Engineered heart tissue measuring platform, software, and components for the same

Abstract

Disclosed herein are embodiments of tissue measurement platforms, software, and components for the same. In at least one embodiment, a measurement chamber comprises: a sample tray, one or more light sources, and one or more cameras configured to capture images and / or video of a plurality of tissue samples when loaded onto the sample tray. In at least one embodiment, the measurement chamber is operatively coupled to an evaluation unit and is configured to evaluate the plurality of tissue samples in parallel.

Description

ENGINEERED HEART TISSUE MEASURING PLATFORM, SOFTWARE, AND COMPONENTS FOR THE SAMECROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of priority of United States Provisional Patent Application No. 63 / 616,646, filed December 31, 2023, the disclosure of which is hereby incorporated by reference herein in its entirety.FIELD OF INVENTION

[0002] The present disclosure generally relates to a measuring platform including software, data analytics solutions, and tools for production, examination, treatment, evaluating, and handling of three-dimensional engineered tissues, in particular muscle tissues, such as heart tissues.BACKGROUND

[0003] Drug development is a time- and cost-intensive process. With the recently passed FDA Modernization Act 2.0, researchers are encouraged to rely on human based in vitro models. With these models, the translation of data obtained during development is increased, animal experiments can be substantially reduced, and candidates can be developed time- and cost- effectively. To gain reliable data with human in vitro models, these models require a high level of reproducibility and thus, robustness. Furthermore, data integrity is key to ensure reliability of results.SUMMARY OF THE DISCLOSURE

[0004] The following presents a simplified summary of various aspects of the present disclosure in order to provide a basic understanding of such aspects. This summary is not an extensive overview of the disclosure. It is intended to neither identify key or critical elements of the disclosure, nor delineate any scope of the particular embodiments of the disclosure or any scope of the claims. Its sole purpose is to present some concepts of the disclosure in a simplified form as a prelude to the more detailed description that is presented later.

[0005] Certain objects of the present invention include a device, a system, and a method for examining tissue samples, in particular muscle tissue samples such as heart tissue, where a series of image recordings is taken in a short time and made available for evaluation.

[0006] According to aspects of the present invention, it is possible to record, store, evaluate and process images or image sequences, in particular video sequences of tissue, especially ofmoving tissue located in an examination space of a examination device or analyzer. Advantageously, the recorded images show movements of the tissue, which are analyzed using data processing devices and methods. Favorably, from the type, speed, acceleration and / or amplitude of movement of the tissue, in particular muscle tissue such as heart muscle tissue, certain properties of the tissue can be inferred or determined.

[0007] In a particular embodiment of the present invention, it is envisaged that, prior to the visual examination of a plurality of tissue samples, as is described in greater detail herein, at least some of the samples are brought into contact with a medium, such as an active pharmaceutical agent or an at least partially toxic medium, and that, based on the results of the visual examination of the samples, a conclusion is or can be drawn regarding the influence that the respective medium exerts on the samples. In this context, it is conceivable that pattern recognition programs and data processing units on which these pattern recognition programs can be executed are used to evaluate the movements of the examined tissue. It is also conceivable that suitable comparative data or comparative data sets stored in an internal or external data memory are used to evaluate the recorded tissue movements.

[0008] One aspect of the invention relates to a examination device with an examination chamber in which the samples to be examined are arranged, preferably in the recesses or wells of a microtiter plate, and where the samples are illuminated and image series of the illuminated samples are recorded.

[0009] Preferably, a plurality of samples are arranged in the wells of a microtiter plate and are illuminated at least temporarily from below (i.e., through the bottom of the microtiter plate) which is transparent to the radiation used for illumination. Furthermore, at least one image recording unit, in particular a camera, is arranged in the examination device, which records a sequence of images of the illuminated samples. Preferably, the at least one image recording unit and the illumination unit are arranged in relation to one another in such a way that optical axes running between the image recording unit and the various samples are not obscured by the illumination unit. A particularly suitable arrangement with two image recording units and one illumination unit, which may also be made in multiple parts, as is described in greater detail herein.

[0010] Furthermore, it is advantageous if the examination chamber is or can be sealed in a gas-tight manner, at least during the examination. According to one aspect of the present invention, it is possible to adjust and / or change the properties of the atmosphere within the examination chamber, such as the gas composition, the air content and / or the moisture content within the examination chamber during the examination, especially during the recording of a series of images of a plurality of illuminated samples. For example, gases can be introduced intothe examination chamber that have a targeted influence on the samples or prevent contact with air. Furthermore, it is advantageous if the examination device has a temperature control device, in particular with a heating element, in order to be able to adjust the temperature inside the examination chamber in a targeted manner.

[0011] Another aspect of the invention relates to the processing, storage and evaluation of the image series recorded in the examination chamber. For this purpose, suitable data storage units and data processing units are provided that are integrated into the examination device or can at least be temporarily connected to the examination device via a wireless or wired data connection and suitable interfaces. As is explained in greater detail herein, this makes it possible to edit the recorded images or image series, in particular to generate a video sequence that is particularly suitable for evaluation. Preferably, the data processing unit can be used to execute programs for image processing, image evaluation, and pattern recognition. Likewise, the data processing unit and the programs that can be executed on it are designed in such a way that information about the recorded images, image series, or video sequences produced from them and / or about properties of the examined samples, at least one for the treatment of at least sample, or of the movements performed by the individual samples, such as the type of movement, speed, acceleration, vibration frequency, or amplitude, can be output on an output unit, such as a display or monitor. Furthermore, it is advantageous if an input unit is provided, via which targeted inputs can be made that are used by the data processing unit and the device control or device regulation to set the necessary process parameters during the examination, in particular for taking images, in the examination chamber.

