Multimodal biophysical characterization apparatus, systems, methods, and applications

Abstract

This application relates to the field of cell biology, and more specifically to a multimodal biophysical characterization apparatus, a multimodal biophysical characterization system, a multimodal biophysical characterization method, and its applications. This application enables multimodal, high-resolution, high-throughput, high-sensitivity, low-cost, low-damage, and configurable physical characterization of single cells or multicellular aggregates, and allows for the application of different types and intensities of stimuli (e.g., mechanical, electrical, or optical stimulation) to the sample under measurement to better mimic the in vivo microenvironment, and allows for manipulation (e.g., labeling, fixation, ablation, cutting, extraction, or sorting) of samples at specific locations or regions, and can be combined with other characterization methods (e.g., protein staining, histochemical staining, or single-cell sequencing) for comparative analysis. The multimodal biophysical characterization apparatus according to this application is suitable for fields such as synthetic biology, diagnostics, drug discovery, early tumor screening, cell therapy, and precision medicine.

Description

Technical Field

[0001] This application relates to the field of cell biology, and more specifically, to a multimodal biophysical property evaluation apparatus, a multimodal biophysical property evaluation system, a multimodal biophysical property evaluation method, and its applications.

Background Art

[0002] Cells are the basic units of life, and cell behavior, including adhesion, migration, differentiation, apoptosis processes, and dynamic changes in different physiological and pathological processes, as well as interactions with small and macromolecular substances, have important significance in understanding and controlling life phenomena. The physical characteristics of cells or multicellular aggregates, such as cellular forces (also called cellular mechanical forces or cellular traction forces) and cellular stiffness (rigidity), play a crucial role in maintaining life activities and regulating biological functions. For example, cellular forces can influence processes such as cell morphology, migration, proliferation, differentiation, and signal transduction, and, along with other biochemical signals, play a critical regulatory role in processes such as embryonic development, stem cell differentiation, immune processes, wound repair, and cancer metastasis, and are therefore targets for the treatment of many diseases. Several studies have found that there are enormous differences in the maximum cellular mechanical forces of normal cells, benign and malignant tumor cells. Cellular stiffness refers to the ability of a cell to deform under external force and is usually used to describe the flexibility or rigidity of a cell. Cellular stiffness is of great significance in tumor identification, evaluation, and therapeutic monitoring. In cancer research, cellular stiffness is widely used to identify and isolate tumor cells, such as circulating tumor cells (CTCs). Tumor cells typically have higher stiffness compared to normal cells. Changes in cell stiffness are generally considered an important characteristic of tumor cell invasion and metastasis, and cell stiffness can also be used to assess the malignancy of tumors, aiding in the development of more accurate treatment plans. At the same time, cell stiffness is central to immune evasion mechanisms. Furthermore, the characterization and study of cell stiffness have significant implications for the mechanisms of inflammation, the diagnosis and treatment of hematological disorders, the diagnosis and monitoring of liver diseases, and drug screening and development. In short, cell stiffness can objectively reflect changes in the internal structure and composition of cells, and the study of cell stiffness is advantageous in solving problems in basic biology and developing applied fields such as clinical diagnosis, drug discovery, early tumor screening, and cell therapy. Therefore, characterizing the type, state, and behavior of cells and multicellular aggregates, especially high-throughput and accurate characterization of cellular mechanical forces and cell stiffness, is a crucial challenge in fields such as biotechnology, cell biology, and physical biology, and there is a need to perform accurate, highly efficient, and comprehensive physical characterization of cells or multicellular aggregates of different types and states.

[0003] Currently, there are several main methods for characterizing cells and multicellular aggregates: physical methods, etc.: These methods use instruments to measure the mechanical or electrical properties of cells, such as tensile force, adhesion force, elastic modulus, and electrical impedance. While these methods can reflect the morphology, function, and metabolic state of cells, existing technologies typically require expensive equipment and data processing techniques. Examples include atomic force microscopy (AFM), traction force microscopy (TFM), electrical impedance spectroscopy (EIS), and microfluidic chips (MFCs). However, these methods have limitations, such as complex operation, low throughput, high cost, and difficulty in long-term and real-time monitoring of cells due to throughput and phototoxicity. There are also biochemical methods, which use reagents and labels to measure cells. These methods can sensitively detect the state of cells and their binding to macromolecules, but they may also affect their inherent properties and functions. Examples include fluorescence resonance energy transfer (MRI), biotin-avidin systems, and enzyme-linked immunosorbent assays. While chemical methods can sensitively detect the binding status between cells and molecules, they usually require labeling or modification of the target, which can interfere with its normal function. Therefore, it is extremely necessary, urgent, and valuable to propose systems and methods that, based on existing technologies, non-invasively and label-free monitor the physical state of cells and multicellular aggregates, predict the differentiation and behavior of cells and multicellular aggregates, and ultimately characterize cells and multicellular aggregates in real time, with high throughput and low cost, non-invasively and label-free, including cellular mechanical forces, cell hardness and softness, and simulations of their behavior after external stimuli. [Overview of the project]

[0004] Therefore, there is a need to overcome the aforementioned shortcomings of existing technologies and provide a solution that enables real-time, high-throughput, and low-cost quantitative measurement and monitoring of cellular mechanical forces and / or cell stiffness without requiring expensive equipment, thereby meeting the demand for next-generation tools and methods for the physical characterization of cells and multicellular aggregates in applications such as basic biology problems, the development of clinical diagnostics, drug discovery, early tumor screening, and cell therapy.

[0005] To achieve the above objective, in a first aspect, the present application provides a multimodal biophysical characterization apparatus, and the multimodal biophysical characterization apparatus is Base and, The system includes a micropillar or micropillar array consisting of one or more micropillars, which are mounted on the base and capable of undergoing a change of state in response to cellular mechanical forces and / or magnetic forces, The aforementioned change of state includes at least one of: pulling, movement in any direction, and deformation; The micropillar includes a bottom end connected to a base, sides, a columnar body enclosed by the sides, and a top end far from the base and facing the bottom end. A light-reflecting layer is provided at the top end, sides, and / or any position within the columnar body of the micropillar, and a magnetic material is provided at the top end, sides, and / or any position within the columnar body of the micropillar. Preferably, a magnetic metallic light-reflecting layer is provided at the top end of the micropillar.

[0006] The base in this application may be any regular or irregular surface, including a plane, inclined surface, curved surface, or irregular curved surface. This allows for customization of the three-dimensional morphology of the surface to which cells or multicellular aggregates adhere, and also provides a three-dimensional adhesion environment. Optionally, the lengths of the micropillars on the inclined or curved surface may also vary, which may result in different micropillar hardnesses. The lengths, aspect ratios, and hardnesses of micropillars on the same base in this application may also vary, expanding the measurable range and allowing for the application of different hardness stimuli to the organism being measured. Magnetic materials in this application refer to materials that react in any way to a magnetic field, and include, but are not limited to, ferromagnetic materials, paramagnetic materials, diamagnetic materials, ferrimagnetic materials, antiferromagnetic materials, and superparamagnetic materials.

[0007] When the multimodal biophysical characterization apparatus provided in this application characterizes cells or multicellular aggregates, the micropillars can penetrate the cells or multicellular aggregates. The penetrating forces include, but are not limited to, the gravity of the cells or multicellular aggregates, the attractive adhesion caused by substances having mechanisms that promote cell adhesion, and applying pressure to the micropillars or cells or multicellular aggregates. In particular, after the micropillars penetrate the cells or multicellular aggregates, magnetic materials at the top, sides, and any position within the columnar body of the micropillars are subjected to magnetic force, pulling on the micropillars and causing at least one type of activity or deformation in each direction.

[0008] In this application, "several" does not mean multiple quantities, but rather one or more.

[0009] Furthermore, a light-reflecting layer is installed at the top of the micropillar, and an anti-reflective layer is installed on the side surface and / or base surface of the micropillar. By installing them in this manner, the signal-to-noise ratio of the optical signal in biological characterization is significantly improved, and the measurement results can be made more accurate.

[0010] Furthermore, the multimodal biophysical characterization apparatus further includes a plurality of cell restriction structures mounted on the base, each cell restriction structure including one or more restriction surfaces, the restriction surfaces being planar or curved, the restriction surfaces being perpendicular to the plane on which the base is located, being connected to the base or integrally molded with the base, and the height of the restriction surfaces being greater than that of the micropillars, and enclosing a predetermined number of micropillars inside.

[0011] Furthermore, a substance that interacts with cells is placed on at least one of the base, micropillar, and limiting surface, and the substance that interacts with cells is, It contains one or more of the following: substances that promote cell adhesion, substances that interact with cell surface receptors, bioactive substances, and bioinactivating substances.

[0012] Preferably, the cell adhesion-promoting substance includes, but is not limited to, one or more of the following substances: (1) extracellular matrix proteins, including, but not limited to, collagen, fibronectin, vitronectin, laminin, or elastin; (2) extracellular matrix mimics, including, but not limited to, polypeptides containing RGD adhesion sequences; (3) substances having a cell adhesion-promoting mechanism, including, but not limited to, polylysine; and substances having interactions with cell surface receptors, including, but not limited to, antibodies.

[0013] Furthermore, an anti-cell adhesion substance is placed on at least one of the base, micropillar, and limiting surface. This placement further limits the adhesion range of cells or multicellular aggregates, ensuring they remain within a predetermined measurement area, controlling morphology including cell size, shape, and tropism, and can be used to better control the adhesion, migration, and differentiation of cells or multicellular aggregates.

[0014] Furthermore, the cell-interacting substance is placed on the apex of the micropillar, and the multiple micropillars on which the cell-interacting substance is placed form a first predetermined pattern, and / or the anti-cell-adhesion substance is placed on the apex of the micropillar, and the multiple micropillars on which the anti-cell-adhesion substance is placed form a second predetermined pattern. The first predetermined pattern may be the same as or different from the second predetermined pattern, and such naming in this application is solely for the purpose of distinguishing and identifying the patterns that are formed as a whole by the micropillars on which the cell-interacting substance and the anti-cell-adhesion substance are placed, respectively.

[0015] Furthermore, a fluorescent substance is installed in the extension of the micropillar from its apex to its base. This extension includes the sides of the micropillar and the solid portion within the columnar body enclosed by the sides. By installing it in this way, the objective of more intuitively calibrating cells or multicellular aggregates and amplifying the signal, thereby improving the signal-to-noise ratio, can be achieved. At the same time, the cells or multicellular aggregates to be characterized can be stained with a fluorescent substance of a different color, and the hardness of the cells or multicellular aggregates can be determined by the depth of penetration, thereby enhancing the characterization signal.

[0016] Furthermore, the base and / or micropillars are made of conductive material. The purpose of using conductive material for the base and / or micropillars is to enable electrical stimulation during the characterization process of cells or multicellular aggregates, as well as to achieve the objective of receiving electrical signals from cells.

[0017] Specifically, the multicellular aggregates of this application are coupled to a multimodal biophysical characterization apparatus in various ways, two of which are, for example: The first coupling method involves placing a culture medium on a micropillar of a multimodal biophysical characterization device, transplanting cells into the culture medium on the micropillar, and culturing them to obtain multicellular aggregates; in another embodiment, this coupling method allows for real-time monitoring of the cell culture process while cellular mechanical force information is output in a visualized format, and can be applied to assessing the effects of chemical, biological, and physical external stimuli such as culture medium and drugs on cell growth; Second binding method: Multicellular aggregates cultured directly are attached to micropillars of a multimodal biophysical characterization system for detection.

[0018] To achieve the above objective, in a second aspect, the present application provides a multimodal biophysical characterization system, the multimodal biophysical characterization system is A multimodal biophysical characterization apparatus according to one or more claims 1 to 8, A light signal emitting device for emitting a predetermined ray, An optical signal detection device for detecting light rays reflected from the aforementioned light reflection layer, The system includes a magnetic field generating device for generating a magnetic field and causing a magnetic force interaction with the magnetic material, The light beam emitted by the optical signal emitter passes through the incident light path and irradiates the light reflection layer, and the light beam reflected by the light reflection layer enters the optical signal detection device via the reflected light path.

[0019] In addition to the optical signal emitter, optical signal detector, and magnetic field generator, the multimodal biophysical characterization system of the present application can be configured by employing only one multimodal biophysical characterization device described in the first aspect of the present application to measure the cellular mechanical force of a cell or multicellular aggregate and its single-sided hardness, or by employing two or more multimodal biophysical characterization devices described in the first aspect of the present application to measure the cellular mechanical force of a cell or multicellular aggregate and its double-sided or multi-sided hardness.

[0020] Furthermore, the multimodal biophysical property evaluation system further includes an optical signal analysis device, and the optical signal analysis device is used to analyze the optical signal detected and acquired by the optical signal detection device.

