Systems and methods for in vitro mechanical testing

Abstract

In vitro systems and methods can be used to detect one or more mechanical properties of one or more samples. A rotator can be configured to rotate around a rotational axis to produce centrifugal force that deforms at least one sample located in a deformation area. The sample can be pushed towards a deformation wall that is perpendicular to direction of the centrifugal force. At least one measurement sensor can be configured collect data related to shape changes of the at least one sample as it deforms or relaxes after deformation to reveal the one or more mechanical properties. The one or more mechanical properties are related to material sciences, life sciences, pharmaceutical sciences, or drug screening.

Description

NONPROVISIONAL APPLICATIONSYSTEMS AND METHODS FOR IN VITRO MECHANICAL TESTINGGOVERNMENT SUPPORT

[0001] This invention was made with government support under 5R01 HL145031 -04 and 5R01AR081529-02 awarded by the National Institutes of Health. The government has certain rights in the invention.Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 685,998, filed August 22, 2024, entitled “MECHANICAL TESTING PLATFORMS FOR TISSUES AND MATERIALS”. The entirety of this provisional application is hereby incorporated by reference for all purposes.Technical Field

[0003] This disclosure relates generally to mechanical testing of samples, and more specifically to systems and methods that can use mechanical testing in vitro to detect one or more mechanical properties of one or more samples.Background

[0004] Mechanical properties, also referred to as mechanical phenotypes or mechanotypes, can be studied with mechanical testing in the fields of material sciences, life sciences, or drug screening. As an example, mechanical testing can reveal changes in tissue mechanical properties, which have been shown to be reliable indicators of cumulative injuries in fibrotic diseases, aging, and cancers and those resulting from drug-induced secondary (side) effects. Accordingly, the changes in tissue mechanical properties have increasingly been used clinically (also referred to as mechanotyping) for non-invasive differential diagnosis. However, mechanotyping has not been used for in vitro drug efficacy and safety screening. This stems from a lack of “fit-for-purpose” platforms for high-throughput, tissue mechanotyping. Bringing a drug to market now costs an estimated $1 -2.8 billion, with late-stage clinical failures mainly driven by efficacy and safety issues. This underscores the need for improved in vitro drug screening platforms to identify unsuccessful candidates early.

[0005] Additionally, changes in one or more mechanical properties (e.g., stiffness) have been shown to indicate the presence and / or progression of many medical conditions. In fact, stiffness has become a non-invasive diagnostic marker for disease state that is studied in vivo using imaging modalities like ultrasound (US) and magnetic resonance (MR). Stiffness remains overlooked and completely untapped for in vitro drug screening and the study of diseases themselves due in large part to difficulties measuring stiffness in long-term, high-throughput experiments. Current in vitro studies, including atomic force microscopy and rheoology, are not easy to use, lack easy routes for repeated measurements, have a low throughput, and a relatively expensive. Additionally, US and MR, while useful in vivo studies, cannot be used with in vitro tissues because of challenges regarding spatial resolution and measurement noise.Summary

[0006] Described herein are systems and methods that can use mechanical testing in vitro to detect one or more mechanical properties of one or more samples.

[0007] In an aspect, the present disclosure can include an in vitro system to detect one or more mechanical properties of one or more samples. The system can include a rotator configured to rotate around a rotational axis to produce centrifugal force that deforms at least one sample located in a deformation area. The sample is pushed towards a deformation wall that is perpendicular to direction of the centrifugal force. At least one measurement sensor can be configured collect data related to shape changes of the at least one sample as it deforms or relaxes after deformation to reveal the one or more mechanical properties. The one or more mechanical properties are related to material sciences, life sciences, or drug screening.

[0008] In another aspect, the present disclosure can include a method for detecting one or more mechanical properties of one or more samples in vitro. The method can include rotating a rotator around a rotational axis at a rotational speed to produce a centrifugal force; causing at least one sample in a deformation area on the rotator to deform, wherein the at least one sample is pushed towards a deformation wall that is perpendicular to a direction of the centrifugal force; and collecting data related to shape changes of the at least one sample as it deforms or relaxes after deformation to reveal the one or more mechanical properties. The one or moremechanical properties are related to material sciences, life sciences, or drug screening.Brief Description of the Drawings

[0009] The foregoing and other features of the present disclosure will become apparent to those skilled in the art to which the present disclosure relates upon reading the following description with reference to the accompanying drawings, in which:

[0010] FIG. 1 is a block diagram of a system that can use mechanical testing in vitro to detect one or more mechanical properties of one or more samples;

[0011] FIG. 2 is a block diagram of an example of a sample located within a deformation area of the rotator of FIG. 1 when there is no force @t0 and when there is force @t1 ;

[0012] FIG. 3 is a process flow diagram of a method for using mechanical testing in vitro to detect one or more mechanical properties of one or more samples;

[0013] FIG. 4 is an illustration of the centrifugal mechanical testing (CeMeT) platform assembly;

[0014] FIG. 5 is an illustration of the design and fabrication details of microchips and the disc for the CeMeT platform;

[0015] FIG. 6 is an illustration of accuracy, precision, and error on the CeMeT platform;

[0016] FIG. 7 shows a working mechanism of CeMeT for mechanotyping of 3D cardiac organoids with experimental results;

[0017] FIG. 8 shows mechanical characterization of hydrogel beads of different densities;

[0018] FIG. 9 includes representative images showing hydrogel beads before, during, and after the experiments;

[0019] FIG. 10 is a comparison of experimental and numerical simulation results;

[0020] FIG. 11 shows mechanotyping of human hiPSC-derived cardiac organoids;

[0021] FIG. 12 shows the relationship between deformation and estimated Young’s modulus;

[0022] FIG. 13 is a comparison of the deformation of healthy cardiac organoids of three different sizes;

[0023] FIG. 14 shows the size-dependent variation in Young’s modulus of cardiac organoids measured using the CeMeT platform;

[0024] FIG. 15 shows mechanotyping of cardiac organoids to diagnose the adverse side effects of pergolide;

[0025] FIG 16 shows how normalized roundness changes differently in control and pergolide-treated groups upon increasing RCF;

[0026] FIG. 17 is an illustration of the working principle of the CeMeT platforms;

[0027] FIG. 18 is an illustration of the CeMeTLS platform; and

[0028] FIG. 19 is an illustration of a multi-ring high-throughput CeMeTDS design for differential RCF analyses and GPU integration.Detailed DescriptionI. Definitions

[0029] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0030] As used herein, the singular forms “a,” “an,” and “the” can also include the plural forms, unless the context clearly indicates otherwise.

[0031] As used herein, the terms “comprises” and / or “comprising,” can specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups.

[0032] As used herein, the term “and / or” can include any and all combinations of one or more of the associated listed items.

[0033] As used herein, the terms “first,” “second,” etc. should not limit the elements being described by these terms. These terms are only used to distinguish one element from another. Thus, a “first” element discussed below could also be termed a “second” element without departing from the teachings of the present disclosure. The sequence of operations (or acts / steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

[0034] It will be understood that when an element is referred to as being "on," "attached" to, "connected" to, "coupled" with, "contacting," etc., another element, itcan be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, "directly on," "directly attached" to, "directly connected" to, "directly coupled" with or "directly contacting" another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0035] As used herein, the term “platform” refers to a setup to measure one or more mechanical properties, also referred to as “mechanical phenotypes” or “mechanotypes”, of a sample. The one or more mechanical properties of the sample can be related to material science, life sciences, pharmaceutical sciences, and / or drug screening.

[0036] As used herein, the term “material science” refers to the study of the structure, properties and behavior of one or more materials.

[0037] As used herein, the term “life sciences” refers to the study of living organisms. Specifically, while studying living organisms, scientists can investigate disease, aging, and injury progression / regression.

[0038] As used herein, the term “pharmaceutical sciences” refers to the study of chemicals compounds used as active pharmaceutical ingredients; specifically, the effects of the chemical compounds on living organisms.

[0039] As used herein, the term “time independent mechanical properties” refer to properties of materials that describe their immediate response to an applied load and does not depend on how long the load is applied or the rate at which it is applied; e.g., Young’s modulus (stiffness), yield strength, ultimate tensile strength, hardness, and toughness.

[0040] As used herein, the term “time dependent mechanical properties” refer to properties of materials that describe how materials’ responses to applied loads change with the duration of loading, the rate of loading, or repeated loading cycles; e.g., creep, stress relaxation, fatigue, and viscoelasticity.

[0041] As used herein, the term “drug screening” refers to a preclinical identification of drug candidates.

[0042] As used herein, the term “sample” refers to one or more one, two, and / or three dimensional structures of synthetic, natural, and / or living material.

[0043] As used herein, the term “rotator” refers to a geometry that has at least one deformation area to hold at least one sample. The rotator rotates around a rotational axis at a rotational speed (e.g., in RPM) and generates a force that deforms and / or compresses the sample against a deformation wall. One example force that can be generated is centrifugal force. However, other forces can be generated (e.g., inertial force if the samples are deformed controllably).

[0044] As used herein, the term “deformation area”, also referred to as a “compression area”, refers to a location on the rotator where a sample is held, deformed, and / or compressed. In some instances, the deformation area can be connected to one or more channels and / or one or more in lets / outlets via tunnels to feed the sample with media and other substances. The deformation area can have one or more walls in any regular or irregular shape (e.g., rectangle, circular, etc., or a combination of thereof, as well as any three-dimensional geometry).

[0045] As used herein, the term “channel” refers to connecting tunnels between a reservoir (or inlet / outlet) storing the sample and the deformation area and / or deformation wall. The channel can be a microchannel where at least one of the characteristic lengths is less than a millimeter.

[0046] As used herein, the term “deformation wall”, also referred to as a “wall”, “deformation plate” or “compression plate”, refers to one of the walls of the deformation area, perpendicular to the force, that the sample within the deformation area can deform again due to the force.

[0047] As used herein, the term “hurdle” refers to any shape that changes the deformation of the sample within the deformation area and / or a channel leading to the deformation area. The hurdle can be, but is not limited to, a tip, a pointy tip, a round tip, a pin, a pillar, and / or a geometry that changes a cross-sectional area of the sample.

