due to one or more of different pH values, different dilution rates including salinity, and different fluid compositions.

11. The cell body according to any one of claims 1 to 10, wherein in preparing the cell body, the functionalized wall portion comprises a tumor cell.

12. The cell body of claim 11, wherein in preparing the cell body, the cell body comprises a T cell genetically engineered to express a T cell receptor for an antigen present on the tumor cell.

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