[0021] Furthermore, the multimodal biophysical property evaluation system further includes a data processing device, and the data processing device is used to perform arithmetic processing on the optical signal to obtain results of cell mechanical force and / or cell hardness and softness (including but not limited to numerical values of size, directionality, and change trends) and spatial distributions. Furthermore, the data processing device can also provide prediction information on cell behavior and differentiation direction based on the results and spatial distributions of cell mechanical force and / or cell hardness and softness.

[0022] Furthermore, the multimodal biophysical property evaluation system further includes a pressure transducer, and the pressure transducer is used to apply pressure to cells. The pressure transducer includes but is not limited to weights, automated pressure devices, probes, etc. The pressure transducer can include a pressure sensor and sense the magnitude of the pressure applied simultaneously to control an appropriate pressure value.

[0023] Furthermore, the optical signal emitting device has a light source. The light source includes a first light source installed at the bottom of the base, and / or a second light source installed on the side of the base, and / or a third light source installed far from the base at the top opposite to the bottom end. The light rays emitted by the first light source, and / or the second light source, and / or the third light source reach the micropillar.

[0024] In this application, the first light source, the second light source, and the third light source do not limit the installation order or type of the light source, but are only for distinguishing light sources at different positions. Preferably, the second light source employs a waveguide illumination light source.

[0025] Furthermore, the multimodal biophysical property evaluation system further includes a device for causing movement or deformation, and the device for causing movement or deformation is used to cause deformation in the multimodal biophysical property evaluation system to apply a mechanical force to the multimodal biophysical property evaluation system. Preferably, the device for causing movement or deformation includes, but is not limited to, a tension device and / or a gas charging and discharging device. The tension device is used to apply an appropriate mechanical tensile force to the base to control the stretching movement or deformation of the base in the horizontal direction, and the gas charging and discharging device is used to form movement or deformation on the surface of the base.

[0026] Furthermore, the multimodal biophysical property evaluation system is configured with a double-sided structure or a multi-sided structure by two or more of the multimodal biophysical property evaluation devices. The outside of the double-sided structure or multi-sided structure is the base, and the inside forms a three-dimensional accommodation cavity for cells by surrounding them. By installing in this way, a three-dimensional close contact environment can be provided for the cells, and furthermore, the multi-faceted and diverse physical properties of cells or cell aggregates within the three-dimensional scale can be evaluated, which is more accurate and closer to the in-vivo environment.

[0027] Furthermore, when a double-sided or multi-sided structure is configured by two or more of the multimodal biophysical property evaluation devices, the cell restriction structures located on different multimodal biophysical property evaluation devices are adapted.

[0028] As a third aspect, the present application provides a method for evaluating the physical properties of cells using the multimodal biophysical property evaluation system described in the second aspect of the present application. The method includes: a step of emitting a predetermined light beam using the light signal emitting device; and a step of detecting the light beam after the action of the multimodal biophysical property evaluation device using the light signal detecting device.

[0029] The method provided in this application employs micropillars equipped with magnetic materials and light-reflecting layer materials (one or more of the following may also be installed: fluorescent material, anti-reflective layer material, and magnetic metallic light-reflecting layer), controls the spacing between the micropillars, and applies substances with mechanisms of action that promote cell adhesion, such as antibodies or extracellular matrix, or substances with mechanisms of action that promote cell adhesion, such as F127, to specific locations. This allows for autonomous switching between measuring cellular mechanical force and measuring cell rigidity, and, if necessary, simultaneously measuring cellular mechanical force and cell rigidity. It enables simultaneous measurement of cellular mechanical force on both sides or multiple sides, and further enables measurement of cell rigidity at specific locations in a cell or multicellular aggregate, as well as simultaneous measurement of cellular mechanical force and cell rigidity. This method is the first to link the active mechanical force of cells within the cellular space with the characteristics of cell rigidity.

[0030] Furthermore, the method further includes the step of generating a magnetic field of a predetermined direction and intensity using the magnetic field generating device, Optionally, the process may further include the step of applying different types and intensities of stimuli to the sample being characterized before, during, or after physical characterization. The stimulus is optionally selected to be at least one or a combination of one or more physical, chemical, and biological stimuli. Optionally, the physical, chemical, and biological stimuli include one or more combinations of stimulus types from among drugs, mechanical force, hardness, biochemistry, electric fields, flow fields, chemotactic induction, and radiation.

[0031] In particular, before, during, or after the physical characterization of cells or multicellular aggregates, operations including but not limited to labeling, fixation, ablation, dissection, extraction, and sorting can be performed on target samples at specific locations or regions, and comparative analyses can be conducted in conjunction with other characterization methods, including but not limited to protein staining, histochemical staining, and single-cell sequencing.

[0032] Furthermore, the method further includes an optical signal analysis step: using the optical signal analyzer, compare and analyze reflected light rays before and after the fluid flows through a multimodal biophysical characterization device, and within a certain deformation range, the deformation and the attenuation of the light reflection signal are linearly related, and the state of the fluid can be calculated from the strength of the light reflection signal caused by the deformation of the micropillar, thereby enabling fluid velocity calibration; and / or, using the optical signal analyzer, compare and analyze reflected light rays before and after the magnetic field generator generates a magnetic field of a predetermined direction and intensity, and obtain information on the cellular mechanical force and / or cellular stiffness of the sample to be characterized. The cellular mechanical force information includes the magnitude, direction, frequency, distribution, and dynamic changes over time of the cellular mechanical force at a specific location in a cell or multicellular aggregate, and the cellular stiffness information includes the magnitude, spatial distribution, and dynamic changes over time of the stiffness of different layers at a specific location within a cell or multicellular aggregate.

[0033] In a fourth aspect, the present application provides applications of the multimodal biophysical characterization apparatus described in the first aspect in the fields of synthetic biology, cell therapy, drug discovery, characterization of intercellular interactions, early tumor screening, or precision medicine, preferably the application includes obtaining at least one of the magnitude, spatial distribution, and dynamic change trends of cellular mechanical forces and cell stiffness through the multimodal biophysical characterization apparatus to determine or predict whether transfection has been successful; determining or predicting whether a sample to be characterized is a cell that highly expresses a certain bioactive substance; typing for normal or pathological cells; determining or predicting the state and differentiation direction of cells; typing for sensitivity to drugs and treatments; predicting the behavior of cells, including but not limited to state, differentiation, proliferation, migration, and apoptosis; and determining or predicting interactions between cells or multicellular aggregates and bioactive substances and between (multi)cells and (multi)cells.

[0034] Unlike existing technologies, the above technical solution has the following advantages: (1) High throughput, low cost: Compared with existing AFM, TFM, MFC and conventional micropillar arrays, the technical solution of this invention eliminates reliance on expensive equipment, significantly simplifies the operating process, does not require imaging with a high-resolution confocal microscope, and allows for high-throughput cell monitoring simply by monitoring the intensity of reflected light, all at a low cost. (2) Enables multimodal biophysical characterization: By adding a magnetic metal reflective layer and magnetic material to the top of the micropillar and changing parameters such as the coating composition characteristics, spacing, and motion logic of the micropillar, it is possible to switch between measuring cellular mechanical force and cellular stiffness, or to measure cellular mechanical force and cellular stiffness simultaneously, enabling a more flexible and accurate comprehensive characterization of the physical characteristics of cells. (3) The properties of the micropillar, such as hardness, softness, and motion mode, can be changed dynamically and in real time, the measurement range can be optimized dynamically and in real time, and various conditions can be configured to apply different stimuli: By adding components such as magnetic materials, light sources, electrodes, and pressure transmitters, dynamic control and adjustment can be performed on the micropillar to adapt to different measurement demands and conditions, and at the same time, physical or chemical stimuli can be applied to cells or multicellular aggregates, and the effects of different conditions and stimuli on the physical characteristics of cells or multicellular aggregates can be observed. (4) Diverse operating modes are possible: By installing devices such as cell restriction devices and sandwich structures, operations such as morphological control, pressure application, positional labeling, and sample extraction can be performed on cells or multicellular aggregates, and further comparative analysis can be performed by combining them with other characterization methods (protein staining, histochemical staining, single-cell sequencing, etc.). (5) High resolution, high sensitivity, and suitable for long-term monitoring: Real-time monitoring is possible for single cells and multicellular aggregates, and the resolution is high; deformation signals of micropillars are amplified only through light reflection signals, improving the sensitivity of deformation monitoring; real-time monitoring: Since fluorescence is not required and phototoxic effects on cells or multicellular aggregates by lasers can be avoided, it is suitable for long-term monitoring and can be used to study the long-term response of cells or multicellular aggregates to drugs.

[0035] (6) Configurable: The system's configuration and functions can be flexibly configured according to the usage scenario. For example, the magnetic field can produce different effects when used in the following three scenarios: First, the magnetic field can be used to drive the micropillars into deformation to measure cell stiffness; or, a tensile force parallel to the micropillars can be applied, which is equivalent to increasing the stiffness of the micropillars; and the measurement range of the micropillars can be adjusted and optimized, and / or mechanical or motion stimulation can be applied to the cells in real time.

[0036] In general, this invention enables multimodal, high-resolution, high-throughput, high-sensitivity, low-cost, low-damage, and configurable physical characterization of single-cell or multicellular aggregates, and allows for the application of different types and intensities of stimulation (e.g., mechanical, electrical, or optical stimulation) to the sample under test to better mimic the in vivo microenvironment, as well as the application of manipulations (e.g., labeling, fixation, ablation, dissection, extraction, sorting, etc.) to samples at specific locations or regions, and can be combined with other characterization methods (e.g., protein staining, histochemical staining, single-cell sequencing, etc.) for comparative analysis. The multimodal biophysical characterization apparatus provided by this invention is suitable for fields such as synthetic biology, diagnostics, drug discovery, early tumor screening, cell therapy, and precision medicine. [Brief explanation of the drawing]

[0037] [Figure 1] This is a schematic diagram of a multimodal biophysical characterization apparatus in a specific embodiment 1 of the present invention. [Figure 2] This is a schematic diagram of a specific embodiment of the present invention in the direction A-A' (where the light-reflecting layer is installed at the top of the micropillar); [Figure 3] This is a schematic diagram of a specific embodiment of the present invention in the direction A-A' (where the light-reflecting layer is installed on the side of the micropillar); [Figure 4] This is a schematic diagram of a specific embodiment of the present invention in the direction A-A' (where the light-reflecting layer is installed at any position within the columnar body of the micropillar); [Figure 5] This is a schematic diagram of a specific embodiment of the present invention in the direction A-A' (where the light-reflecting layer is installed on the top and side of the micropillar); [Figure 6] This is a schematic diagram of a specific embodiment of the present invention in the direction A-A' (where the light-reflecting layer is installed at any position and on the side of the column body of the micropillar); [Figure 7] This is a schematic diagram of a specific embodiment of the present invention in the direction A-A' (where the magnetic metallic light reflective layer is installed at the top of the micropillar); [Figure 8] This is a schematic diagram showing an anti-reflective layer installed on the side of a micropillar in a specific embodiment of the present invention. [Figure 9] This is a schematic diagram showing an anti-reflective layer installed on the surface of a base in a specific embodiment of the present invention. [Figure 10] This is a schematic diagram showing an anti-reflective layer installed on the side of a micropillar and on the surface of a base in a specific embodiment of the present invention. [Figure 11] This is a schematic diagram showing a magnetic material installed on the side of a micropillar in a specific embodiment of the present invention. [Figure 12] This is a schematic diagram showing a specific embodiment of the present invention in which the magnetic material is placed on top of the light-reflecting layer at the top of a micropillar. [Figure 13] This is a schematic diagram showing a specific embodiment of the present invention in which the magnetic material is placed at an arbitrary position within the columnar body of a micropillar. [Figure 14] This is a schematic diagram showing a specific embodiment of the present invention in which the magnetic material extends from the top to the bottom of the micropillar and is located above the light-reflecting layer. [Figure 15]This is a schematic diagram showing a specific embodiment of the present invention in which the magnetic material extends from the top to the bottom of a micropillar and is located above the light-reflecting layer, and an anti-reflective layer is installed on the surface of the base. [Figure 16] This is a schematic diagram showing a specific embodiment of the present invention in which the magnetic material is installed on the columnar body of a micropillar, positioned above the light-reflecting layer, and an anti-reflective layer is installed on the surface of the base. [Figure 17] This is a schematic diagram showing a multimodal biophysical characterization apparatus having a restrictive structure in a specific embodiment of the present application for measuring a single cell. [Figure 18] This is a schematic diagram showing a multimodal biophysical characterization apparatus having the restrictive structure described in a specific embodiment of the present invention, with a cell adhesion promoting substance placed on the surface of the base, for measuring a single cell. [Figure 19] This is a schematic diagram showing a multimodal biophysical characterization apparatus having the restrictive structure described in a specific embodiment of the present invention, wherein a cell adhesion promoting substance is placed on the surface of the base and an anti-cell adhesion substance is placed on the restrictive surface, and which measures a single cell. [Figure 20] This is a schematic diagram showing that the fluorescent material of this invention is installed on the columnar body (below the top) of the micropillar and located below the light-reflecting layer. [Figure 21] This is a schematic diagram showing the fluorescent material of the present invention installed at the top of a micropillar and combined with a light-reflecting layer material. [Figure 22] This is a schematic diagram showing the fluorescent material of the present invention installed on the side of a micropillar. [Figure 23] This is a schematic diagram showing the fluorescent material of the present invention installed at any position within the columnar body of a micropillar. [Figure 24] This is a schematic diagram showing the measurement of bilateral hardness of a single cell using a bilateral structure consisting of two multimodal biophysical characterization devices in a specific embodiment of the present invention. [Figure 25] This is a schematic diagram showing the measurement of the polyhedral hardness of a single cell using a polyhedral structure consisting of three multimodal biophysical characterization devices in a specific embodiment of the present invention. [Figure 26]This is a schematic diagram showing how a double-sided cell restriction structure, consisting of two multimodal biophysical characterization devices, is fitted and measured in a specific embodiment of the present invention. [Figure 27] This is a schematic diagram of a multimodal biophysical characterization system (with the light source installed below the base) in a specific embodiment of the present invention. [Figure 28] This is a schematic diagram of a multimodal biophysical characterization system (with light sources installed on the side and / or above the base) in a specific embodiment of the present invention. [Figure 29] This figure shows an example of measuring a multicellular aggregate by constructing a bimodal structure using the multimodal biophysical characterization device provided in this application. [Figure 30] This figure shows the method for fabricating a multimodal biophysical characterization device, the characterization results obtained using an electron microscope, and a schematic diagram of the principle of mechanical calculation. [Figure 31] This figure shows a method for calibrating a multimodal biophysical characterization device using a flow field, along with experimental results (there is a linear relationship between the deflection of micropillars and the attenuation of the light reflection signal within a certain interval). [Figure 32] This figure shows the experimental results of a multimodal biophysical characterization system simultaneously characterizing the mechanical force and hardness of tumor cells (a correspondence exists between cellular mechanical force and hardness). [Figure 33] This figure shows the experimental results obtained by measuring hardness using a magnetic field and performing cross-validation by Z-scanning with a microscope in a specific embodiment of the present invention using a multimodal biophysical characterization apparatus. [Figure 34] This figure shows the experimental results of tumor tissue characterization and drug testing under multimodal biophysics in a specific embodiment of the present invention. [Figure 35] This figure shows the experimental results of characterizing the activation of immune cells under multimodal biophysics in a specific embodiment of the present invention, and comparing and verifying it with the gold standard. [Modes for carrying out the invention]