[0048] As used herein, the term “hole” refers to a hollow place in the deformation are and / or channel leading to the deformation area of any geometry or shape.

[0049] As used herein, the term “motor” refers to a machine, tool, equipment, etc., that can rotate the rotator as governed by a controller.

[0050] As used herein, the term “brake” refers to a mechanism and / or device to stop and / or pause the rotator. The brake can stop and / or pause the rotator upon instruction from the controller.

[0051] As used herein, the term “controller” refers to one or more devices that regulate the rotator’s rotational speed and / or cyclic motion’s frequency. The controller can be run electrically, but alternatively or additionally can be a mechanical controller that uses spring torsion and / or gravity.

[0052] As used herein, the term “measurement sensor” refers to one or more devices that collect data related to shape changes of at least one sample as it deforms or relaxes after deformation to reveal the one or more mechanical properties. In some instances, analysis can be done by the sensor. In other instances, analysis can be done by another device, which can be remote from the sensor.II. Overview

[0053] In the fields of material sciences, life sciences, pharmaceutical sciences, and / or drug screening, platforms for robust, in vitro mechanotyping are absent from the market despite a pressing need for their use (e.g., in preclinical use and / or secondary safety testing). Described herein are in vitro mechanotyping platforms that measure mechanical properties of one or more samples and can be used in the fields of material sciences, life sciences, pharmaceutical sciences, and / or drug screening. The platforms can each have a rotator configured to generate a force. For example, the rotator can rotate around a rotational axis to produce centrifugal force. The force can deform at least one sample located in a deformation area on the rotator. The sample can be pushed towards a deformation wall that is perpendicular to direction of the force and deform against the deformation wall. At least one measurement sensor can collect data related to shape changes of the at least one sample as it deforms or relaxes after deformation to reveal the one or more mechanical properties (which may be related to material sciences, life sciences, or drug screening.III. Systems

[0054] There is a pressing need for systems (also referred to as platforms) for robust, in vitro mechanotyping in the fields of material sciences, life sciences, and / or drug screening. Described herein are in vitro mechanotyping platforms (shown, for example in FIG. 1 ) that measure mechanical properties of one or more samples and can be used in the fields of material sciences, life sciences, pharmaceuticalsciences, and / or drug screening._FIG.1 illustrates an example of a system 100 that can use mechanical testing in vitro to detect one or more mechanical properties of one or more samples. It should be understood that the example shown in FIG. 1 is simply for ease of illustration and other examples / configurations are contemplated. It should be understood that unless otherwise stated, elements of the systems described herein generally operate as widely known in related fields.

[0055] The system 100 can include a rotator 102, a geometry that can be configured to rotate around a rotational axis (also referred to as the center of rotation or rotation center) at a rotational speed. The rotator 102 can be a disc of any shape and / or size that is able to rotate at the rotational speed. In some instances, the disc can be symmetrically shaped. The rotational speed of the rotor 102 can be a number of revolutions in a defined time course. For example, the rotational speed of the rotator 102 can be from 0.01 revolutions per minute (RPM) and 5000000 RPM. As another example, the rotational speed of the rotator 102 can be from 1 RPM to 1000000 RPM. Although not illustrated, one or more motor can be used to drive the rotator to rotate around the rotational axis (located at the center of rotation) at the rotational speed. The motor can be a machine, tool, equipment, or the like, that can rotate or apply cyclic motion to the rotator 102. The motor can be operated according to electrical, piezoelectric, magnetic, mechanical, manual, or the like, methods. In some instances, the motor can be controlled by one or more controllers (not illustrated), which may be electrical and / or mechanical (using spring torsion and / or gravity). For example, the controller can regulate rotation of the rotator 102 (e.g., speed, frequency, or the like). Although not illustrated, in some instances, rotation of the rotator 102 can be stopped by one or more brakes. The brake can stop and / or pause the rotator within a time (0.001 microseconds to 1 hour) that may be controlled by the controller but can be manually applied. In other words, the brake can operate according to electrical, mechanical, and / or manual control.

[0056] In some example, a holder device (not shown) can clasp at least the rotator 102 to a platform (not shown) that facilitate use of the system 100. It should be understood that the rotator 102 can rotate freely under the excitement of the motor. The holder device can be made with three-dimensional printing, additive manufacturing, machining, molding, assembly, or the like. For example, the holder device can be a tool where the deformation area and / or wall is placed.

[0057] The rotation of the rotator 102 can generate and / or apply a force and / or pressure to one or more samples to deform the one or more samples. The force and / or pressure can cause the sample to engage in cyclic motion that causes the sample to follow one or more periodic trajectories. As an example, the force can be centrifugal force. It should be noted, however, that the force and / or pressure can be any type of force and / or pressure that can be applied to change a mechanical property of the sample when rotated on a rotator 102. The one or more samples can be of regular and / or irregular geometries of one, two, or three-dimensional structures. Example regular geometries include a sphere, prism, cube, cone, pyramid, or the like. Each sample can be a one, two, or three-dimensional material that is deformable under force and / or pressure. For example, each sample can include one or more cells, one or more tissue, one or more polymer beads, one or more composite materials, one or more hydrogels, one or more spheroids, one or more organs, and / or one or more organoids. Each sample can include one or more elastic, inelastic, viscoelastic, partially elastic, thermoplastic, thermoset, and / or plastic material. The material can be synthetic, natural, and / or living.

[0058] The rotator 102 can be configured to hold (or be a resting place for) one or more samples (included within in elements X and, in some instances, Y, but it should be illustrated that any number of samples are contemplated). There can be a pivotal distance between the axis of rotation and the location of the one or more samples. In some examples, every two samples located on the rotator 102 can have an equal and opposite pivotal distance. In some instances, the sample center can overlap or partially overlap the center of rotation. In other instances, the sample center and the center of rotation can be separate without overlapping. The force has a purpose of creating stress on the one or more samples to deform at least a portion of the one or more samples. The deformation can lead to mechanical characterization of one or more mechanical properties the sample and eventual mechanotyping.

[0059] The one or more mechanical properties can be related to material sciences, life sciences, and / or drug screening. The one or more mechanical properties can be static or dynamic. For example, the one or more mechanical properties include, but are not limited to, strength, compressibility, elongation, bending, hardness, elasticity, toughness, tensile, strength, yield strength, elongation, fatigue strength, corrosion, and / or plasticity. In some instances, the one or moremechanical properties can include and / or be related to stiffness, elasticity, and / or viscoelasticity. Examples of the mechanical property can include an elastic property, an inelastic property, a viscoelastic property, a partially elastic property, a thermoplastic property, a thermoset property, and / or a plastic property.

[0060] The sample can be located in a deformation area on the rotator 102 (illustrated as part of X 106 and, in some instances, Y) so that the sample can deform under application of a force and / or pressure. Generally the term deformation area refers to the area where deformation or compression of the sample occurs. The deformation area can have one or more walls (also referred to as deformation walls, compression plates, or the like) (illustrated as part of X 106 and, in some instances, Y) that the sample can touch during deformation. The walls can be arranged in any regular and / or irregular shape arranged in any three-dimensional geometry. In some instances, the walls must be perpendicular to the direction of the force (e.g., centrifugal force). Examples of regular shapes can include rectangle, circle, rectangular prism, other prisms, sphere, disk, cylinder, cube, cuboid, polyhedral, cone, pyramid, or the like.

[0061] It should be noted that the force pushes the sample towards the wall and the sample is deformed based on the force pushing the sample into the wall. In some instances, the deformation area can have one or more hurdles and / or one or more holes. Hurdles, also referred to as deformation hurdles, refers to any shape (e.g., a tip, a pointy tip, a round tip, a pin, a pillar, and / or a geometry that changes a cross- sectional area of the sample) that can change the cross-sectional area and / or the deformation more generally of the sample. The size (height, length and / or width) of the hurdle geometry can differ between 1 nm and 1 m in any dimension. Moreover, the number of hurdles can be from 1 to 1000000 in a single portion of the deformation area, more specifically, 5 to 10000, even more specifically, 7 to 500. Holes, also referred to as deformation holes, can refer to any geometry or shape taken out of the wall that allows the sample to be deformed under the force. The size (e.g., diameter, side length, other characteristic length, etc.) of a hole can be from 1 pm to 1 meter. The number of holes can be from 1 to 1000000, more specifically from 1 to 500, and even more specifically from 1 to 10.

[0062] As an example, the rotator 102 can include a symmetrical shaped mechanism, which in some instances can be shaped like a disc, a rectangular prism,and / or a polygonal prism. The symmetrical shaped mechanism can be configured to rotate around the center of rotation at the rotational speed to generate a force. In this example, the force can be a centrifugal force. However, the force may be inertia if the samples are deformed controllably. In this example, the rotator 102 also includes at least one chip mounted on the rotator. The at least one chip can be configured to hold the sample in the deformation area with the wall. The chip can include soft, stiff, opaque, transparent, or the like, sub-parts. The sub-parts can be at various locations when placing multiple of them on chip to apply varying forces to samples so that more tests may be done using a single setup.

[0063] The sub-parts can include a channel, a microchannel, a milichannel, a deformation area, one or more inlets, one or more outlets, or the like. The terms inlet and outlet can refer to a reservoir that introduces the samples, media, and / or other substances, such as drugs, molecules, dyes, vitamins, minerals, etc. The inlet / outlet design can vary (e.g., circles with diameters of 100 pm to 1 m, rectangles / squares with side lengths of 100 pm to 1 m). Other geometric shapes, such as triangles, trapezoids, hexagons, polygons, and different regular and irregular geometries may be utilized. The height of the inlets and outlets can be created by extruding the bottom geometry of the chip. The height can be any value from 1 pm to 1 m. All the bottom geometry and the extruded height can be extended to any regular and / or irregular three-dimensional geometry. The inlets and outlets can be considered reservoirs and may be used to introduce samples and media.

[0064] The terms channel, milichannel, and microchannel refer to lengths / sizes / shapes. A channel refers to the connecting tunnels that are connected to the deformation area and / or wall where the samples are deformed and / or one or more inlets / outlets. The channel can have reservoirs to introduce the sample, media, and / or other substances, such as drugs, molecules, dyes, vitamins, minerals, etc. It some instances, one or more channels can have one or more hurdles. Dimensions (e.g., length of the opening from the bottom to the top, area within, etc.) of the channel can be between 0.1 micrometers and 999 centimeters. The dimensions can be selected in some instances based on sample size and / or other measurement requirements. For example, the channels can be rectangular, circular, or the like. Microchannels and / or milichanels (microchannels have a characteristic length and / or diameter of less than 1 millimeter, milichannels have a characteristic length and / ordiameter less than 1 centimeter) can be connecting tunnels. In some instances, the wall can be at the end of the channel, milichannel, and / or microchannel. In other instances, the location of the wall can be independent of the location of the channel, milichannel, or microchannel.

[0065] FIG. 2 shows an example of the contents of element X 106 with the sample (S) being compressed as the rotator rotates. The sample (S) is shown at an initial time (tO) and then at a subsequent time (t1 ). At to, there is no rotation of the rotator and no force (F=0, as shown). The sample (S) is in the deformation area (DA) against the wall (DW). At t1 , the rotator rotates and a force is applied (F>0, as shown). The sample (S) is compressed against the wall (DW). The visible compression of the sample (S) can be detected and subsequently converted to the associated one or more mechanical properties.

[0066] Referring again to FIG. 1 , the system 100 can also include at least one measurement sensor 104. The measurement sensor 104 can include at least an imaging mechanism and a measurement mechanism. The imaging mechanism can collect analog and / or digital visual data from samples before, during, and after the measurement. The imaging can be done through imaging modalities including, for example, a camera, a high-speed camera, a fluorescence microscope, a confocal microscope, laser imaging (using a photodiode, for example), and the like. As an example, a photodiode can be a semiconductor diode that may be used to analyze photon counts in different samples, as well as borders and / or geometries when light (e.g., laser light) is applied thereto. The imaging data can reveal one or more physical changes (e.g., deformation, compression, or the like) to the sample, as well as recovery from the physical changes - together the physical changes and recovery can be referred to as shape changes. The visual data can be converted to a different data type (e.g., binary data) for further processing. The measurement mechanism can collect the data related to the shape changes (in its original and / or converted form) and based on the data can reveal the one or more mechanical properties. The measurement mechanism can perform the analysis and assessment of the shape changes. The measurement mechanism, for example, can be operated according to one or more of the following mechanisms: optical, interferometric, spectrometric, fluorometric, inductive, capacitive, magnetic, acoustic, or the like. The measurement data can be used for further analysis, including but not limited to size, aspect ratio,area, volume, mass density, viscosity, elasticity, stiffness, viscoelasticity, circularity, sphericity, roundness, and cylindricity.

[0067] In some instances, the measurement sensor 104 can include one or more sources. The one or more sources can generate triggering signals (also referred to as stimuli) on the samples. The sources can include one or more mechanical sources, electrical sources, magnetic sources, optical sources, acoustic sources, or the like. One example of an optical source can be a light source able to produce coherent and / or non-coherent light with a wavelength from 100 nm to 2000 nm. Another example of an optical source can include a laser source that can be dynamic and / or fixed to generate directed laser light with a wavelength from 100 nm to 2000 nm.

[0068] In other instances, the measurement sensor 104 can include one or more sensors. Each of the one or more sensors can be configured to collect data and create a signal to be analyzed for measurement purposes. The sensors can include but are not limited to CMOS (complementary metal-oxide-semiconductor) or CCD (charge-coupled device) sensors, optic sensors, acoustic / ultrasound sensors, magnetic sensors, inductive sensors, confocal sensors, laser sensors, capacitive sensors, mechanical sensors, and the like. CCD and CMOS cameras are known as high-speed cameras. High speed cameras can refer to and / or utilize sensors that can collect imaging data with exposure times from 0.01 ns to 1 second and a resolution from 0.01 megapixels to 1000 megapixels and a frame rate from 1 frame per second to 100 million frames per second.

[0069] The measurement sensor 104 can, in some instances, use both the one or more sources together with the one or more sensors. In instances where the measurement sensor 104 can use the one or more sources and the one or more sensors, the sources can use one or more objectives between the sources and sensors, The one or more objectives are optical devices that magnify signals (e.g., an optical signal) that include the samples. For example, the magnification can be greater than or equal to 1x. for example a value from 1x to 100x (but it should be noted that values greater than 100x are anticipated).

[0070] In some instances, a signal analyzer 108 can be used to reveal the one or more mechanical properties. The signal analyzer 108 can refer to any device that can be used to analyze one or more signals collected by the platform 100 during experiments. In these instances, an aspect of the measurement sensor 104 can becoupled to the signal analyzer 108. The signal analyzer can be configured to analyze the data to reveal the one or more mechanical properties. The signal analyzer 108, for example, can be a computing device that includes a processor or a microprocessor. The signal analyzer 108 can be connected to the measurement sensor 104, or another element of the platform 100, via a wired and / or wireless connection. Additionally, data can be transferred from the signal analyzer 108 and / or other parts of the platform 100 to a data storage location (e.g., cloud or database storage).

[0071] In one example, the system can include multiple light sources and camera that are positioned in fixed locations relative to the rotator. In this example, the cameras and the light sources can rotate together with the rotator. The integrated or mounted chips can hold at least one sample during deformation and the cameras can capture real-time images of the deformation and relaxation of the at least one sample, thereby enabling measurement of one or more time-dependent properties. In one example, a time-dependent property can include viscoelastic properties of the at least one sample.

[0072] In another example, the system can include a laser coupled with photodiode arrays and / or optical pickup units. The laser coupled with photodiode arrays or optical pickup units can be used as a measurement sensor, such that at least one sample is mapped bitwise to detect deformation. Signals from the photodiode arrays or optical pickup units are processed in real time to determine the position and the shape of the sample relative to a deformation wall.IV. Methods

[0073] Another aspect of the present disclosure can include a method 300 for using mechanical testing in vitro to detect one or more mechanical properties of one or more samples. The system 100 of FIG. 1 , and the example shown in FIG. 2, for example, can be used to execute the method 300. Unless otherwise stated, the methods described herein generally follow the usual practices widely known in related fields. Moreover, examples of how elements of the method can operate and / or what the elements can be are described in the Systems section above.

[0074] At 310, a rotator can be rotated around a rotational axis (also referred to as the center of rotation or rotation center) at a rotational speed. The rotator 102 can be a disc of any shape and / or size that is able to rotate at the rotational speed. The rotating can be based on a motor setting the rotational speed to a value between0.01 RPM and 500000 RPM. The rotation can produce (generate and / or apply) a force. At 320, at least one sample in a deformation area on the rotator to deform. In some instances, the deformation of multiple samples can occur.simultaneously on a symmetrically (around rotational axis of the rotator and / or the force axis in between samples) distributed two or more chips. For example, the deformation area can be on one or more chips. At 330, data related to shape changes of the at least one sample can be collected as the at least one sample deforms and / or relaxes after deformation to reveal one or more mechanical properties. The one or more mechanical properties can be related to material sciences, life sciences, and / or drug screening. The deformation of the at least one sample can be analyzed to reveal the mechanical property. At least a portion of the analyzing occurs via a computer program remote from the rotator.

[0075] In some instances, the force can be a centrifugal force. However, in some instances, the deformation can be due to a compression test and / or a compression-like test. With this test, the force can be a deformation or squeezing force applied to the one or more samples. The deformation and / or squeezing rate and the force amount can be correlated. In other instances, the force can be a tensile test, a tensile-like test, a tension test, and / or a tension-like test. The tensile test refers to an expansion and / or bulging of samples when tension is applied to samples.V. ExperimentalExperiment 1

[0076] The CeMeT platform represents a significant advance in 2D and / or 3D tissue mechanotyping, offering rapid and precise mechanical characterization of living tissues. Studies described herein demonstrate its capability to detect subtle mechanical changes in cardiac organoids and establish 2D and / or 3D tissue mechanotyping as a powerful approach for early detection of drug-induced off-target effects. Beyond this current application, the platform serves as a versatile foundation for investigating disease mechanisms and progression in 2D and / or 3D tissue models. This versatility facilitates a wide range of applications, from fundamental research to the development of therapeutics for conditions such as fibrotic diseases and cancer.Methods

[0077] CeMeT Platform: The Setup, Operation, and Data Acquisition

[0078] The CeMeT platform setup features microfluidic chips (referred to as “chips” herein) on a custom-made disc, which was then mounted on an optical table via a 3D-printed disc holder and two optical posts. A DC motor, connecting to a controller, rotates the disc (FIG. 4). The rotational speed was measured with an external tachometer. A microscope equipped with a high-speed camera was used for imaging the CeMeT chips during operation.

[0079] The chips were designed for optical clarity, sturdiness, and easy handling while avoiding air bubble trapping, and were made up of two layers: the top and the bottom layers (FIG. 5). The top layer features symmetric Y-shaped channels to remove air bubbles easily when injecting media and ensure symmetric force distribution over the chips during centrifugation. The compression plates, towards which the samples were compressed due to FCENT, were at the tip of Y-shaped chips (FIG. 5, photograph). The height and width of the compression plates were 1 .2 mm x 2 mm for organoids and 3 mm x 3 mm for hydrogel beads to prevent samples from contacting side walls while allowing samples to deform without constraint. The bottom layer was designed to provide a base for the top layer, and its thickness of 1 .5 mm was dictated by the nuts (h = 1 ,2mm) used for assembly of the two layers and the overall chip. The overall dimensions of the chips were 25 mm x 14.5 mm x 3.4 mm for organoids and 25 mm x 14.5 mm x 5.25 mm for hydrogel beads. Both layers were fabricated via stereolithography using a polycarbonate-like material (Accura 60), which allows the printing of fine details and provides optical clarity. A UV glue (AA 3926, Loctite) was deployed around the channel walls by capillary effect and hardened by applying UV light (RH-UV2000, Railhead Gear) in close vicinity for 5 min to avoid any medium leakage during the experiments under high RCF. The assembled chips provided high sturdiness due to the high stiffness (3500±400 MPa) of Accura 60, UV-glue bonding, and screw / nuts. The chips were fixed on the disc using a cover via screws and nuts.