[0038] To provide a detailed explanation of the technical content, structural features, objectives, and effects of the technical solution, specific examples and attached drawings will be provided below.

[0039] Where the term "Examples" appears in the text, it means that certain features, structures, or characteristics described in conjunction with the Examples may be included in at least one Example of the Application. The term "Examples" appearing throughout the Specification does not necessarily refer to the same Example, nor does it particularly limit the independence or relevance between other Examples. In principle, in the Application, unless there is a technical contradiction or conflict, each technical feature referred to in each Example can be combined in any way to form a corresponding implementable technical solution.

[0040] In the description of this application, the term "and / or" is an expression used to describe a logical relationship between objects, indicating that three relationships may exist. For example, A and / or B represents three situations: A exists, B exists, and A and B exist simultaneously. In addition, the character " / " in the text generally indicates that the preceding and following related objects are in an "or" logical relationship.

[0041] In this application, terms such as "first" and "second" are used simply to distinguish one substance or operation from another, and do not necessarily require or imply that there is any actual relationship between these substances or operations, such as quantity, hierarchy, or order.

[0042] In the absence of further restrictions, the “includes,” “contains,” “possesses,” or other similar expressions used in this Application are intended to be non-exclusive, and these expressions do not preclude the existence of further elements in a process, method, or product containing the elements described herein. Therefore, a process, method, or product containing a set of elements may include not only those limited elements, but also other elements not explicitly listed, or elements specific to such a process, method, or product.

[0043] Similar to the understanding in the "Examination Guidelines," in this application, expressions such as "greater than," "less than," and "exceeding" are understood to exclude the number; expressions such as "greater than or equal to," "less than or equal to," and "within" are understood to include the number. Furthermore, the meaning of "multiple" in the description of embodiments in this application is two or more (including two), and similar expressions related to "many" are understood in the same way. For example, this is the case unless there is a separate, clear and specific limitation such as "multiple sets" or "multiple orders."

[0044] Cellular traction force, also known as cellular traction force, refers to the force that a cell exerts on its surrounding environment through its cytoskeleton and adhesive structures. This force can cause deformation and displacement between the cell and the external matrix, thereby influencing biological processes such as cell morphology, movement, signal transduction, and gene expression. In tumor development, changes in cellular traction force are associated with many important biological processes; therefore, studying cellular traction force is of crucial importance in tumor identification. Specifically, this includes: Tumor cell migration and metastasis: Tumor cells control their movement and migration within the external matrix by regulating mechanical forces. Changes in mechanical forces cause changes in cell morphology and movement patterns, thereby influencing tumor cell metastasis and invasion.

[0045] Tumor microenvironment: Cell-matrix interactions in the tumor microenvironment are regulated by mechanical forces, influencing important biological processes such as cell growth, proliferation, angiogenesis, and immune evasion, thereby influencing tumor growth and metastasis.

[0046] Tumor treatment: The mechanical forces of tumor cells are associated with their sensitivity and resistance to chemotherapy and radiotherapy. Therefore, studying cellular mechanical forces can provide new perspectives and methods for the personalization and targeted therapy of tumors.

[0047] Overall, cellular mechanical forces serve as important biological indicators in the tumor development process, helping us to gain a deeper understanding of tumor mechanisms, identify tumors, and provide new perspectives and methods for tumor prevention, treatment, and personalized medicine.

[0048] Beyond tumors, the characterization and study of cellular mechanical forces have significant implications for many other diseases and biological processes. Specifically, these include: Cell migration and development: Cellular mechanical forces are important factors that control cell migration and tissue development, playing a crucial role in cell migration and morphological changes in biological processes such as embryonic development, tissue regeneration, and repair.

[0049] Nervous System: Mechanical forces between neurons and glial cells can influence neuronal growth and connectivity, playing a crucial role in the normal development and maintenance of nervous system function, and are also associated with certain neurological disorders such as spinal cord injury and neurodegenerative diseases.

[0050] Immune system: The mechanical forces of immune cells such as lymphocytes, monocytes, and macrophages can influence their movement within the body and their recognition and elimination of pathogens, playing a crucial role in maintaining the normal function of the immune system and fighting disease.

[0051] Overall, the characterization and study of cellular mechanical forces are of great significance not only for tumor identification and treatment, but also for many other biological processes and diseases. They help us to gain a deeper understanding of the mechanisms of biological processes and the onset and development of diseases, and to provide new ideas and methods for the treatment and prevention of related diseases.

[0052] Currently, tools for studying cellular mechanical forces can be broadly classified into three categories: microfluidic technology, microscopy technology, and micro-nanochips. For example, traction force microscopy (TFM) uses an optical microscope to observe the morphology and movement of cells and calculates the magnitude and direction of cellular mechanical forces by measuring the displacement of the matrix surrounding the cells. Alternatively, a confocal microscope can be used to image the relative displacement caused by the deformation of micro-nanostructures in the Z-axis direction and calculate the force acting at that point. However, existing measurement techniques require special experimental conditions and equipment, are highly dependent on microscopes, are relatively complex and expensive to construct and operate, and require a certain level of experimental experience and technical ability. Furthermore, image data must be analyzed and processed to obtain the magnitude and direction of cellular mechanical forces. This process requires a series of mathematical calculations and image processing, demanding advanced data processing skills and computational capabilities. Thirdly, traction force microscopy, for example, requires the placement of special labels such as microbeads or fluorescent markers around the cells. The presence of these labels can affect the behavior of cells such as growth, differentiation, and migration, thereby potentially affecting the reliability of the measurement results. More importantly, existing measurement methods suffer from low throughput and high cost, as well as insufficient time and low resolution. Most studies only measure static or stationary cells, and current technology still makes real-time, quantitative, and long-term monitoring and analysis of changes in mechanical forces during cell movement or deformation difficult.

[0053] Cellular rigidity, also known as cellular stiffness, refers to a cell's ability to deform under external force and is commonly used to describe the flexibility or stiffness of a cell. Cellular rigidity is of significant importance in tumor identification, evaluation, and treatment monitoring. In cancer research, cellular rigidity is widely used to identify and isolate tumor cells, such as circulating tumor cells (CTCs). Tumor cells typically have higher rigidity compared to normal cells. This is because the structure of tumor cells, including the cytoskeleton, nucleus, and stroma, is abnormal, and the expression and distribution of cytoskeletal proteins are also altered. High rigidity in tumor cells allows them to more easily pass through the extracellular matrix, enter the circulatory system, and migrate to other sites to form metastatic lesions. Therefore, changes in rigidity are considered an important characteristic of tumor cell invasion and metastasis, and cellular rigidity can also be used to assess the malignancy of tumors, aiding in the development of more accurate treatment plans. Furthermore, cellular rigidity is central to immune evasion mechanisms. Therefore, using cell stiffness as an indicator for identifying tumor cells can effectively distinguish between tumor cells and normal cells, playing a crucial role in the early diagnosis and treatment of tumors. Currently, several detection methods based on cell stiffness, such as atomic force microscopy and cell microfluidic chips, are being developed. Since cell stiffness can also change after treatment, it can also be used to monitor the effectiveness of treatment. The following are some aspects of how cell stiffness can be applied at different stages of tumor development: Diagnosis: Cell stiffness can be used to distinguish between tumor cells and normal cells by measuring cell stiffness. Since tumor cells are usually stiffer than normal cells, cell stiffness can be a diagnostic tool used for the early diagnosis and differential diagnosis of tumors.

[0054] Prognosis: The stiffness of tumor cells is associated with their malignancy. Studies have shown that tumor cells with high stiffness are more invasive and metastatic. Therefore, measuring cell stiffness can predict the prognosis of a tumor and provide a basis for selecting a treatment plan.

[0055] Treatment: Cell stiffness can be a monitoring indicator in tumor treatment. Some treatments can cause changes in the stiffness of tumor cells. Therefore, by measuring changes in cell stiffness, the effectiveness of treatment can be monitored and the treatment plan can be adjusted.

[0056] New drug screening: Cell stiffness can also be used to screen for new drugs. Since some drugs may affect cell stiffness, measuring changes in cell stiffness can be used to evaluate the efficacy and safety of a drug.

[0057] Beyond tumors, the characterization and study of cell stiffness has many other applications and significances in biology and medicine. Examples include: Inflammation: Inflammation is a cellular and tissue reaction. Inflammation causes disruption of the cell membrane and changes in permeability, which affects cell stiffness. Therefore, measuring cell stiffness can be used to study the mechanisms of inflammation.

[0058] Blood disorders include conditions such as decreased deformability of red blood cells and anemia, and these conditions are usually related to the deformability and stiffness of red blood cells. Therefore, measuring the stiffness of red blood cells can be used in the diagnosis and treatment of these conditions.

[0059] Liver Diseases: Liver tissue stiffness can be used to diagnose and monitor liver diseases such as cirrhosis and hepatic fibrosis. These diseases harden liver tissue, causing it to lose elasticity and thereby affecting liver function.

[0060] Cell movement and migration: Cell stiffness is related to cell movement and migration. Therefore, measuring cell stiffness can be used to study cell movement and migration mechanisms, such as cancer cell metastasis and inflammatory cell migration.

[0061] Drug Screening and Development: The potency and toxicity of a drug may be influenced by cell stiffness. Therefore, measuring cell stiffness can be used in drug screening and development to evaluate the efficacy and toxicity of a drug. In short, cell stiffness has extensive applications in biology and medicine and can be used in the study of diverse physiological and pathological processes and diseases, providing important information for the diagnosis and treatment of diseases.

[0062] In this application, "multimortal" refers to multiple forms of physical information, such as cellular force (cellular traction force or cellular mechanical force), cellular stiffness (rigidity), or cellular mechanical force and cellular stiffness.

[0063] In this application, "multicellular aggregate" refers to a group of cells formed by the aggregation of two or more cells, i.e., a multicellular aggregate; a multicellular aggregate includes, but is not limited to, cell groups obtained by in vitro or intracellular culture, such as tumor multimers, and includes, for example, tumor spheroids, organoids, and living tissues. Generally, "cell" as used in this application includes a single cell and a multicellular aggregate formed by the aggregation of two or more cells.

[0064] Example 1 A type of multimodal biophysical characterization apparatus, system, and measurement method This embodiment provides a type of multimodal biophysical characterization apparatus that enables the measurement of multimodal biophysical information of cells by influencing the intensity of the light reflection signal using a light reflection layer, a magnetic material, or a magnetic metal light reflection layer.