[0080] The disc, diameter of 168 mm and houses up to 8 chips, was manufactured using Poly(methyl methacrylate) (PMMA) via computer numerical control (CNC) milling (FIG. 5). The distance from the compression plate of the chips to the center of rotation was 76.5 mm. The disc features cutouts to secure the chips and avoid sliding at high rotational speeds. A tachometer (TA150-NIST, Triplett) was used to measure the rotational speed of the disc using laser reflectance. The disc was painted black using spray paint (Rust-Oleum), and a strip of reflective tape wasapplied on the painted disc at a single location to accurately measure the rotational speed with the tachometer. A custom disc holder was designed and fabricated via 3D printing to make sure that the disc rotates freely, and that the compression plate area of each chip lies perfectly above the microscope objective.

[0081] The disc was rotated via a DC motor (775 Gear Motor, Puly), and the rotational speed was adjusted by modulating the voltage using a DC motor controller (RR-PWM-3A, RioRand). An inverted microscope (Zeiss Axiovert 200M, Carl Zeiss Microscopy) equipped with 10x and 4x objective lenses and a high-speed camera (Phantom v7.3 4800 was O, Vision Research) were employed for video and image acquisition. Videos and images were post-processed with Fiji (National Institute of Health) and a custom-made MATLAB code. Data was plotted using commercially available graphing and data analysis software (GraphPad Prism, GraphPad Software, Inc.) and an open-source programming language (RStudio programming language).

[0082] Image Analysis

[0083] A semi-automated analysis method was developed to post-process acquired images using a custom-made MATLAB code. The analysis was based on adaptive thresholding to detect the sample in each frame of the high-speed video recording. The method required the user input to manually mark five anchor points on the border of the samples, where zooming capability was provided to assist users with a more accurate selection of the anchor points. A 5x5 window was placed at each anchor point on the periphery of the sample. The 90th percentile of the pixel intensities from all five windows was used as the threshold for detecting the edges of the sample. An octagon was constructed based on the five anchor points and was used to select the region of interest and to mask out the dark region outside the sample (e.g., the chamber walls). The segmented sample was then processed with a morphological opening function followed by a flood-fill operation to fill any holes in the segmented region. The final segmented sample was computed as the convex hull of the segmented region.

[0084] Hydrogel Beads

[0085] Polyacrylamide (PAA) hydrogel beads can be synthesized using a monomer (acrylamide), a crosslinker (Bis-acrylamide), and redox initiators (APS: ammonium persulfate), and a catalyst (TEMED: tetramethylethylenediamine). Their stiffness can be adjusted from 0.3 kPa to 300 kPa by varying the monomer amountin the pre-polymer solution, making them highly versatile as testing materials for different stiffness requirements. Accordingly, three PAA hydrogel formulations were prepared with increasing monomer amounts while keeping the amount of other chemicals the same in the pre-polymer solution based on a previous study with slight modifications. The hydrogel formulations were named based on their density: HpZow Hpmed, and Hp / iig / i.

[0086] Specifically, acrylamide (Sigma Aldrich, A8887) was used as the monomer and was prepared as a stock solution at a concentration of 40% (w / v) and diluted to 30% (w / v) and 25% (w / v) as needed. N,N'-Methylenebisacrylamide (Bisacrylamide, Sigma Aldrich, M7279), the crosslinker, was prepared at a concentration of 2% (w / v). Ammonium persulfate (APS, Sigma Aldrich, 248614) and tetramethylethylenediamine (TEMED, Sigma Aldrich, T7024) were used as redox initiators and catalysts, respectively, with APS prepared as a stock solution of 25% (w / v). After mixing these solutions in the proper volumetric proportions, the prepolymer mixture was manually dripped into silicone oil (Millipore Sigma, 107742) as droplets (diameter of 1 .6 mm) using a syringe and needle. The droplets were incubated at room temperature for >1 hour to ensure full polymerization. Hydrogel beads were stored in silicone oil to prevent humidity exposure and water absorption, ensuring consistent mechanical properties during preparation and experimentation.

[0087] Rheometry

[0088] Hydrogel solutions of different monomer concentrations were injected into 3D-printed cylindrical molds using a 1 mL syringe and left to polymerize at room temperature for >1 hour. The Young’s moduli of these hydrogel samples (diameter of 20 mm, height of 3.5 mm) were then measured via a rheometer (Discovery HR20, TA Instruments) using the compression testing mode at a 10 pm / s step rate. The testing results showed a linear relationship for the stress-strain data for each hydrogel tested. Thus, the slopes of these stress-strain plots were considered as Young’s modulus for each hydrogel formulation.

[0089] Estimation of Young’s Modulus of the Samples: Hertz’s Theory of Contact

[0090] Hertz's theory of contact was employed to estimate Young’s modulus of elastic samples under compression. Hertz's theory of contact describes the mechanical behavior of elastic bodies when they come into contact under a load, providing a way to calculate deflection, contact area, and stress distribution thatoccur when two curved surfaces (such as spheres or cylinders) were pressed together. Hertz's theory of contact can, thus, be used to estimate the stiffness (Young’s modulus or Elastic modulus) of materials, particularly for elastic solids under small deformations compressed towards a rigid wall.

[0091] The effective radius was derived from the geometry of the contact area, while the elastic property comprises Young's moduli and Poisson's ratios of the interacting bodies, simplified due to the rigidity of one surface. Deflection was calculated from the axis length change of the sample, and these elements were integrated into a final equation to determine Young’s modulus. The analysis assumes small deformations and uses previously established formulations to connect these parameters for material characterization.

[0092] Elastic Recovery Testing

[0093] Elastic recovery testing characterizes the ability of a material to recover its original geometry after several cyclic loadings. Specifically, the minor axis of the sample, the axis perpendicular to the compression plate, was recorded before the deflection. A cyclic loading of 1500 RCF was performed ten times. Specifically, first, the load was applied for 30 seconds. Once the load was removed, the minor axis of the sample was measured again after a 2-minute recovery time. This procedure was repeated 10 times. The elastic recovery was calculated as Elastic Recovery = (Minor Axisaf ter / Minor AxiSbeforep \ 00.

[0094] Human pluripotent stem cell culture, generation, and maintenance of cardiac organoids

[0095] WTC-11 was a commercially available wild-type human male iPSC line (Coriell Institute: #GM25256). WTC-Cas9 (Control) was derived from WTC-11 by inserting CAG-rtTA:TetO-Cas9 (Addgene #73500) into the AAVS1 locus to yield a dox-inducible Cas9 hiPSC line. Additionally, the commercially available WTC-11 TNNI1 -GFP line (AICS-0037 cl.172) was used.

[0096] Cardiac organoids were generated as published previously using a spinner flask-based suspension system (Pfeiffer CELLspin Spinner System, Cole Palmer®. For experimental and drug assessment using the CeMeT platform, an average of 12 organoids were transferred to a T25 flask and incubated in a humidified incubator at 37°C with 5% CO2 for 72 hours. Cardiac organoids were maintained in a culture media of RPMI-1640 (HyClone), containing 2% supplement (B-27, 50X, Sigma Aldrich) and 1% Pen-Strep.

[0097] Immunohistochemical analysis

[0098] For immunohistochemical analysis, cardiac organoids were cryosectioned and stained with primary antibodies and staining. Phalloidin (Invitrogen, Cat # A22287, Lot # 2326923, Dilution 1 to 400), Hoechst (Life Technologies, Cat # H1399, Lot # 1924446, Dilution 1 to 2500), and GFP (WTC-11 TNNI1 -GFP line, AICS-0037 cl.172) were employed to stain and visualize F-actin in cytoskeleton, nucleic acid in cell nucleus, TNNI1 in the filaments of endogenous sarcomere, respectively. For marking and imaging apoptotic cells in the cardiac organoids, Caspase-3 (Cell Signaling, Cat # 9662S, Lot # 19, Dilution 1 to 400), tagged with a secondary fluorescent antibody (Alexa Fluor® 555, Invitrogen, Cat # A31572, Lot # 2339822, Dilution 1 to 400) was utilized. Sections were imaged using a confocal microscope (Olympus FV-3000) and analyzed with Fiji (National Institutes of Health).

[0099] The normalization of Caspase 3+ nuclei counts was performed in two steps. First, the area of each sample was normalized to the mean area of control organoids. This normalized area was then used to standardize the nuclei counts in pergolide-treated organoids.

[0100] Mean Fluorescence Intensity

[0101] Mean fluorescence intensity (MFI) was analyzed to determine the TNNI1 and Phalloidin signals. The periphery of cardiac organoids was chosen as the region of interest (ROI). The signal threshold was set to 1 to cover all signals. The total fluorescence signal value was divided by pixel number in the ROI. This was repeated for each drug dose.

[0102] Cytochalasin D treatment and Paraformaldehyde fixation of cardiac organoids

[0103] Cardiac organoids were treated with Cytochalasin D (CytoD, Sigma Aldrich), which causes disruption of actin filaments and inhibition of actin polymerization, to soften cardiac organoids. Stock CytoD solution was prepared in dimethyl sulfoxide (DMSO, D8418, Sigma Aldrich) at a concentration of 2 mM. The stock solution was, then, diluted to the final CytoD concentration of 0.2 pM in the culture media. Then, cardiac organoids were incubated in a humidified incubator at 37°C with 5% CO2 for 72 hours. For paraformaldehyde (PFA) fixation, an equal number of organoids were placed in a 2 mL Eppendorf tube, washed twice with phosphate buffered saline (PBS) and treated with 4% PFA for 30 minutes. FollowingPFA treatment, the organoids were washed again with PBS and stored at 4°C until further experimentation.