[0065] Refer to Figure 1, which shows a type of multimodal biophysical characterization device 1. A micropillar array is formed by a base 11 and a plurality of micropillars 12 installed on the base 11, thereby constituting the multimodal biophysical characterization device 1, or micropillar chip. Each micropillar 12 consists of a bottom end 120 connected to the base 11, a side 121, a columnar body 122 enclosed by the side, and a top end 123 that is far from the base 11 and opposite the bottom end 120. Specifically, when observed from the A-A' direction of the multimodal biophysical characterization apparatus 1 shown in Figure 1, the top 123 of the micropillar 12 of the present invention has a light-reflecting coating 13 (Figure 2); or the side surface 121 of the micropillar 12 has a light-reflecting coating 13 (Figure 3); or any position within the column of the micropillar has a light-reflecting coating (Figure 4); or both the top 123 and the side surface 121 of the micropillar 12 have a light-reflecting coating (Figure 5); or even all of the top 123, side surface 121, and any position within the column 122 of the micropillar 12 have a light-reflecting coating (Figure 6). The top 123 of the micropillar 12 has a coating of a magnetic metal (e.g., iron, cobalt, nickel, etc.) (in other embodiments, it is possible to substitute with other magnetic materials, and the position where the magnetic material is installed may be on the side surface or inside the column of the micropillar, as long as the micropillar can generate a magnetic force under a magnetic field). The coating having the magnetic metal 131 can function as a light-reflecting layer 13. In a preferred embodiment, the magnetic metal light-reflecting layer 13 is installed on the top end 123 of the micropillar 12 (Figure 7).

[0066] It should be noted that although the term "coating" is used in this embodiment, this merely indicates that the light-reflecting layer 13 in this embodiment can be manufactured by a coating process. This does not necessarily limit the manufacturing of the light-reflecting layer 13 to a coating process; it may also be manufactured by, for example, sputtering or vapor deposition.

[0067] A single multimodal biophysical characterization system consists of one multimodal biophysical characterization device 1, an optical signal emitter, an optical signal detector, and a magnetic field generator. The optical signal emitter is used to emit a predetermined light ray, the optical signal detector is used to detect the light ray reflected from the optical reflection layer 13, and the magnetic field generator is used to generate a magnetic field and create a magnetic effect with the magnetic metal 131. The light ray emitted by the optical signal emitter passes through the incident optical path and irradiates the optical reflection layer 13, and the light ray reflected by the optical reflection layer 13 enters the optical signal detector via the reflected optical path.

[0068] When measuring the multimodal biophysical characteristics of cells using the multimodal biophysical characterization system described above (Figures 27 and 28), the specific operating steps are as follows: S1. The cells (or cell multimers, or single cells and cell multimers simultaneously) of the target for measurement (characterization) are cultured on the multimodal biophysical characterization apparatus shown in Figure 1. After culturing for 1 to 2 days, the cells of the target for measurement (characterization) are allowed to fully adhere to and grow on the multimodal biophysical characterization apparatus; S2, a predetermined ray is emitted using an optical signal emitter; S3. A light signal detection device is used to detect the light rays after the action of the multimodal biophysical characterization device. In this embodiment, "action" may refer only to the reflection effect produced by a light-reflecting layer installed at either the top or side of the micropillar of the multimodal biophysical characterization device for a given light ray, or it may refer to the reflection effect produced by light-reflecting layers installed at both the top and side of the micropillar for a given light ray. Preferably, the magnetic metal light-reflecting layer installed at the top of the micropillar may also refer to the reflection effect produced by the magnetic metal light-reflecting layer installed at the top of the micropillar under the magnetic force of a magnetic field of a specific direction and intensity generated by a magnetic field generator, causing the micropillar to tilt in one direction, pierce the target cell, deform, be pulled, or oscillate, while simultaneously causing the magnetic metal light-reflecting layer to produce a reflection effect on the light ray during the deformation process of the micropillar. Therefore, by using the system of this embodiment, it is possible to measure the mechanical force of the target cell alone, measure the hardness of the target cell alone, or measure the mechanical force and hardness of the target cell in combination.

[0069] It should be noted that the cells to be measured in this embodiment may be single cells, multicellular aggregates, or a combination of single cells and multicellular aggregates.

[0070] Example 2 Refer to the multimodal biophysical characterization apparatus (A-A' direction view) shown in Figures 8, 9, and 10. The difference from Embodiment 1 is that a micropillar array is formed by a base 11 and a plurality of micropillars 12 installed on the base 11, thereby constituting the multimodal biophysical characterization apparatus 1. The micropillar 12 consists of a bottom end 120 connected to the base 11, a side 121, a columnar body 122 enclosed by the side, and a top end 123 far from the base 11 and opposite the bottom end 120. The top end 123 of the micropillar 12 has a magnetic metallic light reflective layer 13 that reflects light, and an anti-reflective layer 14 is installed on the side of the micropillar (Figure 8). Alternatively, the anti-reflective layer 14 is installed on the surface of the base (Figure 9). Or, the anti-reflective layer 14 is provided on both the side 121 of the micropillar 12 and the surface of the base (Figure 10).

[0071] In other different embodiments, a light-reflecting layer 13 can be provided on one or more of the top and sides of the micropillar, and a magnetic material 131 can be placed on the top, sides, and any position within the column of the micropillar, and various combinations can be made with the anti-reflective layer 14 on one or more of the sides of the micropillar and the surface of the base (Figures 11, 12, 13, 14, 15, and 16). This achieves the objective of reflecting light to the micropillar or a specific part of the micropillar and generating a magnetic force.

[0072] Example 3 Please refer to Figure 17. The difference from Examples 1 and 2 is that the multimodal biophysical characterization apparatus of this embodiment further includes a cell restriction structure. The cell restriction structure includes one or more restriction surfaces 15, the restriction surfaces 15 being perpendicular to the plane on which the base 11 is located, connected to the base 11 or integrally molded with the base 11, and the height of the restriction surfaces 15 being greater than the micropillars 12 and enclosing a predetermined number of micropillars 12 inside.

[0073] The function of the cell restriction structure in this embodiment is to detect the isolation of single cells, the overlap of two single cells, or the isolation of multicellular aggregates, that is, to avoid contact or adhesion between cells or between multicellular aggregates at the time of detection, and to restrict the size, shape, and morphology of cells, thereby facilitating high-throughput testing. Depending on different needs, the number of restricting surfaces 15 or the enclosed shape of the cell restriction structure may vary. For example, the restricting surfaces 15 included in the cell restriction structure may be a single cylindrical surface, or three planes whose starting and ending points meet to form a triangular cross-section and enclose a certain number of micropillars, or four planes perpendicular to each other with their starting and ending points meeting to form a rectangular shape and enclose a certain number of micropillars, or N planes whose starting and ending points meet to enclose an N-sided polygon, or a single curved surface with a nearly circular cross-section. In other words, the cross-sectional shape of the restricting surface 15 is a controllable closed shape, and its area (or can be understood as the number of micropillars that can be accommodated in that space) is also controllable. In actual embodiments, depending on the manufacturing process, the cell restriction structure may be a structure integrally molded with the base 11, or a structure adhered to the base 11.

[0074] Example 4 Please refer to the multimodal biophysical characterization apparatus shown in Figure 18. Based on the multimodal biophysical characterization apparatus of Examples 1-3, a cell-interacting substance 16 is placed on at least one of the base 11, micropillars 12, and limiting surfaces 15. The cell-interacting substance 16 includes one or more of the following: a substance that promotes cell adhesion, a substance that interacts with cell surface receptors, a bioactive substance, and a bioinactivating substance. Therefore, for the multimodal biophysical characterization apparatus in this example, the cells to be measured are susceptible to the effects of the adhesive action because at least one of the base 11, micropillars 12, and limiting surfaces 15 is coated with a cell-interacting substance (adhesion promoter), and the cells to be measured enclose some or all of the micropillars.

[0075] Example 5 Refer to Figure 19. Based on the multimodal biophysical characterization apparatus of Example 4, an anti-cell adhesion substance is placed on at least one of the base 11, micropillar 12, and limiting surface 15. Thus, the cell-interacting substance (adhesion promoter) 16 and the anti-cell adhesion substance 17 in this example can be intentionally placed on the base 11, micropillar 12, and limiting surface 15 according to the actual needs at the time of measurement, thereby further limiting the cell adhesion range, ensuring that it fits within the area and shape of the measurement target, better controlling cell migration and differentiation, and controlling the size, shape, and tropism characteristics of the cells. This allows for the measurement of cellular mechanical force by controllingly inducing the cells or multicellular aggregates to remain on the micropillars without being impaled, or by inducing specific parts of the cells or multicellular aggregates to be impaled by the micropillars to measure cell stiffness.

[0076] Example 6 Based on the multimodal biophysical characterization apparatus of Example 5, a substance that interacts with cells is placed on the top of a micropillar, and multiple micropillars on which the cell-interacting substance is placed form a first predetermined pattern, and / or a substance with anti-cell adhesion properties is placed on the top of a micropillar, and multiple micropillars on which the anti-cell adhesion properties are placed form a second predetermined pattern. Specifically, by micrometer printing technology, substances having a cell adhesion mechanism of a specific pattern, such as (1) extracellular matrix proteins including but not limited to collagen, fibronectin, vitronectin, laminin, or elastin; (2) extracellular matrix mimics including but not limited to polypeptides containing RGD adhesion sequences; (3) substances having a cell adhesion-promoting mechanism including but not limited to polylysine; and substances having interactions with cell surface receptors including but not limited to antibodies, can be printed to promote cell adhesion in these areas. The so-called predetermined pattern may be in the shape of a triangle, quadrilateral, polygon, circle, ellipse, etc. The effects of the predetermined patterns include: firstly, controlling cell and intercellular contact through patterns composed of materials with these cell adhesion mechanisms, thereby facilitating high-throughput data acquisition. Secondly, achieving dimensionality reduction in data processing by unifying cell shapes, thereby reducing the difficulty of analysis. Thirdly, controlling cell size, shape, tropism, and differentiation state by limiting cell adhesion areas with anti-cell adhesion materials such as BSA and F127, and further controlling actin filaments to regulate the cell's mechanical state, thereby achieving the requirements of specific technically demanding scenarios. In actual operation, the patterns of the first predetermined pattern and the second predetermined pattern may be identical or different.

[0077] Example 7 Please refer to Figures 20, 21, 22, and 23. The difference from the multimodal biophysical characterization apparatus provided in Example 1 is that the fluorescent substance 18 is placed on the extension from the apex to the base of the micropillar. It may be on the side of the micropillar near the apex, or within the column enclosed by the side near the apex, or on the side near the base, or within the column enclosed by the side at the base, or at any position within the column enclosed by the sides of the micropillar. The presence of the fluorescent substance can achieve the objective of more intuitively calibrating cells or multicellular aggregates, amplifying the signal, and improving the signal-to-noise ratio. At the same time, the cells or multicellular aggregates to be measured can be stained with a different colored fluorescent substance, and the hardness of the cells or multicellular aggregates can be determined by the depth of penetration.

[0078] In Examples 1-7 of the present application, the material of the micropillar 12 is polydimethylsiloxane (PDMS), and in some other key embodiments of the present application, the material of the micropillar 12 may be some other polymer material, such as silicon-based polymers, photoresist polymer materials, conductive polymer materials, temperature-sensitive polymer materials, etc.

[0079] Example 8 Please refer to Figure 24. Two multimodal biophysical characterization devices provided in this application are set up facing each other to form a double-sided structure. The outer part is the base, and the inner part forms a three-dimensional containment cavity surrounding cells or multicellular aggregates. The volume or height of the three-dimensional containment cavity can be adjusted according to the actual requirements. For example, if the cells to be measured are single cells on opposite sides, the height of the three-dimensional containment cavity can be controlled between 5 nm and 2 mm. If the cells to be measured are multicellular aggregates or biological tissues, the height and volume can be adjusted to be larger accordingly, with the height even reaching 5 cm and the volume reaching 30 cm³.

[0080] This embodiment provides an apparatus and method for simultaneously measuring the physical properties of both sides of a type of cell. S1: Prepare one micropillar tip. The top of the micropillar has a metallic reflective coating and is covered with extracellular matrix. The sides of the micropillar and the space between the micropillar are coated with an anti-reflective coating and an anti-cell adhesion coating.

[0081] S2: Culture the lung cancer cell line A549 on this chip and wait until it is fully attached.

[0082] S3: Prepare a separate chip and coat only the apex of the micropillar with extracellular matrix protein, while coating the rest with anti-cell adhesive F127 to facilitate measurement of cellular mechanical forces.

[0083] S4: The chip from step S3 is inverted and placed on top of the chip containing cancer cells, so that the restriction structures are aligned with each other and both types of cells are in contact with each other.

[0084] S5: The device is equipped with a light source (optical signal emitter) and a reflected light receiver (optical signal detector) at the top and bottom, respectively, allowing for simultaneous detection of reflected signals from the upper and lower chips to characterize the cellular mechanical force and hardness.

[0085] S6: Monitor changes in the cellular physical properties of both cell types in real time and establish a correlation between the trends in changes in cellular mechanical force and hardness / softness.

[0086] Figure 29 shows an example of measuring a multicellular aggregate by constructing a bimodal structure using the multimodal biophysical characterization apparatus provided in this application.

[0087] In other different embodiments, two or more multimodal biophysical characterization devices are configured in a multifaceted structure (Figure 25), allowing for simultaneous measurement of the magnitude and spatial distribution of cellular mechanical force and cell stiffness for multiple faces of a target cell or different cells within a multicellular aggregate.

[0088] Another objective of this embodiment is to culture cells in a three-dimensional, close-contact environment with a multifaceted structure and to flexibly measure the cellular mechanical forces and / or hardness of each facet of the cell.

[0089] Experimental steps: S1. Prepare a base with a micro-pillar structure, and apply a metallic reflective coating to the top of the micro-pillar and an anti-reflective coating to the sides of the micro-pillar.

[0090] S2. Two bases with micropillar structures are placed on the left and right sides of the base from step S1, forming a cistern-like structure inside. The micropillars are coated with an extracellular matrix protein that promotes cell adhesion only at the top, and with the anti-cell adhesion-promoting F127 on the remaining parts, so that cells come into contact only with the top of the micropillars, making it easier to measure the cellular mechanical force.

[0091] S3. Extracellular matrix is ​​applied around and between the micropillars at the bottom of the cisterna structure, and the base is extended to adjust the spacing between the micropillars, so that the micropillars penetrate the cells and make it easier to characterize the hardness and softness of the cells.

[0092] S4. Distribute the cell suspension uniformly within the cisternoid structure so that the cells adhere to multiple surfaces of the micropillars.

[0093] S5. Place the apparatus in a cell culture incubator to provide the cells with a suitable growth environment and culture them for a set period of time (e.g., 48 hours) to allow the cells to fully adhere to multiple surfaces of the cisternoid structure.

[0094] S6. During the experiment, an appropriate magnetic field is applied to the micropillars at the bottom of the vessel structure from outside the apparatus to deform the micropillars at the bottom of the vessel structure. The micropillars at the bottom of the vessel structure are then illuminated using a light source, and the reflected light signal is collected. By analyzing the changes in the intensity and frequency of the reflected light signal, the hardness and softness of the cell bottom surface are observed.

[0095] S7. Micropillars on the sides of the cisternoid structure are irradiated using a light source, and reflected light signals are collected. Mechanical forces on the sides of the cells are observed by analyzing the attenuation and dynamic changes of the reflected light signals.

[0096] S8. Analyze experimental data and summarize the relationship between the physical properties of cells in a three-dimensional close-contact environment and their biological properties such as growth and migration.

[0097] In some more preferred embodiments, the cell restriction structures are fitted together (Figure 26), i.e., two opposing cell restriction structures form a cavity to accommodate the target cells to be measured.

[0098] Example 9 The difference from the multimodal biophysical characterization system provided in Embodiment 1 of the present invention is that an optical signal analyzer can be connected to it, and the optical signal can be analyzed to improve the timeliness and operability of the processing.

[0099] Example 10 The multimodal biophysical characterization system provided in this embodiment connects a data processing device to a base to which an optical signal analyzer is connected. The data processing device is used to process the optical signals to obtain results and spatial distribution information of cellular mechanical forces and / or cellular hardness. This eliminates the need for complex manual calculations and improves the accuracy and measurement efficiency of data processing. At the same time, it provides predictive information on cell behavior and differentiation direction based on the results and spatial distribution of cellular mechanical forces and / or cellular hardness.

[0100] Example 11 The multimodal biophysical characterization system provided in this embodiment further includes a pressure transducer, which is used to apply pressure to cells. The pressure transducer may also include pressure sensing and pressure feedback, which measure specific pressure values ​​and adjust the magnitude of the applied force. If no substance with a mechanism of action that promotes cell adhesion is placed on the micropillar, external pressure can be applied to direct the cells and cause them to move relative to the micropillar in order to facilitate the measurement of cellular mechanical force and / or cell stiffness. These pressure transducers include, for example, weights, automated pressure devices, probes, etc.

[0101] Example 12 The multimodal biophysical characterization system provided in this embodiment further includes a device for inducing motion or deformation, which is used to apply mechanical force to the multimodal biophysical characterization system. Since the base can be manufactured from a material having certain flexibility and elasticity, the device for inducing motion or deformation that can be employed includes, but is not limited to, a tensioning device or a filling / exhausting device. The tensioning device is used to apply appropriate mechanical tensile force to the base to control the horizontal stretching motion or deformation of the base, and the filling / exhausting device is used to create motion or shape changes on a curved surface in the base.

[0102] Example 13 The difference in the multimodal biophysical characterization system provided in this embodiment lies in the fact that the base and / or micropillars are made of conductive material, which is intended to enable electrical stimulation during the characterization process of cells or multicellular aggregates, as well as to achieve the objective of receiving biological electrical signals.

[0103] Example 14 The difference in the multimodal biophysical characterization system provided by this embodiment is that the optical signal emitter has light sources, which include a first light source located at the bottom of the base, and / or a second light source located on the side of the base, and / or a third light source located at the top, far from the base and opposite the bottom. The rays emitted from the first and / or second and / or third light sources reach the micropillar. The types of the first, second and third light sources located at the bottom of the base do not need to be specifically defined, and such arrangement can improve the overall measurement throughput, reduce the volume of the system, and enhance the portability of the system. In a preferred embodiment, the second light source can employ waveguide illumination, which further reduces the volume of the system and, by adding an evanescent wave illumination mode, can significantly improve the signal-to-noise ratio.

[0104] Example 15 The difference in the multimodal biophysical characterization system provided in this embodiment lies in the addition of a magnetic field generator, which generates a magnetic field of a predetermined direction and intensity in the multimodal biophysical characterization device. The magnitude and direction of the magnetic field can be adjusted according to the actual needs, for example, all micropillars can be tilted in an orderly manner in one direction. When the cell stiffness is relatively high, the magnetic field can suppress the tilt of the micropillars, reducing the tilt amplitude and allowing for the detection of more light reflection signals; conversely, when the cell stiffness is relatively low, the tilt amplitude of the micropillars becomes larger, and the detectable light reflection signals become weaker. The stiffness of the cell can be determined by utilizing the magnitude of the applied magnetic field and the strength of the light reflection signals.

[0105] Example 16 Measurement of cell nuclear hardness using two independent methods: magnetic materials method and fluorescence method. The objective of this embodiment is to measure the hardness of cell nuclei using two independent methods: magnetic materials and fluorescence. Measurement using magnetic materials is simpler, does not require an expensive confocal microscope, and its accuracy is confirmed by cross-validation using fluorescence.

[0106] S1: Provides a chip whose surface is covered with PDMS nanomagnetic micropillars. The micropillars contain a fluorescent material and have an extracellular matrix coating.

[0107] S2: Macrophage strain THP-1 is cultured on the chip, and the THP-1 cells are brought into contact with the chip by controlling the height of the culture medium.

[0108] S3: Lipopolysaccharide (LPS) is added to the culture medium to activate THP-1 cells and convert them into M1 macrophages.

[0109] S4: After several days of culture, the magnetic field is turned on using the magnetic materials method, and the attenuation of reflected light is measured. After turning off the magnetic field, the attenuation value and frequency of the light reflection signal of each micropillar are measured, and the hardness of the cell nucleus at each position is calculated sequentially.

[0110] S5: Cells are fixed onto a chip using paraformaldehyde, and the cell nuclei are stained using DAPI.

[0111] S6: Using fluorescence imaging, a confocal microscope is used to perform a Z-axis tomographic scan from the top to the bottom of the micropillars to analyze the depth of the DAPI signal of the cell nucleus between the micropillars. When the stiffness of the cell nucleus is low, the depth of sinking of the cell nucleus into the space between the micropillars is greater; conversely, when the stiffness of the cell nucleus is high, the depth of sinking of the cell nucleus is shallower.

[0112] S7: This experiment revealed that the depth of cell nucleus sinking increases after activation of macrophage THP-1. This indicates that the activation process softens the hardness of the cell nucleus. The results measured by magnetic materials and fluorescence methods were identical (Figure 33), and the accuracy of cell nucleus hardness measurement by magnetic materials was confirmed by cross-validation using fluorescence.

[0113] Example 17 This embodiment demonstrates an apparatus and method for measuring the hardness, viscosity, and viscoelasticity of a specific location within a type of cell. By combining the strength of magnetic metals and light reflection signals, cell stiffness can be measured. The specific operating steps are as follows: S1: Prepare one micropillar tip. The surface of the micropillar is covered with a magnetic metal coating (e.g., iron, cobalt, nickel, etc.) which functions as a reflective layer. Apply an anti-reflective coating to the sides of the micropillar, and at the same time coat the area above the magnetic metal layer and between the micropillars with extracellular matrix.

[0114] S2: Seed cells onto micropillar tips and culture them. After a culture period of 1 to 2 days, ensure that the cells have fully attached and grown on the tips.

[0115] S3: A PDMS block of a specific mass is placed on the cell, or a constant voltage is applied to a fixed piezoelectric material, thereby applying downward pressure to the cell and prompting the micropillar to penetrate the cell to a certain depth.

[0116] S4: A magnetic field is applied to tilt all micropillars in an orderly manner in one direction. Then, the magnetic field is turned off, and the oscillations as the deformed micropillars return to their original positions are observed. Depending on the hardness, viscosity, and viscoelasticity of the cells, the characteristics of the light reflection signal produced during oscillations change, including the dynamic changes, the magnitude of the extreme values, and the frequency. Harder cells limit the tilt amplitude of the micropillars, thereby producing stronger light reflection signals; conversely, less hard cells increase the tilt amplitude of the micropillars, producing weaker light reflection signal extreme values. The higher the viscosity, the slower the oscillations. By measuring the characteristics of these light reflection signals, the physical characteristics of the cells, such as hardness, viscosity, and viscoelasticity, can be calculated.

[0117] Example 18 This embodiment provides a device technology for measuring the hardness of a type of multicellular spheroid, utilizing magnetic metals and the intensity of light reflection signals to measure the hardness of the multicellular spheroid. The specific steps are as follows: S1: A micropillar tip is provided. The top of the micropillar is coated with a magnetic metal (e.g., iron, cobalt, nickel, etc.) which functions as a reflective layer; the sides of the micropillar are provided with an anti-reflective coating; and the extracellular matrix is ​​coated above the magnetic metal layer and between the micropillars.

[0118] S2: Multiple-cellular spheroids are cultured on this micropillar chip and cultured for 1 to 2 days to allow the multi-cellular spheroids to fully adhere to the chip and grow.

[0119] S3: Adjust the spacing between micropillars so that the multicellular spheroids are embedded in the micropillars to a certain depth by gravity.

[0120] S4: A magnetic field is used to tilt all micropillars in an ordered direction. When the hardness of the multicellular spheroid is relatively hard, the tilt of the micropillars is suppressed, reducing the tilt amplitude of the micropillars and allowing for the detection of more light reflection signals; conversely, when the hardness of the multicellular spheroid is relatively soft, the tilt amplitude of the micropillars becomes larger, and the detectable light reflection signal becomes weaker. Therefore, this device can determine the hardness of the multicellular spheroid by utilizing the strength of the light reflection signal.

[0121] Example 19 This embodiment introduces a device technology for measuring tissue hardness, utilizing magnetic metals and the intensity of light reflection signals to measure tissue hardness. The specific steps are as follows: S1: Prepare a micropillar tip. The top of the micropillar is covered with a magnetic metal coating (e.g., iron, cobalt, nickel, etc.) which functions as a reflective layer. Apply an anti-reflective coating to the sides of the micropillar and coat the top of the magnetic metal layer and the space between the micropillars with extracellular matrix.

[0122] S2: Biological tissue is cut into thin sections approximately 200 μm thick using a tissue microtome and spread directly onto a micropillar tip. Culture medium is added, and after 1 day of incubation, the tissue is completely adhered to the tip.

[0123] S3: A constant voltage is applied to a piezoelectric material fixed above the PDMS block or tissue, applying downward pressure to the tissue and encouraging the micropillar to penetrate the tissue to a certain depth.

[0124] S4: A magnetic field is used to tilt all micropillars in an ordered direction. When the tissue block is hard, the tilt of the micropillars is suppressed, reducing the tilt amplitude of the micropillars, thereby allowing for the detection of a stronger light reflection signal; conversely, when the tissue block is soft, the tilt amplitude of the micropillars becomes larger, and the detectable light reflection signal becomes weaker. Therefore, this device can measure the hardness of the tissue by utilizing the strength of the light reflection signal (Figure 34).