[0104] Cardiac Organoid Functionality Experiment

[0105] The electrical and mechanical responses of cardiac organoids following CeMeT mechanotyping were assessed To demonstrate the non-destructive nature of the CeMeT platform. Experimental organoids were transferred to microfluidic chips and rotated at 3000 RCF on the CeMeT platform for 1 minute to simulate the maximal compression forces of the mechanotyping protocol. Control organoids were simultaneously removed from incubation but were not loaded onto the CeMeT platform. Control and experimental organoids were then incubated at 37°C for 90 minutes with an excess of 2pM Cal-520 AM in RPMI-1640 media. Stained organoids were washed three times in live cell imaging solution and transferred to the organoid chip with a 1 -mL wide bore pipette tip.

[0106] The electrophysiological recordings were conducted using a custom-built pacing setup. This pacing setup incorporates a fluorescence imaging module and parallel carbon rod electrodes embedded in a PDMS-based chip to enable simultaneous optical recording and electrical pacing of cardiac organoids. Briefly, organoids were placed in open microwells (1 .2 mm diameter) connected to electrode channels, and field stimulation was applied using a function generator (SDG6022X, Siglent Technologies NA, Inc.). The fluorescence videos were collected on the Nikon Ti2E inverted microscope at an average frame rate of 40 fps. During imaging, each of 5 channels containing 5-18 individual organoids was captured, either during spontaneous beating or while being electrically stimulated at 0.5-1 .5 Hz.

[0107] Calcium Image Analysis

[0108] Videos were saved as uncompressed .avi files and analyzed using NIS Elements AR software. In each video, fluorescent intensity over time was quantified in one ROI per organoid, excluding those that were unresponsive to electrical pacing. Fluorescent intensity data was exported to a spreadsheet and loaded into a custom R script which selects four peaks following the establishment of a regular beating frequency and identifies Fmax for each peak. The script calculates frequency from the average time between four consecutive maxima, identifies F0 for the segment, and calculates F / FO across the spectra. A LOWESS curve with a medium smoothing parameter was fitted in Graphpad Prism 10 to the normalizedfluorescence vs. time data for every organoid in each pacing frequency (0.5, 1 .0, and 1 .5 Hz) and treatment group (CeMeT or control).

[0109] The unpaced (spontaneous beating) frequency was calculated from the time between two consecutive peaks among organoids exhibiting spontaneous contractions. Two-way ANOVA with post-hoc Sidak’s multiple comparisons test was performed to determine statistically significant differences between observed contraction frequencies among control and CeMeT treatment groups at each electrical pacing frequency.

[0110] Preparation and administration of Pergolide to cardiac organoids

[0111] A stock solution of pergolide was prepared by dissolving pergolide mesylate (USP, 1510845) in DMSO to a concentration of 10 mM. Solutions with final concentrations of 1.25, 2.5, 5, 10, 20, 40, and 50, 80, 160, and 320 pM pergolide were prepared by diluting the stock solution in RPMI 1640 culture media. Four organoids per dose were treated with each pergolide dose for 72 hours.

[0112] Numerical Simulations

[0113] Numerical simulations were conducted using COMSOL Multiphysics 6.2. Briefly, finite element simulations were performed to estimate the Young’s modulus of spherical hydrogels under centrifugal forces.

[0114] Hydrogels were modeled as linearly elastic, isotropic materials with a Poisson’s ratio of 0.49, and contact with a rigid wall was simulated using a penalty method. A grid with 12,473 elements was used and validated that the results were independent of the number of elements by performing additional simulations with different numbers of elements. Hydrogel deformation was quantified by roundness (R = D1 / D2), where D1 and D2 were the major and minor axes of the deformed sphere and normalized force Fn = VFcent / EA, where A was the maximum cross-sectional area, and E was the Young’s modulus. An error analysis was conducted to evaluate potential variability introduced by experimental image edge-detection methods.

[0115] Stress and deformation calculation

[0116] The disk revolutions per minute (RPM) were measured using a tachometer (TA150-NIST, TRIPLETT) aided by reflective tape placed on the disk. The measured RPM was then converted into relative centrifugal force (RCF) using RCF = (7?PM / 1 OOO)2X r x 1.1 18. Stiffness measurements involved applying RCF ranging from 250 RCF to 3000 RCF for organoids and 500 RCF to 5500 RCF forhydrogels and rigid polyethylene microspheres (WPMS, Cospheric LLC). At each RCF, (4 x cross-sectional area) / (p x Major Axis2), where the major axis was the longest axis encompassing the sample. Then, the roundness values were computed using MATLAB code according to the equation. The normalized roundness was defined as NR, roundness relative to initial roundness. Hydrogel beads with NR of >0.85 and organoids with NR of >0.77 were employed to limit shape effect in calculations. The deformation was defined as D = 1 - Normalized Roundness. The stress was calculated as follows, Stress =Force / Surface Area, which leads to a more explicit form as follows: Stress = RCF x g x Vx (pSamPie- pmedtum) / Surface Area, where RCF, g, V, psample, pmedium, and surface area were relative centrifugal force, the gravitational constant, sample volume, sample density, medium density, and cross-sectional area of the samples, respectively.

[0117] Error, Accuracy, and Precision Calculations

[0118] The accuracy and precision of the CeMeT Platform were evaluated using polyethylene (PE) beads (Item # WPMS-1 .10355-425um, WPMS-1 .10 710-850um, WPMS-1 .10 1 .00-1 ,18mm: Cospheric LLC). The PE beads were expected to not exhibit any measurable deformation in CeMeT under the maximum RCF of 5500 due to their high stiffness (Young’s modulus of 200 MPa). Prior to the experiment (RCF=0), the roundness of the PE beads was measured and normalized to their initial values, setting R0 = 1 .

[0119] Statistical Analysis

[0120] Before statistical analysis of the CeMeT experiments, outliers were removed from each group. The data normalization was explained in the related figures. Results were presented as either mean±SD or meantSEM. Sample size of each experimental and control group was shared in the related figure caption. The statistical methods used to assess significant differences along with post-hoc tests were shared in the figure captions. The statistical analysis was performed using graphing and data analysis software (GraphPad Prism, GraphPad Software, Inc.). Results

[0121] The CeMeT platform measured the micro-biomechanical changes in tissues due to drug exposure, diseases, and aging (FIG. 7, element a). It comprised a custom-made disc, a direct current (DC) electric motor, a high-speed camera, and an inverted microscope (FIG. 7, element b, and FIG. 4). The disc featured eightrecess structures to house eight custom CeMeT microfluidic chips (FIG. 5). Each chip holds one sample for mechanotyping by applying a centrifugal force on the disc and analyzing the subsequent deformation and relaxation events. The centrifugal force (FCENT) in the radial direction of the disc was generated by modulating the rotational speed (w) of the disc via a motor controller and monitoring it with a tachometer. Increasing co results in higher FCENT, and relative centrifugal force (RCF) on the sample; thus, higher sample compression as the sample gets pushed against the compression plate of the chips (FIG. 7, element c, and FIG. 5).

[0122] The current CeMeT platform achieved a maximum co of 8019 RPM, equivalent to an RCF of 5500. This RCF was sufficient to compress and analyze samples with Young’s moduli up to ~32 kPa, covering a wide range of tissues. The CeMeT Platform can collect image data in under 10 seconds. Using this platform, a series of validations were performed and foundational experiments on the deformation response of synthetic hydrogel, polyacrylamide (PAA), beads under centrifugal loading (FIG. 7, element d). A series of drug tests were conducted by mechanotyping multicellular cardiac organoids to demonstrate the utility of the platform (FIG. 7, element e). These studies were augmented by both theoretical analyses and numerical simulations (FIG. 7, element f).

[0123] The experimental studies presented herein used high-speed video recordings of sample deformation on the CeMeT platform under various RCFs, which were then analyzed using a custom image processing and analysis approach to assess parameters related to the deformation mechanical response of the samples. Specifically, changes in roundness were used as a measure of sample deformation wherein the following definition for roundness, R = (4 x cross-sectional area) / (it * major-axis2). R can vary between 1 , for a perfectly round sample, to 0, for highly irregular or non-round samples. In the following, to compare samples with slightly different sizes and initial roundness, normalized roundness was defined as NR = Rexp I RQ, where Rexp and RQ were the experimental and initial roundness values. The initial roundness values, R, were measured as Hp / ow : 0.968 ± 0.009, HpmerZ : 0.962 ± 0.020, and Hphigh 0.919 ± 0.040 for hydrogel beads; and Control: 0.894 ± 0.049, CytoD-treated: 0.894 ± 0.037, and PFA-fixed: 0.848 ± 0.043 for cardiac organoids.

[0124] The accuracy and precision of the CeMeT platform were first evaluated, where both the imaging hardware and software can contribute to erroneous measurements. To this end, polyethylene (PE) beads with Young’s modulus of 200 MPa were used. These PE beads were not expected to deform measurably, even under the highest RCF on the CeMeT platform. Here, accuracy was defined as the ratio between Rexp, i.e., at RCF>0, and RQ values as Accuracy {%) = (ReXp / RQ) x 100; and the precision as the standard deviation of the accuracy (%). The error was =then defined as Error (%) = 100 - Accuracy (%) (FIG. 6, element a). Measurements at RCF of 1500 with PE beads with diameters of 390 pm, 780 pm, and 1090 pm show accuracies of 98.5%, 97.5% and 99.6% (FIG. 6, element b). Corresponding precision values for these measurements were ±0.84, ±0.32, and ±0.4 for the 390 pm, 780 pm, and 1090 pm beads, respectively. The high accuracy, i.e., low error, and the high precision were both maintained throughout the RCF range of 500 to 5500, with no statistically significant differences across different bead sizes (FIG. 6, element c).

[0125] Mechanical Characterization of Hydrogel Beads

[0126] The mechanical characterization of spherical hydrogel beads with different densities was performed to further validate the CeMeT platform and establish a framework for the mechanotyping of samples (FIG. 8). The three hydrogel formulations were classified according to their mass density as HpZow Hpmed, and Hphigh and showed visibly observable differences in deformation at an RCF of up to 5500, which was the maximum RCF on the CeMeT platform (FIG. 8, element a, and FIG. 9). The observed deformations for all formulations indicate a change from a near-perfect sphere to an oblate spheroid. The rheometry measurements reveal an increasing Young’s modulus, E, with increasing p, with a range of ~5 kPa to ~32 kPa.