[0125] Example 20 This embodiment provides a method for separating drug-resistant and non-resistant cells by measuring a type of cell stiffness. The specific steps are as follows: S1: Prepare a micropillar tip. The top of the micropillar is covered with a magnetic metal coating (e.g., iron, cobalt, nickel, etc.) which functions as a reflective layer. Apply an anti-reflective coating to the sides of the micropillar and coat the top of the magnetic metal layer and the space between the micropillars with extracellular matrix.

[0126] S2: Lung cancer cell line HCC827 is cultured on a double-sided sandwich chip composed of micropillar chips. On one side of the chip (upper), a cell adhesion-promoting layer is applied only to the apex of the micropillars, while an anti-adhesion layer is applied to the remaining areas to prevent cells from growing between the micropillars and to measure the cellular mechanical forces. On the other side (lower), a cell adhesion-promoting layer is applied to the apex, periphery, spaces, and base of the micropillars, inducing the micropillars to penetrate the cells under the action of cell adhesion-promoting substances and gravity. After 1 to 2 days of culture, the cells are allowed to fully adhere to the chip and grow.

[0127] S3: A pressure transducer is used to apply a constant pressure to the cells, allowing the micropillars on the lower chip to penetrate the cells more effectively. A magnetic field is applied, and the magnetic field is used to tilt all the micropillars in an orderly direction. The strength of the reflected light signal is measured to estimate cell stiffness. Simultaneously, the mechanical force of the cells is measured through the reflected light from the upper micropillars.

[0128] S4: Record the position coordinates of each cell. Add the EGFR-TKI drug and use a magnetic field to tilt all micropillars in an ordered direction. Measure the intensity of the light reflection signal to estimate cell stiffness. Observe the stiffness of all cells every 30 minutes. Simultaneously monitor the mechanical force of the cells through the light attenuation of the micropillars on the upper tip.

[0129] S5: Since cellular mechanical force and cell viability are directly related, the viability and dynamic changes of each cell can be calculated based on the cellular mechanical force and its dynamic changes. Therefore, under the action of a magnetic field, the magnitude of the tilt amplitude of the micropillar on which the cell is located causes a corresponding increase in the intensity of the light reflection signal, thereby allowing the calculation of the stiffness and dynamic changes of each cell and establishing the relationship between cellular mechanical force and cell stiffness. A decrease in cellular mechanical force indicates a decrease in cell viability under drug action, i.e., a sensitive cell. No significant decrease in cellular mechanical force means that there is no significant change in cell viability under drug action, suggesting a drug-resistant cell. Thus, the characteristics of cell stiffness of sensitive and resistant cells can also be obtained.

[0130] S6: A photoactivating fluorescent dye is added, and the photoactivating fluorescent dye is excited using a UV laser to induce red fluorescence in the resistant cells of interest.

[0131] S7: All cells are detached from the chip using trypsin, and then fluorescent cells are selected using a flow cytometer.

[0132] S8: The isolated cells are cultured in a culture medium containing EGFR-TKI, and it is confirmed that the selected cells have EGFR-TKI resistance. The resistance results of the confirmed cells are then used in supervised or semi-supervised machine learning.

[0133] Example 21 This embodiment provides a device technology for measuring the mechanical properties of two cells after their interaction. The specific steps are as follows: S1: Prepare one micropillar tip. The top of the micropillar has a metallic reflective coating and is covered with extracellular matrix. Anti-reflective coatings and anti-adhesion coatings are applied to the sides of the micropillar and between the micropillar and the micropillar itself.

[0134] S2: Culturing lung cancer cell line A549 on the chip and waiting until it is fully adhered.

[0135] S3: The endothelial cell line HUVEC is cultured on a separate chip and allowed to adhere completely. Alternatively, immune cells, such as T cells or NK cells, can be cultured on the chip and used to monitor tumor-immune cell interactions. Alternatively, cells can be cultured on a base containing only the restriction device and without the micropillar, enabling measurement of cells on only the other side.

[0136] S4: The chip from step S3 is inverted and placed on top of the chip containing cancer cells, so that the restriction mechanisms are aligned with each other and both types of cells are in contact with each other.

[0137] S5: The device is equipped with a light source and a reflected light receiver on the top and bottom, respectively, allowing for simultaneous detection of reflected signals from the upper and lower chips to determine the magnitude of the cell's mechanical properties.

[0138] S6: Monitor changes in the cellular dynamics of both cell types in real time and characterize cell-cell interactions.

[0139] Example 22 This embodiment provides an apparatus and method for measuring the mechanical force and / or hardness of cells by causing deformation or mechanical movement of the apparatus by stretching a type of base, thereby applying stretching and / or mechanical stimulation to cells, and simultaneously utilizing reflected light.

[0140] S1: Prepare a base with a micropillar structure, and apply a metallic reflective coating to the top of the micropillar and an anti-reflective coating to the sides of the micropillar.

[0141] S2: Extracellular matrix proteins are applied to the top of the micropillars to encourage cells to adhere to the top of the micropillars.

[0142] S3: Distribute the cell suspension evenly on the base so that the cells adhere to the top of the micropillars.

[0143] S4: Place the device in a cell culture incubator, provide the cells with a suitable growth environment, and culture for a set period of time (e.g., 24-48 hours) to allow the cells to fully adhere to the micropillars.

[0144] S5: Using a precision electric arm or similar device, stretching operations are performed on the base to deform or induce mechanical movement in the micropillar, thereby achieving stretching and / or mechanical stimulation of the cells. In the experiment, the amplitude and speed of stretching can be adjusted as needed to simulate different degrees of mechanical stimulation. Alternatively, inflation and deflation operations are performed on the bottom of the deformable base to change the curvature to which the cells adhere and to provide stretching and movement stimuli to the cells.

[0145] S6: When stretching and mechanically stimulating, a light source is used to illuminate the micropillar and the reflected light signal is collected. By analyzing the intensity and dynamic changes of the reflected light signal, the mechanical force and / or hardness during the cell stretching and mechanical stimulation process is measured in real time.

[0146] S7: Analyze experimental data and study the relationship between changes in the physical properties of cells under different stretching and mechanical stimulation conditions, and their biological properties such as growth, migration, and differentiation.

[0147] Example 23 This embodiment provides an apparatus and method for characterizing the physical properties (cellular mechanical force and hardness) of a type of cell in its initial state, relaxed state after preliminary stretching, and stretched state. The specific steps are as follows: S1: Prepare three bases with a micropillar structure. Apply a metallic reflective coating to the top of the micropillars and an anti-reflective coating to the sides of the micropillars. Apply extracellular matrix protein to the top of the micropillars to encourage cells to adhere to the top of the micropillars.

[0148] S2: One of the bases is stretched to the desired extent beforehand, and then the cell suspension is uniformly distributed onto the pre-stretched base. After the cells have completely adhered to the top of the micropillar, the base is released and contracted, thereby relaxing the cells.

[0149] S3: Distribute the cell suspension uniformly onto another base so that the cells adhere to the top of the micropillars. After the cells have fully adhered, stretch them against the base to elongate them.

[0150] S4: Distribute the cell suspension uniformly onto the third base and leave it untreated.

[0151] S5: Three devices are placed in separate cell culture incubators to provide cells with an appropriate growth environment. Micropillars are then irradiated onto cells in three different stretched states (S1, S2, S3) using a light source, and reflected light signals are collected. By analyzing the intensity and dynamic changes of the reflected light signals, the mechanical forces on cells in different states are measured in real time.

[0152] S6: Using a pressure transducer, micropillars are inserted into cells, a magnetic field is applied, and the hardness and softness of the cells are characterized by detecting the light reflection signal.

[0153] S7: Analyze experimental data to compare changes in physical properties in the initial state of cells, the relaxed state after preliminary stretching, and the stretched state, as well as the relationship between cell stiffness and cellular mechanical force.

[0154] Example 24 This embodiment provides a method for changing the equivalent stiffness of a micropillar by using a magnetic field parallel to a type of micropillar, thereby providing dynamic mechanical stimulation (stiffness) to cells, and optimizing the measurement range of the micropillar so that the force of the object being measured falls within the measurement range of the micropillar sensor. The specific steps are as follows: S1. Prepare a base with a micro-pillar structure, and apply a metallic reflective coating to the top of the micro-pillar and an anti-reflective coating to the sides of the micro-pillar.

[0155] S2. Extracellular matrix proteins are applied to the top of the micropillars to encourage cells to adhere to the top of the micropillars.

[0156] S3. Distribute the cell suspension evenly on the base so that the cells adhere to the top of the micropillars.

[0157] S4. Place the apparatus in a cell culture incubator, provide the cells with a suitable growth environment, and culture for a set period of time (e.g., 24-48 hours) to allow the cells to fully adhere to the micropillars.

[0158] S5. During the experimental process, applying a magnetic field parallel to the micropillar imparts an upward tensile force to the micropillar, making it less susceptible to lateral deformation, which is equivalent to increasing the rigidity of the micropillar. By adjusting the strength of the magnetic field, dynamic rigidity stimulation to the cells can be achieved, while simultaneously optimizing the measurement range of the micropillar so that the force of the object being measured falls within the measurement range of the micropillar sensor.

[0159] S6. When applying dynamic rigidity stimulation, a light source is used to illuminate the micropillar, and the reflected light signal is collected. By analyzing the intensity and dynamic changes of the reflected light signal, the mechanical force and / or stiffness of cells under different rigidity stimulation conditions are measured in real time.

[0160] S7. Analyze experimental data and study the relationship between changes in the physical properties of cells under different rigid stimuli and their biological properties such as growth, migration, and differentiation.

[0161] Example 25 Method for measuring the interaction between immune cells and molecules and the cell activation process by characterizing cellular mechanical force and hardness. This embodiment provides a method for evaluating the interaction between immune cells (e.g., T cells) and molecules (e.g., CD3 antibodies) and the cell activation process by measuring changes in the mechanical force and stiffness of immune cells (e.g., T cells). The expression of green fluorescent protein is used as an indicator of cell activation and is cross-validated with the results of measurements of cellular mechanical force and stiffness.

[0162] S1, prepare a mechanical chip, and the apex of its micropillars is covered with CD3 antibody; on the other hand, the sides of the micropillars and the spaces between the micropillars are treated with Pluronic F127 to prevent adhesion.

[0163] S2, an NFAT reporter (eGFP) Jurkat recombinant cell line is added to the chip, and the CD3 antibody attracts T cells to come into contact with the chip. When the T cell line is activated by the CD3 antibody, it expresses green fluorescent protein.

[0164] S3 uses a multimodal biophysical characterization system to monitor the mechanistic changes in cells and the expression of green fluorescent protein. By analyzing changes in the mechanical forces of cells at different time points, it is possible to study the interactions between immune cells and molecules, as well as the cell activation process.

[0165] S4. Using a pressure transducer, micropillars are inserted into cells, a magnetic field is applied, and the stiffness of the cells is characterized by detecting the light reflection signal.

[0166] In S5, we will analyze experimental data and monitor changes in cellular mechanical force and stiffness during the interaction between immune cells and molecules, as well as during the cell activation process. Simultaneously, we will observe the expression of green fluorescent protein and cross-validate it with the measured cellular mechanical force and stiffness to evaluate the activity of cells under different activation conditions.

[0167] Experiments revealed that cellular mechanical forces detect cell activation more quickly and sensitively, and that during the dynamic change of cellular mechanical forces, mechanical forces are activated around the cell before cell activation. Specific characteristics can be used to predict the cell state and activation time (Figure 35).

[0168] Example 26 A method for identifying activated and inactivated T cells and determining their activation ratio by measuring cellular mechanical force and stiffness. This embodiment provides a method for identifying activated and inactivated T cells and determining their activation ratio by measuring cellular mechanical force and stiffness. This method can be used to characterize cell therapy samples and predict the success rate of cell therapy.

[0169] S1, NFAT reporter (eGFP) Jurkat cells were cultured on a mechanical chip, the volume of culture medium was controlled, and the T cells were brought into contact with a multimodal biophysical characterization device to measure the magnitude of the mechanical force on inactive cells.

[0170] S2. Add CD3 antibody, CD28 antibody, and cytokines to the culture medium to activate T cells; when T cells are activated by the antibodies, they express green fluorescent protein.

[0171] S3 monitors changes in the mechanical force of each T cell and the expression of green fluorescent protein in real time.

[0172] S4. Using a pressure transducer, micropillars are inserted into cells, a magnetic field is applied, and the stiffness of the cells is characterized by detecting the light reflection signal.

[0173] When S5 and T cells are activated, cellular mechanical forces are gradually increased, and it was found that this has a positive correlation with green fluorescence.

[0174] S6. Changes in cellular mechanical force and stiffness during the activation process of each T cell are entered into a database. Using machine learning methods, a model is constructed that can determine the degree of T cell activation by utilizing the characteristics, spatial distribution, and temporal dynamic changes of cellular mechanical force and stiffness.

[0175] S7. T cells are isolated from donor peripheral blood mononuclear cells (PBMCs) using CD4 and CD8 antibodies.

[0176] S8. The isolated T cell pool is cultured on a mechanical chip, the amount of culture medium is controlled, and all T cells are brought into contact with the mechanical chip.

[0177] In S9, the distribution of the mechanical force of all T cells is recorded, and computational analysis is performed using the previously constructed model to determine the ratio of activated to inactivated T cells in the donor body. This value can be used to predict the probability of success of cell therapy.

[0178] Example 27 Microfluidics and mechanical chips: Applications of microfluidics and mechanical chips for monitoring in vitro culture of CAR-T cell therapy. This embodiment provides a technology for applying microfluidics and mechanical chips to in vitro culture monitoring of CAR-T cell therapy, aiming to more effectively monitor and evaluate the growth, activation, and therapeutic effects of CAR-T cells in an in vitro environment.

[0179] S1, the apex of the micropillars of the multimodal biophysical characterization instrument chip is coated with CD3 and CD28 antibodies; on the other hand, the sides of the micropillars and the spaces between the micropillars are treated with anti-adhesion treatment using Pluronic F127.

[0180] S2, the chip is laid at the bottom of the microfluidic device.

[0181] S3, T cells isolated from donor peripheral blood mononuclear cells (PBMCs) are injected into a microfluidic device to activate the T cells. Simultaneously, the cellular mechanical forces are monitored to determine whether all T cells have been fully activated.

[0182] After S4 and T cells are fully activated, a lentivirus is added to induce transduction, causing the T cells to express a chimeric antigen receptor with an antibody-specific sequence (sc-fv) that can recognize cancer cell surface proteins.

[0183] In S5, transduced T cells (CAR-T) are removed from the microfluidic fluid and culture amplification continues.

[0184] S6. A PDMS microwell thin film is laid on a mechanical chip, and tumor cells, tumor multicellular spheroids, or tumor organoids are cultured on the mechanical chip containing the microwells. The cells grow in an orderly manner within the microwells.

[0185] In S7, CAR-T cells are added to a mechanical chip containing tumor cells, and the cellular mechanical force of the tumor cells is monitored in real time. When CAR-T cells effectively kill tumor cells, the cellular mechanical force of the tumor cells decreases significantly.

[0186] This embodiment allows us to more effectively monitor and evaluate the cellular dynamics of CAR-T cells during activation, amplification, and tumor cell killing processes in an in vitro environment, thereby providing more accurate data support for CAR-T cell therapy.

[0187] Example 28 Application of microfluidics and mechanical chips to in vitro culture monitoring of CAR-T cell therapy. This embodiment provides a technology for applying microfluidics and mechanical chips to in vitro culture monitoring of CAR-T cell therapy, aiming to more effectively monitor and evaluate the growth, activation, and therapeutic effects of CAR-T cells in an in vitro environment.

[0188] S1, the tops of the micropillars of the multimodal biophysical characterization apparatus are coated with CD3 and CD28 antibodies; on the other hand, the sides of the micropillars and the spaces between the micropillars are treated with Pluronic F127 to prevent adhesion.

[0189] S2. A multimodal biophysical characterization device is laid at the bottom of the microfluidic device.

[0190] S3, T cells isolated from donor peripheral blood mononuclear cells (PBMCs) are injected into a microfluidic device to activate the T cells. Simultaneously, the cellular mechanical forces are monitored to determine whether all T cells have been fully activated.

[0191] After S4 T cells are fully activated, CRISPR-Cas9 technology is used to modify the T cell receptor (TCR) of the T cells to express one that can recognize the antigen of cancer cells.

[0192] In S5, the modified TCR-T cells are extracted from the microfluidic fluid and culture and amplification continues.

[0193] S6. A PDMS microwell thin film is laid on a mechanical chip, and tumor cells, tumor multicellular spheroids, or tumor organoids are cultured on the mechanical chip containing the microwells. The cells grow in an orderly manner within the microwells.

[0194] In S7, TCR-T cells were added to a mechanical chip containing tumor cells, and the cellular mechanical force of the tumor cells was monitored in real time. When the TCR-T cells effectively killed the tumor cells, the cellular mechanical force of the tumor cells decreased significantly.

[0195] This embodiment allows us to more effectively monitor and evaluate the cellular dynamics of TCR-T cells during activation, amplification, and tumor cell killing processes in an in vitro environment, thereby providing more accurate data support for TCR-T cell therapy.

[0196] Example 29 Determining the efficacy of antibody-drug conjugates in treating floating cancers by monitoring changes in cellular mechanical forces using a multimodal biophysical characterization system. This embodiment provides a method for monitoring changes in cellular mechanical forces using a multimodal biophysical characterization system and determining the therapeutic effect of antibody-drug conjugates on floating cancer cells.

[0197] S1, an antibody-drug conjugate is coated onto the top of the micropillar of the multimodal biophysical characterization apparatus, and the sides of the micropillar and the spaces between the micropillars are treated with Pluronic F127 to prevent adhesion.

[0198] S2, blood cancer cells are cultured on this device. The antibody coating attracts the blood cancer cells to the device, allowing them to come into contact with it.

[0199] S3 monitors the cellular mechanical force of blood cancer cells in real time by detecting the intensity of light reflection signals; when the antibody-drug conjugate effectively inhibits cell growth or induces apoptosis, the cellular mechanical force decreases significantly.

[0200] By monitoring changes in cellular mechanical forces using a multimodal biophysical characterization system, we can better understand the therapeutic effects of antibody-drug conjugates against parasitic cancers. Real-time monitoring of cellular mechanical forces helps to better evaluate the therapeutic effects of antibody-drug conjugates, thereby optimizing drug design and treatment planning.

[0201] Example 30 Determination of the therapeutic effect of antibody-drug conjugates on adhesive cancers by monitoring changes in cellular mechanical force and stiffness using a multimodal biophysical characterization system. This embodiment provides a method for evaluating the therapeutic effect of antibody-drug conjugates on adhesive cancers by monitoring changes in cellular mechanical force and stiffness.

[0202] S1, the apex of the micropillar of the multimodal biophysical characterization apparatus is coated with extracellular matrix, and the sides of the micropillars, the spaces between micropillars, and the base are treated with Pluronic F127 to prevent adhesion.

[0203] S2. Lung cancer cells are cultured on a multimodal biophysical characterization device, and after one day of culture, the cells are allowed to adhere completely to the device.

[0204] S3. Add the antibody-drug conjugate to the culture medium.

[0205] S4 monitors the cellular mechanical force and stiffness of lung cancer cells in real time by detecting the intensity of light reflection signals. When the antibody-drug conjugate effectively inhibits cell growth or induces apoptosis, the cellular mechanical force and stiffness decrease significantly.

[0206] Monitoring changes in cellular mechanical force and stiffness using multimodal biophysical characterization systems can provide important evidence for evaluating the therapeutic effects of antibody-drug conjugates on adhesive cancers.

[0207] Example 31 Determining the therapeutic effect of antibody-drug conjugates by monitoring changes in cellular mechanical forces in biological tissues. This embodiment aims to provide a method for evaluating the therapeutic effect of antibody-drug conjugates on tumor cells by monitoring changes in cellular mechanical forces in biological tissues.

[0208] In S1, the apex of the micropillar of the mechanical chip is coated with extracellular matrix, and the sides of the micropillar and the spaces between the micropillars are treated with Pluronic F127 to prevent adhesion.

[0209] S2. Using a tissue microtome, fresh tumor tissue is cut into 200-micrometer thick sections.

[0210] In S3, thin sections of biological tissue are laid on a mechanical chip and cultured for one day to allow the tissue to fully adhere to the chip. It is known that the cellular mechanical force of tumor cell blocks is stronger than that of non-tumor blocks.

[0211] S4. Add the antibody-drug conjugate to the culture medium.

[0212] S5. By detecting the intensity of the light reflection signal, the cellular mechanical force of tumor cell blocks and non-tumor cell blocks is monitored in real time. When the antibody-drug conjugate effectively inhibits cell growth or induces apoptosis, the cellular mechanical force decreases significantly.

[0213] S6. Furthermore, by comparing the changes in cellular mechanical forces between tumor cell blocks and non-tumor cell blocks, it is possible to determine whether the antibody-drug conjugate can specifically kill tumor cells and to evaluate its effects on normal cells.

[0214] This embodiment provides important evidence for evaluating the therapeutic effect of antibody-drug conjugates on tumor cells by monitoring changes in cellular mechanical forces in biological tissues.

[0215] Example 32 Method for fabricating a type of multimodal biophysical characterization device This embodiment provides a method for fabricating a type of multimodal biophysical characterization device, which achieves the reflection of optical signals by coating the tops of micro-nanopillars with a metallic reflective layer.

[0216] S1, a single layer of photoresist is spin-coated onto the micro-nanopillar array. This step protects the sides of the micro-nanopillars and prevents metal deposition in subsequent processing steps.

[0217] S2. The tops of the micro-nanopillars are exposed using oxygen plasma etching technology. This step ensures that the metal coating is applied only to the tops of the micro-nanopillars and does not affect the sides.

[0218] S3. A metal coating is deposited using an electron beam deposition apparatus. This step forms a metallic reflective layer on the top of the exposed micro-nanopillars, enabling the reflection of optical signals. If a magnetic metal is used, a magnetic metal reflective layer can be formed on the top of the micro-nanopillars, enabling both the reflection of optical signals and a response to magnetic fields. The process can be repeated multiple times to deposit coatings of different metals or with different functions (Figure 30).

[0219] S4. The photoresist is removed. This step removes any remaining photoresist and ensures that the metal layer covers only the tops of the nanopillars. The fabricated chip samples are then characterized using a scanning electron microscope to confirm that the metal coating has been successfully applied (Figure 30).

[0220] Through the above fabrication process, we can obtain micropillar tips having a metal coating or magnetic metal top, which can be used to achieve reflection and detection of optical signals and response to magnetic fields.

[0221] Example 33 Method for converting the deformation of a micropillar and the applied force. This embodiment provides a method for converting deformation and the applied force, based on the deformation of the micropillar and Hooke's Law, and taking into account the effect of deformation at the bottom of the micropillar.

[0222] S1, calculate the ideal spring constant kbend for the micropillar: kbend=(3 / 64)*π*E*D4 / L3 Here, E is Young's modulus, D is the diameter of the micropillar, and L is the length of the micropillar.

[0223] Calculate the gradient coefficient Ttilt(ν) for S2: Ttilt(ν)=a*(1+ν) / (2*π)*{2*(1-ν)+(1-1 / (4*(1-ν)))} Here, ν is Poisson's ratio and a is the correction factor.

[0224] S3, calculate the deformation proportionality factor a: a=δbend / (δbend+δshear+δtilt) Here, δbend is the bending deformation of the micropillar, δshear is the shear deformation, and δtilt is the tilt deformation of the bottom of the micropillar.

[0225] S4. Calculate the corrected spring constant kcorr: kcorr=kbend×a S5, Calculate the force F acting on the micropillar: F = kcorr × δ Here, δ is the deformation of the micropillar.

[0226] S6, calculate the deformation δtilt at the top of the micropillar (Figure 30): δtilt=8*Ttilt(ν)*(L / D)2*(4 / π)*F / ED Using the calculation method described above, we can take into account the effect of deformation at the base of the micropillar and calculate the force acting on the micropillar more accurately.

[0227] Example 34 Calibration method for a multimodal biophysical characterization system using a flow field This embodiment aims to provide a method for calibrating a multimodal biophysical characterization system using a flow field.

[0228] S1, the PDMS micropillars have a metallic reflective coating on the top and an anti-reflective coating between and on the sides of the micropillars.

[0229] S2. This micropillar tip is laid at the bottom of the microfluidic pipe (Figure 32).

[0230] In S3, fluids of different flow velocities are injected into the inlet of the microchannel, and laser or visible light is shone onto the chip from below the device. Simultaneously, the light reflection signal generated at the top of the micropillar is measured below the device. The faster the flow velocity, the greater the curvature of the micropillar, and the weaker the light reflection signal. Experiments have shown that within a certain deformation range, there is a linear relationship between the flow velocity and the light reflection signal (Figure 31). Therefore, the fluid state can be calculated from the strength of the light reflection signal generated by the deformation of the micropillar.

[0231] This embodiment provides a method for measuring the flow field and calibrating the flow velocity using reflected light from a micropillar sensor. Within a certain deformation range, there is a linear relationship between deformation and the attenuation of the reflected light signal, and the fluid state can be calculated from the strength of the reflected light signal caused by the deformation of the micropillar.

[0232] Example 35 Characterization of immune-tumor cell interactions and evaluation of drug intervention effects using a multimodal biophysical characterization system. This embodiment aims to provide information on the relationship between the physical properties of tumor cells and their interaction with immune cells, and the impact of drug intervention on this relationship.