[0127] Deformation and Young’s moduli of the different hydrogel beads on the CeMeT platform were assessed. To this end, the normalized roundness (NR) was analyzed at different RCFs (FIG. 8, element b). Hydrogel bead samples tested varied slightly in diameter of 1652±229 pm and initial roundness of >0.85. NR decreased for all hydrogel beads of all densities with increasing RCF, albeit at different rates reflecting the differences in their Young's moduli. These differences were morepronounced and statistically significant at RCF>1000 with a large effect size (r]2=0.86).

[0128] Specifically, the lightest hydrogel beads, HpZoiv, showed the highest change in NR, while the densest, Hphigh, showed the lowest changes in line with their rheometry-measured moduli. Next, Young’s moduli of the hydrogel formulations were determined by applying Hertz’s theory of contact to the images acquired from the CeMeT platform (FIG. 8, element c). Specifically, this theoretical analysis involved images of hydrogel beads deformed at different RCF and yielded Young’s moduli of 1 .042±0.19 kPa, 5.95±1 .43 kPa, and 32.7±10.81 kPa for the hydrogel formulations HpZoiv, HpmecZ, and Hphigh, respectively.

[0129] Numerical simulations were also conducted via finite element analysis (FEA) to analyze the deformation of linearly elastic spheres compressed against a rigid wall under uniform external forces to further validate the experiments in FIG. 8. Applying the Hertz theory of contact, on final shapes provided by these simulations, yielded estimates of Young’s moduli, at 1 .15 kPa, 6.97 kPa, and 37.56 kPa for the hydrogel formulations HpZoiv, HpmecZ, and Hphigh, respectively (FIG. 8, element d). Next, as a validation, a similar iterative reconciliation was conducted between simulations and experiments, using Young’s modulus as a simulation input to match the curves of the NR versus total applied body force until RMSE converged to ±0.05. This alternative approached yielded similar estimates of Young’s moduli 1 .3, 8, and 44 kPa for HpZoiv, HpmecZ, and Hp / iig / i, respectively (FIG. 10, elements a and b).

[0130] Comparing samples at a fixed RCF distinguishes the samples according to their stiffness, yet it does not fully reveal the mechanical properties and material classes of the samples. Thus, the deformation of the hydrogels was analyzed as a function of the applied stresses (FIG. 8, element e). Here, deformation was defined as D = 1 - Normalized Roundness and stress as the ratio of the body forces on a bead and the cross-sectional area of the undeformed hydrogel bead, where forces were corrected for the mass density of different formulations. The lightest hydrogel, HpZoiv, beads showed the highest deformation, whereas the densest hydrogels, Hp / iig / i, showed the lowest deformation at comparable stresses. Importantly, the deformation as a function of the applied stresses showed a linear relationship for all three hydrogel formulations across the stress range, in line with the linear elastic behavior expected for PAA hydrogels.

[0131] Elastic recovery, the ability of a material to return to its original shape after deformation, was a measure of material durability that was not only important to characterize the material state but also critical to repeated and reliable measurements on a single sample over a long time. Cyclic loading-unloading tests were conducted where the deformation and subsequent recovery of hydrogel beads at a loading of 1500 RCF over 10 cycles on the CeMeT platform was analyzed (FIG. 8, element f). Elastic recoveries of 95.6%, 95.5% and 96.7% for the hydrogel formulations HpZow, Hpmed, and Hphigh, respectively, were observed indicating that all hydrogel beads exhibit primarily linear elastic material behavior at the RCF of 1500.

[0132] Mechanotyping of Cardiac Organoids

[0133] Alterations in the mechanical properties, i.e., the mechanotype, of cardiac tissue, can indicate pathological conditions induced by disease or drugs. For instance, increased tissue stiffness often indicates the presence of fibrosis following myocardial injury, and such increases in stiffness can impair relaxation and filling, leading to diastolic dysfunction. Here, a recently established hiPSC-derived cardiac organoid model was used to characterize the mechanical responses of such cardiac organoids under control, fixed, and drug-induced injury conditions on the CeMeT (FIG. 11 ) to establish a foundation for organoid mechanotyping.

[0134] Specifically, cardiac organoids with diameters of 541 ±34 pm were used as the control group. The organoids were treated with 0.2 pM Cytochalasin D (CytoD) for three days, which disrupts the actin filaments and inhibits actin polymerization, resulting in softer organoids. The CytoD treatment affected the actin network in the cardiac organoids compared to the controls (FIG. 11 , elements a and b). Next, 4% paraformaldehyde (PFA) was used to fix the organoids such that they were stiffer than the controls. FIG. 1 1 , element c shows the cross-sectional geometry and the visibly different deformation of three representative organoids from each group at RCF of 1500.

[0135] Upon establishing these experimental groups, the CeMeT platform was used to demonstrate the mechanotyping of 3D organoids (FIG. 11 , elements c-g). The NR across an RCF range of 0 to 3000 was first characterized. This analysis separates the three groups at an RCF of >500, both statistically and in terms of individual organoids (FIG. 11 , element d). At the highest RCF of 3000, the NR for the three organoid groups were 0.68±0.04, 0.42±0.017, and 0.87±0.016 for the control,CytoD-treated and PFA-fixed organoids, respectively. The separation between control and CytoD-treated groups was apparent even at lower RCFs due to the initial rapid decline of NR for CytoD-treated organoids.

[0136] Similar to the characterization of the hydrogels, the deformation was assessed as a function of applied stresses to better understand the mechanotype of the organoids (FIG. 11 , element e). As expected, CytoD-treated organoids showed the highest deformation at all stresses applied with a linear response, albeit with two distinct slopes in their deformation-stress curve. Conversely, both the controls and the stiffer PFA-fixed organoids showed lower deformation at comparable stresses, also with a linear response. These results indicate that the deformation-stress response can also distinguish the organoids according to their mechanotype, similar to the NR-RCF curves in FIG. 11 , element d.

[0137] Both the standard deviation (SD) and the coefficient of variation (CoV) of Young’s modulus were quantified across samples with varying stiffness and deformation ranges to assess the measurement sensitivity of modulus estimation in CeMeT. Measurement sensitivity improved with increasing deformation, as samples undergoing larger shape changes yielded lower SD values (e.g., 0.19 kPa for soft hydrogels vs. 10.81 kPa for stiff hydrogels) (FIG. 12, elements a and b). CoV was lower in stiffer, homogeneous materials but increased in softer, biologically heterogeneous organoids (e.g., 0.17 for PFA-fixed vs. 0.37 for Cytochalasin D- treated) (FIG. 12, element c). These results indicated that the measurement sensitivity of CeMeT was influenced by both the mechanical properties of the sample and the deformation range over which measurements were performed.

[0138] The NR-RCF and deformation-stress response of three different sizes (d=244 pm, d=541 pm and d=885 pm in diameter) of cardiac organoids were assessed to assess the potential effects of sample size on NR and deformation (FIG. 13, elements a and b). These data showed consistent trends between 541 and 244 pm diameter organoids, while the largest group (885 pm) began to separate from the others beyond -250 RCF (FIG. 13, element a). The response for the three sample sizes was similar, up to -50 N / m2in deformation vs stress plot, beyond which they become distinguishable, i.e., larger organoids deform more at the same applied stresses (FIG. 13, element b). When Young’s modulus was calculated using Hertzian theory of contact, a statistically significant size-dependent difference was observed across the three groups (FIG. 14). This difference may reflect underlying biologicalprocesses, as larger organoids could be expected to exhibit more compact structures due to prolonged self-assembly, different stages of maturation, or cellular remodeling and composition.

[0139] Hertz’s theory of contact was used to estimate Young’s moduli of the different experimental groups. Specifically, for this analysis, images obtained below ~50 N / m2were used for CytoD-treated organoids and up to -200 N / m2for the control and PFA-fixed samples to ensure that deformations of all samples were in the linear response regime. This analysis yielded Young’s moduli of 0.62±0.22 kPa, 0.16±0.06 kPa, and 1 .69±0.29 kPa for the control, CytoD-treated, and PFA-fixed organoid groups, respectively (FIG. 11 , element f). Finally, the elastic recovery of the organoids was assessed after ten cyclic loading-unloading tests at an RCF of 1500. The control organoids showed elastic recovery of 90%, while CytoD-treated and PFA-fixed groups showed elastic recoveries of 98% and 97%, respectively, indicating the primarily linear deformation response of the organoids at the RCF of 1500. (FIG. 11 , element g).

[0140] Cardiac Organoid Functionality

[0141] The electrophysiological behavior of cardiac organoids was evaluated before and after mechanical testing to assess whether CeMeT interferes with organoid function. A representative image sequence (FIG. 11 , element h) shows an organoid contracting in response to 1 Hz pacing, confirming electrical capture. Beating frequencies under spontaneous and paced conditions (0.5, 1 .0, and 1 .5 Hz) were shown in FIG. 11 , element i, demonstrating appropriate responsiveness to external stimuli. FIG. 11 , element j compares beating fidelity at each pacing frequency before and after CeMeT testing, showing that organoids maintain contraction / relaxation behavior and electrical responsiveness. These results indicate that CeMeT does not impair electrophysiological function of cardiac organoids.

[0142] Mechanotyping of Cardiac Organoids for Drug-induced Injury

[0143] Pergolide, a dopamine agonist used primarily to treat Parkinson’s disease, also acts as an agonist of serotonin receptors on the interstitial cells of heart valves. These receptors, including 5-HT2B, were involved in promoting proliferation and extracellular matrix deposition. Their prolonged activation by pergolide and other serotonin agonists may lead to fibrosis and valve dysfunction, such as stenosis and regurgitation. Indeed, pergolide was found to cause valvular heart disease (valvulopathy) and was withdrawn from the market in many countries,including the US, around 2007. Here, mechanotyping was used on the CeMeT platform to investigate the effects of pergolide on hiPSC-derived cardiac organoids (FIG. 15).

[0144] Cardiac organoids were first treated with pergolide at different doses for three days and then conducted immunohistochemical analyses to establish an effective dose range where structural changes were observed without significant apoptosis. Specifically, Caspase-3 (CASP3) was stained for and characterized to assess apoptotic response, Troponin 11 (TNNI1) as a marker of the cardiac sarcomere, F-actin as a marker of actin cytoskeletal network integrity, and finally the cell nuclei (FIG. 15, element a). Quantification of Caspase-3 staining revealed that pergolide can be considered relatively safe up to 80 pM (FIG. 15, element b). The F- actin levels decreased rapidly even at this relative safe dose of 80 pM, indicating a disruption of the F-actin cytoskeleton. On the other hand, the TNNI1 levels were well conserved up to 80 pM, indicating intact contractile function for the cardiac organoids (FIG. 15, elements a and c). Both the F-actin and TNNI1 levels were significantly reduced at doses beyond 80 pM. In subsequent experiments, doses up to 80 pM were used.

[0145] As pergolide administration had a significant effect on the F-actin network, it was expected to change the mechanical properties of the cardiac organoids. Thus cardiac organoids treated with 50 pM and 80 pM pergolide were mechanotyped using the CeMeT platform.

[0146] Pergolide-treated organoids, at either dose, showed higher changes in NR than the control group. These changes were statistically significant beyond 1000 RCF with a large effect size (q2=0.32) and 1250 RCF with a large effect size (r]2=0.44) for 80 pM and 50 pM pergolide-treated groups, respectively (FIG. 16, elements a and b). Furthermore, both pergolide-treated groups showed higher deformation than the controls, indicating softening of the organoids (FIG. 15, element d). The control group showed a linear deformation response across all the stresses up to 200 N / m2. In contrast, the pergolide-treated organoids exhibited a linear response up to approximately 125 N / m2, beyond which the deformation plateaued. Young’s moduli, estimated using Hertz’s contact theory using images obtained at RCF of 500, 750, 1000, and 1250, were 0.62±0.22 kPa, 0.45±0.11 kPa, and 0.4±0.07 kPa for control, and 50 pM and 80 pM pergolide-treated organoids, respectively (FIG. 15, element e). These results, which showed highly significantdifferences between pergolide treated and control groups, corroborate the finding that pergolide induces a softening of the cardiac organoids. Finally, the effect of 80 pM pergolide treatment on the elastic recovery of organoids was assessed at an RCF of 1500 via cyclic loading tests, where it was observed no significant differences (FIG. 15, element f).Experiment 2

[0147] The goal is to develop and validate platforms that enable i) cultivating 3D samples, ii) repeatedly assessing their mechanotypes (stiffness, viscoelasticity, etc.) Hi) in high-throughput, and iv) in longitudinal studies of injury and disease. Such mechanotyping platforms are critical to assessing disease / drug-induced tissue mechanotype changes and their reversal in vitro. Two platforms were designed based on a prototype centrifugal mechanical testing (CeMeT) platform (FIG. 17). The working hypothesis for our platforms is that 3D samples can be deformed by compressing them against solid walls in microchips, facilitated by centrifugal forces due to rotation on a disc. Such deformations can then be imaged and analyzed to provide rapid, high-resolution, and time-resolved data on mechanical properties, i.e., mechanotypes. Specifically, CeMeTLS (CeMeT for Life Sciences)(FIG 18) will enable longitudinal hypothesis-driven studies on disease progression; drug exposure, providing time-resolved deformation data at a moderate throughput. In contrast, CeMeTDS (CeMeT for Drug Screening) (FIG. 19) will enable rapid high- throughput testing suited to drug screening.

[0148] CeMeT Platforms

[0149] Practical high throughput mechanotyping platforms for robust in vitro mechanotyping are absent from the market despite a pressing need for their use in preclinical efficacy and secondary safety testing. The CeMeT platforms (FIG. 15, elements a-e) offer a novel yet simple and scalable approach for longitudinal mechanotyping of 3D samples. They provide rapid “compression-like” measurements of sample deformation and recovery under force loading and unloading studies. By generating well-controlled centrifugal forces (FCENT ) on discs housing microfluidic chips (a.k.a “chip” or “CeMeT Chip”), the platforms can compress samples against rigid surfaces perpendicular to the forces (a.k.a “compression plate”). The resulting sample deformation and mechanical response can be measured via integrated i) high-speed (HS) cameras, ii) co-rotating CMOSsensors, or iii) laser scanning via optical pickup units (OPUs) providing different levels of sampling frequency, and acquisition and analysis speeds.

[0150] The CeMeT platforms combine i) the simplicity of centrifugally driven microfluidics on compact discs; ii) custom, portable microfluidic chips that provide long-term sample cultivation to enable longitudinal studies in disease, aging, and chronic drug exposure; and iii) three alternative imaging modalities with different response frequency sampling for different use cases.

[0151] A prototype with 8 CeMeT Chips and an HS camera to mechanotype PAAm hydrogel beads and cardiac organoids of different sizes and stiffnesses; Lattice-Spring - lattice-Boltzmann (LSM-LBM) and COMSOL® Multiphysics simulations were used to predict the deformation of linear elastic solid spheres under a constant body force. Initially spherical objects deform to oblate spheroids under compression, with a qualitative agreement visible between simulations and experiments.

[0152] Development of CeMeTLS Platforms for Life Sciences and Drug Screening

[0153] Studying tissue injury progression and reversal via mechanotyping requires: i) time-resolved mechanical response data to capture mechanotypes beyond stiffness (e.g., viscoelastic behavior) and ii) longitudinal repeated measurements on individual samples. These necessitate i) continuous recording deformation and relaxation dynamics and ii) plug-and-play chips for prolonged 3D tissue cultivation during disease progression and chronic drug exposure.

[0154] CeMeTLS platform (FIG. 18) was developed based on the current CeMeT prototype, which features a microscope-mounted high-speed camera and a custom disc holder with 8 plug-and-play chips where a controlled motor drives centrifugation. Here, our approach is to i) integrate CMOS-based camera modules that co-rotate with the microchips and ii) simultaneously increase device throughput via redesign. We expect to demonstrate that CeMeTLS provides robust, reliable, accurate, precise, and repeatable time-resolved mechanical response data with relative centrifugal force (RCF) up to 10,000 at a medium throughput of 24 chips.

[0155] Drug screening requires high-throughput and rapid data analysis, yet endpoint tissue mechanical assessments such as stiffness would suffice for such purposes. Therefore, the CeMeTDS (FIG. 19) was developed using standard lab-on- a-disc fabrication methods and laser optical pick-up units (OPUs) for rapid and high-resolution data acquisition. Unlike other versions, CeMeTDS uses almost the entire surface area of the discs and position multiple chips along the radial direction to drastically improve throughput and also provide differential RCF analyses on a single disc. The OPU integration will provide rapid data acquisition on end-point deformation and, thus, stiffness. 100 samples can be used on a single platform, providing rapid actionable data for drug efficacy and safety screening with an RCF up to 20,000 for improved measurement sensitivity, robustness and reproducibility.

[0156] The platforms were calibrated and validated with synthetic materials and 3D tissues, and ii) computational simulation and machine learning were used for further validation. A mapping tool with well-characterized microgel beads, discs, and rectangles (d=100-2000 pm, modulus 0.5 - 30 kPa by AFM and / or rheometer) was created. A mapping between AFM stiffness and CeMeT-measured properties was established accompanied by theoretical estimations and ii) a resolution of 0.1 kPa or better for both platforms. Computational models were leveraged based on i) lattice- Boltzmann - lattice Spring, ii) Comsol® models, and ill) machine learning to predict deformation and mechanical response in various material classes (elastic, viscoelastic, poroelastic, and hyperelastic) and shapes (regular and irregular) to support experimental observations. Finally, 3D tissue mechanotyping with 3D spheroids (d=100 and 250 pm) of different tissues was used for validation.

[0157] Specifically, CeMeTLS focuses on medium-throughput, time-resolved measurements of static (stiffness) and dynamic (viscoelasticity, etc.) tissue properties in longitudinal studies (e.g., chronic / repeated drug exposure or disease progression). In contrast, CeMeTDS is designed specifically for high throughput with rapid data acquisition and analysis for evaluating large drug candidate libraries. To meet these distinct demands, CeMeTLS will employ co-rotating CMOS sensors for continuous sampling and plug-and-play microfluidic chips for long-term cultivation of samples, whereas CeMeTDS will use integrated chips for maximum throughput and laser scanning (optical pickup units, OPUs) for rapid data acquisition. Together, these platforms will enable I) minimally invasive and repeated longitudinal tissue mechanotyping; ii) high-throughput mechanotyping based screening (drugs, chemicals, environmental stimuli). These platforms are synergistic, as insights gained from CeMeTLS will guide more efficient high-throughput screens in CeMeTDS - particularly in efficacy testing scenarios.Experiment 3

[0158] The tests conducted below show real world applications of aspects of Experiment 2.Test 1

[0159] Design and Operation of a Centrifugal Mechanical Testing (CeMeT) Platform

[0160] Unlike several micromechanical testing platforms, e.g., atomic force microscope, CeMeT does not utilize a probe to touch or indent samples for mechanical measurement. The compressive load is applied due to the rotator’s rotation and / or cyclic load. To this end, platforms were developed to measure the mechanical properties of the samples. The platforms utilized centrifugal forces to apply controlled stress onto samples and deform them by controlling the compression rate.

[0161] The precise compression rate over the samples was ensured via precisely controlled centrifugal forces, which resulted from the rotator or sample's rotation and / or cyclic motion. Due to the rotation of the sample and / or the platform or cyclic load on the samples, the samples are either compressed, stretched (tensile), sheared, bent, or combinations thereof.

[0162] Fabrication and Operation

[0163] A cylindrical disc was fabricated as a rotator via additive manufacturing. In other embodiments of this example, manufacturing is done by injection molding or CNC machining. The material of the disc was ABS polymer. In some embodiments of this example, disc material can be other polymers, metals, glasses, and plastics, including but not limited to aluminum, PMMA (polymethyl methacrylate), polystyrene, ABS (Acrylonitrile Butadiene Styrene), borosilicate, and polycarbonate.