[0233] S1. Chip preparation: Select a cell mechanics chip with a micropillar structure, and apply an appropriate extracellular matrix (ECM) to the top of the micropillars to promote adhesion and growth of tumor cells and immune cells. Apply an anti-adhesion treatment to the sides of the micropillars and between the micropillars.

[0234] S2. Seeding of tumor cells and immune cells: Tumor cells and immune cells (e.g., NK cells or T cells) are seeded on a mechanical chip and allowed to interact with each other.

[0235] S3. Monitoring the mechanical properties of tumor cells: Using light reflection signals and micropillar deflection, we monitor changes in the cellular mechanical force and stiffness of tumor cells during their interaction with immune cells in real time.

[0236] S4. Observation of the killing effect of immune cells on tumor cells: A positive correlation was found between the mechanical size and hardness of tumor cells, and softer tumor cells (smaller cellular mechanical force) were less likely to be recognized and killed by immune cells.

[0237] S5. Drug intervention: Add the drug JAS, which scleroses tumor cells, to the culture medium and observe the changes in the cellular mechanical force and stiffness of the tumor cells.

[0238] S6. Evaluation of the effects of drug intervention: Under the action of the drug, tumor cells hardened and their cellular mechanical strength increased (Figure 32). The killing effect of immune cells on tumor cells was observed to be enhanced accordingly.

[0239] Summary: This embodiment demonstrates how to characterize immune-tumor cell interactions using a cellular mechanics chip and the impact of drug intervention on this relationship. Studying the mechanical properties of tumor cells can provide a basis for optimizing immunotherapy strategies.

[0240] Example 36 A Multimodal Lung Organ Chip and Method of Using the Same This example aims to provide a multimodal lung organ chip and method of using the same. Cells extracted from normal and tumor lungs are seeded on a system with a double-sided sandwich structure. The base of the chip is made of a flexible material and can simulate the breathing motion of the lung through deformation caused by inflation and deflation. During the process of applying a chemotherapy drug stimulus, changes in cell mechanical force and hardness are monitored using the deflection of micropillars by light reflection signals and magnetic field induction, the killing effect on tumors and the impact on normal cells are measured, and the targeting of the drug is evaluated.

[0241] S1. Prepare a double-sided structure system. The base of the chip is made of a flexible material. Coat the extracellular matrix (ECM) on the top end of the micropillar, and perform an anti-adhesion treatment, such as applying Pluronic F127, on the sides of the micropillar and between the micropillars.

[0242] S2. Seed normal lung cells on one side of the chip and lung tumor cells on the other side, and completely adhere the cells to the chip.

[0243] S3. Place the double-sided chip in a device capable of deformation by inflation and deflation to simulate the breathing motion of the lung. Furthermore, the curved base also simulates the alveolar space in the body and provides a curved adhesion microenvironment for the cells or tissues.

[0244] S4. Add a chemotherapy drug to stimulate the normal lung cells and lung tumor cells. During the stimulation process, utilize the light reflection signal to monitor the interaction between the cells and the micropillars, thereby detecting changes in cell mechanical force in real time. Induce the deflection of the micropillars using a magnetic field, measure the force exerted by the cells on the micropillars, and further evaluate the hardness and dynamic changes of the cells.

[0245] In S5, the deflection data of micropillars induced by light reflection signals and magnetic field induction will be analyzed to compare the effects of chemotherapeutic drugs on normal lung cells and lung tumor cells, measuring the tumor-killing effect and the effect on normal cells. Based on the experimental results, the targeting potential of chemotherapeutic drugs will be evaluated, providing a basis for drug screening and research.

[0246] Example 37: Distinction between tumor and normal tissue and evaluation of drug treatment effects by physical spatial omics monitoring of biological tissue sections. This embodiment presents a type of multimodal tissue physical characterization chip and a method for using it. By monitoring the spatial omics features of the mechanical properties of biological tissue sections, it is possible to distinguish between tumor and normal tissue regions and evaluate the effects of drug treatment.

[0247] S1. Preparation of living tissue sections: A living tissue sample (including tumor and normal tissue) is obtained and prepared into thin sections.

[0248] S2. Measurement of the mechanical properties of tissue sections: The mechanical properties and hardness distribution of tissue sections are measured using an atomic force microscope (AFM) or similar technique.

[0249] S3. Differentiation between tumor and normal tissue: Measuring the mechanical properties of tissue sections: Monitoring mechanical force using the light reflection signal of a mechanical tip, activating a magnetic field to measure the deflection of micropillars embedded in the tissue to determine hardness, and verifying this with an atomic force microscope (AFM) or similar technique.

[0250] S4. Drug treatment: Antitumor drugs are added to the tissue sections and treated for a set period of time.

[0251] S5. Evaluation of drug treatment effect: After drug treatment, changes in mechanical force and hardness at the tumor site were measured. A clear decrease in mechanical force in the tumor area and a more uniform visualization were observed (Figure 34). On the other hand, no clear decrease in hardness was observed, indicating that the response speed of mechanical force was faster than that of hardness.

[0252] Summary: This embodiment demonstrates how monitoring the spatial omics of the mechanical properties of biological tissue sections can be used to differentiate between tumor and normal tissue regions and evaluate the effectiveness of drug treatment. This method may provide a basis for early tumor diagnosis and evaluation of drug therapeutic efficacy.

[0253] It should be explained that although the embodiments described above have already been stated in the main text, this does not limit the scope of the patent protection of this application. Therefore, any changes and modifications made to the embodiments described in the main text, or equivalent structural or process transformations made using the contents of the specification and accompanying drawings of this application, or any application of the above technical solutions directly or indirectly to other related technical fields, are all included within the scope of the patent protection of this application. [Explanation of symbols]

[0254] 1. Multimodal biophysical characterization apparatus, 11. Base, 12. Micropillar, 120. Bottom end, 121. Side view, 122. Column, 123. Top end, 13. Light-reflecting layer, 131. Magnetic material, 132. Magnetic metal light-reflecting layer, 14. Anti-reflective layer, 15. Restricting surface, 16. Substance that interacts with cells, 17. Substance with anti-cell adhesion properties, 18. Fluorescent substance.

Claims

1. A multimodal biophysical characterization apparatus, Base and, The system includes a micropillar or micropillar array consisting of one or more micropillars, which are mounted on the base and capable of undergoing a change of state in response to cellular mechanical forces and / or magnetic forces, The multimodal biophysical characterization apparatus is characterized in that the micropillar includes a bottom end connected to a base, sides, a columnar body enclosed by the sides, and a top end far from the base and facing the bottom end, a light-reflecting layer is installed at the top end, sides, and / or at any position within the columnar body of the micropillar, a magnetic material is installed at the top end, sides, and / or at any position within the columnar body of the micropillar, and preferably a magnetic metallic light-reflecting layer is installed at the top end of the micropillar.

2. The multimodal biophysical characterization apparatus according to claim 1, characterized in that a light-reflecting layer is provided on the top end of the micropillar, and an anti-reflective layer is provided on the side surface and / or base surface of the micropillar.

3. The multimodal biophysical characterization apparatus according to claim 1, further comprising a plurality of cell restriction structures installed on the base, wherein the cell restriction structures include one or more restriction surfaces, the restriction surfaces are planar or curved, the restriction surfaces are perpendicular to the plane on which the base is located, are connected to the base or integrally molded with the base, and the height of the restriction surfaces is greater than that of the micropillars and encloses a predetermined number of micropillars inside.

4. A substance that interacts with cells is placed on at least one of the base, micropillar, and limiting surface, and the substance that interacts with cells is, A multimodal biophysical characterization apparatus according to any one of claims 1 to 3, characterized in that it includes one or a combination of one or more substances selected from among a substance that promotes cell adhesion, a substance that interacts with cell surface receptors, a bioactive substance, and a bioinactivating substance.

5. The multimodal biophysical characterization apparatus according to claim 4, characterized in that an anti-cell adhesion substance is installed on at least one of the base, micropillar, and limiting surface.

6. The substance that interacts with the cell is placed on the top of the micropillar, and the plurality of micropillars on which the substance that interacts with the cell is placed form a first predetermined pattern. and / or The multimodal biophysical characterization apparatus according to claim 5, characterized in that the anti-cell adhesion substance is placed at the top of the micropillar, and the plurality of micropillars on which the anti-cell adhesion substance is placed form a second predetermined pattern.

7. The multimodal biophysical characterization apparatus according to claim 1, characterized in that a fluorescent substance is installed in the extension from the top to the bottom of the micropillar.

8. The multimodal biophysical characterization apparatus according to claim 1, characterized in that the base and / or micropillars are made of a conductive material.

9. A multimodal biophysical characterization system, A multimodal biophysical characterization apparatus according to one or more claims 1, A light signal emitting device for emitting a predetermined ray, An optical signal detection device for detecting light rays reflected from the aforementioned light reflection layer, The system includes a magnetic field generating device for generating a magnetic field and causing a magnetic force interaction with the magnetic material, A multimodal biophysical characterization system characterized in that the light rays emitted by the optical signal emitter pass through the incident optical path and are irradiated onto the optical reflection layer, and the light rays reflected by the optical reflection layer enter the optical signal detection device via the reflected optical path.

10. The multimodal biophysical characterization system according to claim 9, further comprising an optical signal analyzer, wherein the optical signal analyzer is used to analyze an optical signal.

11. The multimodal biophysical characterization system according to claim 10, further comprising a data processing device, the data processing device being used to process the optical signal to obtain results and spatial distribution of cellular mechanical force and / or cellular hardness.

12. The multimodal biophysical characterization system according to claim 9, further comprising a pressure transducer, the pressure transducer being used to apply pressure to a cell.

13. The multimodal biophysical characterization system according to claim 9, characterized in that the optical signal emitting device has a light source, the light source includes a first light source installed at the bottom of the base and / or a second light source installed on the side of the base and / or a third light source installed at the top end far from the base and opposite the bottom end, and the light rays emitted by the first light source and / or the second light source and / or the third light source reach the micropillar.

14. The multimodal biophysical characterization system according to claim 9, further comprising a device for inducing motion or deformation, the device for inducing motion or deformation being used to cause deformation in the multimodal biophysical characterization system and to apply mechanical force to the multimodal biophysical characterization system, preferably the device for inducing motion or deformation comprising a tensioning device and / or a charging / exhausting device.

15. The multimodal biophysical characterization system according to claim 9, characterized in that a bilateral or polyhedral structure is formed by two or more of the multimodal biophysical characterization devices, the outer part of the bilateral or polyhedral structure is the base, and the inner part surrounds and forms a three-dimensional cell containment cavity.

16. The multimodal biophysical characterization system according to claim 15, characterized in that the cell restriction structure is compatible when a double-sided or multifaceted structure is constructed using two or more of the multimodal biophysical characterization devices.

17. A method for performing physical characterization of cells using the multimodal biophysical characterization system described in claim 9, The steps include: emitting a predetermined light ray using the aforementioned optical signal emitting device; A method characterized by comprising the step of detecting the light beam after the action of the multimodal biophysical characterization apparatus using the optical signal detection device.

18. The method further includes the step of generating a magnetic field of a predetermined direction and intensity using the magnetic field generating device, Optionally, the process may further include the step of applying different types and intensities of stimuli to the sample being characterized before, during, or after physical characterization. The stimulus is optionally selected to be at least one or a combination of one or more physical, chemical, and biological stimuli. Optionally, the physical, chemical, and biological stimuli include one or more combinations of stimulus types from among drugs, mechanical force, hardness, biochemistry, electric fields, flow fields, chemotactic induction, and radiation. Optionally, the process may include steps of labeling, fixing, ablating, cutting, extracting, or sorting specific locations or regions of the sample to be characterized, before, during, or after physical characterization. The method according to 17, characterized in that, optionally, comparative analysis is performed before, during, or after physical characterization by a method including protein staining, histochemical staining, or single-cell sequencing.

19. The optical signal analysis step further includes comparing and analyzing reflected light rays before and after a fluid flows through a multimodal biophysical characterization device using the optical signal analyzer, and / or comparing and analyzing reflected light rays before and after a magnetic field generator generates a magnetic field of a predetermined direction and intensity using the optical signal analyzer, in order to obtain information on the cellular mechanical force and / or cellular stiffness of the sample to be characterized. The method according to 18, wherein the cellular mechanical force information includes the magnitude, direction, frequency, distribution, and dynamic changes over time of the cellular mechanical force at a specific location in a cell or multicellular aggregate, and the cellular hardness information includes the magnitude, spatial distribution, and dynamic changes over time of the hardness of different layers at a specific location in a cell or multicellular aggregate.

20. Applications of the multimodal biophysical characterization apparatus according to claim 1 in the fields of synthetic biology, cell therapy, drug discovery, characterization of intercellular interactions, early tumor screening, or precision medicine, wherein the application is, An application comprising obtaining at least one of the magnitude, spatial distribution, and dynamic changes of cellular mechanical force and cellular stiffness through the multimodal biophysical characterization apparatus.

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