[0164] A DC motor regulated by a Pulse Width Modulation (PWM) controller through voltage adjustment was employed. The rotational speed was measured using a tachometer. The data were acquired before, during, and after the experiment using a high-speed camera. The compression test used a cylindrical disc rotating at a rotational speed of 1 to 20,000 RPM. The rotational speed can be between 1- 20000 RPM, including but not limited to 1 , 10, 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 4000, 5000, 6000, 10000, 20000.

[0165] Two chips were placed on the cylindrical disc (dual CeMeT) to keep the balance during disc rotation. The disc had empty areas smaller than the chip to holdthe chip at the desired location to ensure the samples were held at the focal of microscopic imaging during measurement.

[0166] Design, working mechanism & Results

[0167] The CeMeT had i) PDMS u-tube chips (chips), ii) a disc to hold microchips, iii) a motor to drive the rotation, and iv) a controller to modulate the rotational speed (see, e.g., FIGS. 4, 5, 7, and 15). The microchip provided a solid surface (deformation plate) perpendicular to the disc radius to facilitate sample compression. The dual CeMeT device housed two chips (with 100 pm precision) and was symmetrically mounted on a cylindrical disc.

[0168] Paired with an optocoupler sensor, a controller was integrated to measure and control the rotational speed in revolutions per minute (RPM). The rotation of the disc resulted in a relative centrifugal force (RCF) and deformed samples in the microchips. The rotational speed in RPM and the “pivotal distance of the microchip edge (compression surface) to the disc center” (R) dictates the RCF on the 3D samples where RCF can computed as 11 .2 x R x (RPM / 1 ,000). The pressure on a given spheroid sample can be estimated by the simple equation P=F / A. Here, F= m x RCF, A is the cross-sectional area of the 3D spherical sample (nr2), and the mass (m) was estimated from the sample density and measured volume. For a spherical sample with a density of 1000g / m3and a radius (r) of 75 pm at 1000 RCF, the pressure was ~1 kPa, a value that increased linearly with RCF and thus quadratically in RPM.

[0169] The aspect ratio (AR) and roundness of the cardiac organoids were analyzed under varying RCF (g-forces). Live and fixed cardiac organoids were used. The live organoids were fixed with (Paraformaldehyde) PFA 4% to fix the organoids.

[0170] The deformation area of the chips had a flat deformation plate. In some embodiments, the deformation plate is concave or convex. In some embodiments, the measured deformation can be used as compression, tensile, multi-point bending, shear and other deformational testing. In some examples, the deformation plate has holes called deformation holes. In some examples, there are deformation hurdles at the deformation area. Different mechanical tests can be performed with various holes and hurdles and their combination thereof, such as multipoint bending and shear testing.

[0171] Test 2

[0172] A new CeMeT for higher throughput, called the octal CeMeT was designed, fabricated, and tested. The disc design was modified to i) house eight microchips and ii) reach 10000 RPM, allowing i) a moderate throughput and ii) higher forces on samples. These changes also imposed design and material changes to microchips and the disc to cope with increased forces. Thus, more advanced and precise fabrication techniques, such as CNC machining, were used to ensure equal distribution of the weights and accurate balancing of the microchips on the disc. In addition to PDMS, transparent materials, including but not limited to PMMA, borosilicate, polycarbonate, and polystyrene, are used for chip fabrication.

[0173] Test 3

[0174] A cylindrical disc (rotator) with 48 chips was developed to increase the throughput, detection sensitivity, and reproducibility. The device is called High Throughput Centrifugal Testing (HiTCeMeT) Platform (see, e.g., FIG. 17). The disc's rotational speed reached up to 20000 RPM to ensure sensitivity and dynamic range for stiffer samples.

[0175] The HiTCeMeT was fabricated using a standard CD / Blu-ray using a traditional polycarbonate fabrication method, e.g., injection molding. A laser and a photodiode array were incorporated to increase the measurement sensitivity. CD and Blu-ray devices have 800 and 150 nm spatial resolutions, respectively. Thus, these technologies were adapted to increase measurement sensitivity. 780 nm or 405 nm laser diodes and corresponding aligned photodiodes (OPU couple) were integrated into HitCeMeT. The top surfaces of chips were designed as reflecting surfaces so that photodiodes can convert reflected light to binary data “1 ”. The samples in chips interrupt the reflectance, and binary data will be “0”.

[0176] The scanned spatial binary data are correlated with the rotation speed. HiTCeMeT (d=12cm) rotates at a maximum of 20,000 RPM. Using a photodiode with a minimum rise time of 30 ps, feature changes <1 .0pm can be captured. The setup has 100, 250, and 500 Pa resolutions in soft, moderate, and stiff samples at RPMs > 4000 RPM.

[0177] Test 4

[0178] Another platform called the Real Time Centrifugal Mechanical Testing (RTCeMeT) Platform was developed to detect the deformation dynamically (also called CeMeTLS (Time-Resolved Centrifugal Mechanical Testing Platform for Life Sciences Research)) (see, e.g., FIG. 18). A mini camera and a light- emitting diode(LED) source are fixed under and above each chip, respectively. A cylindrical disc with chips was fabricated via 3D printing. The number of chips can be multiple, including but not limited to 1 , 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, 4096, and 8192 chips. Upon the rotation of the cylindrical disc, the samples in the chips are compressed. The visual data are recorded before, during, and after the compression test. This platform ensures the collection of data during deformation.

[0179] From the above description, those skilled in the art will perceive improvements, changes, and modifications. Such improvements, changes and modifications are within the skill of one in the art and are intended to be covered by the appended claims.

Claims

The following is claimed:1 . An in vitro system to detect one or more mechanical properties of one or more samples, the system comprising: a rotator configured to rotate around a rotational axis to produce centrifugal force that deforms at least one sample located in a deformation area, wherein the sample is pushed towards a deformation wall that is perpendicular to direction of the centrifugal force; and at least one measurement sensor configured collect data related to shape changes of the at least one sample as it deforms or relaxes after deformation to reveal the one or more mechanical properties, wherein the one or more mechanical properties are related to material sciences, life sciences, pharmaceutical sciences, or drug screening.

2. The system of claim 1 , wherein the one or more mechanical properties are static or dynamic.

3. The system of claim 1 , wherein the one or more mechanical properties comprise stiffness, Young’s modulus, shear modulus, bulk modulus, Poisson’s ratio, yield strength, ductility, hardness, toughness, creep, fatigue, brittleness, elasticity, and / or viscoelasticity, an elastic property, an inelastic property, a viscoelastic property, a partially elastic property, a thermoplastic property, a thermoset property, and / or a plastic property.

4. The system of claim 1 , wherein at least one light source and at least one high speed camera are positioned at fixed locations and collect visual data during rotation of the rotator which is located in between light source and high speed camera, wherein the rotator comprises one or more integrated or mounted chips that hold the at least one sample during deformation, and the cameras capture images of the deformation and relaxation of the at least one sample at every revolution of the rotator, thereby enabling measurement of time independent mechanical properties of the at least one sample.

5. The system of claim 1 , wherein multiple light sources and cameras are positioned in fixed locations relative to the rotator, such that the cameras and the light sources rotate together with the rotator, wherein the rotator comprises one or more integrated or mounted chips that hold the at least one sample during deformation, and the cameras capture real-time images of the deformation and relaxation of the at least one sample, thereby enabling measurement of time dependent mechanical properties of the at least one sample.

6. The system of claim 1 , wherein a laser coupled with photodiode arrays or optical pickup units is used as the measurement sensor, and the at least one sample is mapped bitwise to detect deformation, wherein signals from the photodiode arrays or optical pickup units are processed to determine position and the shape of the sample relative to a deformation wall.

7. The system of claim 1 , wherein the sample comprises one or more cells, one or more tissue, one or more polymer beads, one or more composite materials, one or more hydrogels, one or more spheroids, one or more organs, and / or one or more organoids.

8. The system of claim 1 , further comprising a motor, controlled by a controller, configured to drive the rotator to rotate around the center of rotation at the rotational speed.

9. The system of claim 7, wherein the rotational speed is between 0.01 RPM and 500000 RPM.

10. The system of claim 1 , wherein the rotator comprises: a symmetrical shaped mechanism configured to rotate around the center of rotation at the rotational speed to generate the centrifugal force; at least one chip, mounted on the rotator, merged with the rotator, or engraved into the rotator, that is configured to hold the sample in the deformation area with the deformation wall.11 . The system of claim 10, wherein the symmetrical shaped mechanism comprises a disc, a rectangular prism, and / or a polygonal prism.

12. The system of claim 10, wherein the at least one chip comprises a microfluidic an / or milifluidic chip with channels and the deformation wall is at an end of at least one channel.

13. The system of claim 10, wherein the deformation area comprises one or more hurdles that changes the deformation of the samples, wherein the one or more hurdles comprise a tip, a pointy tip, a round tip, a pin, a pillar, and / or a geometry that changes a cross-sectional area of the sample, the deformation area, and / or a deformation wall.

14. The system of claim 10, wherein the deformation wall comprises one or more holes.

15. The system of claim 1 , wherein the measurement sensor is coupled to a signal analyzer configured to analyze the data to reveal the one or more mechanical properties.

16. A method for detecting one or more mechanical properties of one or more samples in vitro, the method comprising: rotating a rotator around a rotational axis at a rotational speed to produce a centrifugal force; causing at least one sample in a deformation area on the rotator to deform, wherein at least one sample is pushed towards a deformation wall that is perpendicular to a direction of the centrifugal force; and collecting data related to shape changes of the at least one sample as it deforms or relaxes after deformation to reveal the one or more mechanical properties, wherein the one or more mechanical properties are related to material sciences, life sciences, pharmaceutical sciences, or drug screening.

17. The method of claim 16, further comprising analyzing deformation of the at least one sample to reveal the mechanical property, wherein at least a portion of the analyzing occurs via a computer program remote from the rotator.

18. The method of claim 16, wherein the rotating is based on a motor setting the rotational speed to a value between 0.01 RPM and 500000 RPM.

19. The method of claim 16, wherein the causing further comprises holding the sample in the deformation area of a chip.

20. The method of claim 19, wherein the causing occurs simultaneously on a symmetric number of two or more chips.

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