[0012] In addition, images taken, information about the properties of the samples to be examined, information about the type of pretreatment of the samples and / or information about the movements performed by the samples are stored on suitable internal or external data storage devices and are thus available for further processing and evaluation.

[0013] The present invention thus relates to an examination device with its components, an examination method and a suitable system that comprises the examination device according to the present invention with the components described above and at least some elements for data input, data output, data forwarding, data processing and data storage, which are each suitable for recording and evaluating image series of moving tissue samples. In this context, it is fundamentally irrelevant whether the samples perform spontaneous movements or, as is explained in greater detail herein, are stimulated to move by suitable elements.

[0014] A further aspect of the present invention is that a plurality of tissue samples, which are each attached on both sides to silicone posts of a support element, respectively a silicon rack, as will be explained in greater detail herein, and arranged in the recesses or wells of a microtiterplate filled with a medium, are examined simultaneously. Preferably, a series of image recordings of the samples positioned in the examination chamber of the examination device are generated simultaneously and made available to the evaluation unit. Favorably, the positioning of the microtiter plate with the samples is automated via an input shaft and a handling mechanism, as is also explained in greater detail herein. An advantage of the present invention is that a series of images of a large number of such samples can be recorded simultaneously. Preferably, a video sequence of the samples moving during the recordings is generated from the series of recorded images.

[0015] Another aspect of the present invention is that both the illumination of the samples to be examined in the examination chamber and the image recording are carried out by means of an image recording unit, in particular at least one high-speed camera, preferably two high-speed cameras, from below through the optically transparent base of the microtiter plate. The light unit or light units are arranged in such a way that they do not protrude into the optical axes between the camera and the samples. Preferably, two light units are provided, which are designed as light-emitting diode panels and illuminate the samples at an angle from below.

[0016] The evaluation of the images and / or the generation and evaluation of video sequences, and thus the examination of the movement of the individual samples, takes place by means of a suitable evaluation unit with evaluation software, which in particular has an input unit, a data memory, a data processing unit and an output unit.

[0017] According to a further aspect of the invention, the individual samples are specifically excited to move with the aid of electrical current pulses. Therefore, a pacing unit, as is described in greater detail herein, is provided for the excitation. The pacing board of the pacing unit is connected to electrodes, which are preferably made of graphite. Furthermore, the electrical connection and the conductor tracks of the pacing board are preferably made of gold or coated with gold.

[0018] Another aspect of the present invention provides that fluorescence images of the samples to be examined are also taken. In this case, the samples are treated with suitable dyes (e.g., for calcium transients) and illuminated within the examination chamber in such a way that fluorescence radiation emitted by the samples is excited and fluorescence images are recorded and evaluated. The evaluation takes place in particular taking into account the spatial and / or temporal radiation intensity. The latter is important, for example, for measuring calcium transients during the contraction of heart tissue within the range of a few milliseconds. In the case of the heart, it is particularly important to be able to perform frequency-controlled measurements via electrical stimulation of the cells / tissues. Corresponding elements for recording, storing, and evaluating the fluorescence images are provided. Likewise, theevaluation unit and the programs that can be executed by the evaluation unit are designed in such a way that at least one property of the marked samples can be or is determined on the basis of the properties of the recorded fluorescence images.

[0019] When characterizing tissue, especially muscle tissue samples such as engineered heart tissue (EHT) samples, for functional performance, several endpoints are typically measured to assess their viability, maturity, and physiological relevance, including voltage (action potentials), calcium transients, and contractility. Contractility assessments are often considered as primary endpoint due to their direct relevance to cardiac function and pathophysiology. Voltage and calcium measurements complement contractility analysis by providing mechanistic insights into the electrical and calcium handling properties of engine. It is possible to measure both calcium transients and voltage using fluorescence dyes. These dyes allow for the non-invasive detection of dynamic changes in intracellular calcium and membrane potential (or other targets, like reactive oxygen species) in living cells, including cardiomyocytes within EHTs but also cells / tissues including those of solid organs stained with fluorophores for respective processes of interest.

[0020] Moreover, fluorescence measurements are widely used in various tissues and cell types for studying cell functions, tissue organization, and disease mechanisms. They offer detailed insights into dynamic processes at the cellular and subcellular levels, significantly contributing to the understanding of the biology and pathology of various tissues. Fluorescent dyes are also utilized in tumor and cancer research to investigate cell proliferation, apoptosis, cell migration, and tumor metastasis. In addition, with this extension the device and system according to the present invention could also be utilized in the field of optogenetics.

[0021] A further aspect of the present invention relates to measurement chamber comprising: a sample tray; one or more light sources; and one or more cameras configured to capture images and / or video of a plurality of tissue samples when loaded onto the sample tray. In at least one embodiment, the measurement chamber is operatively coupled to an evaluation unit configured to evaluate the plurality of tissue samples in parallel.

[0022] In at least one embodiment, the evaluation unit is configured to evaluate one or more parameters of the plurality of tissue samples. In at least one embodiment, the one or more parameters are selected from action potential, calcium transients, and contractility.

[0023] In at least one embodiment, the measurement chamber further comprises: a pacing unit operatively coupled to the one or more light sources and the one or more cameras. In at least one embodiment, the pacing unit is configured to generate a stimulation signal to stimulate one or more tissue samples when loaded onto the sample tray.

[0024] In at least one embodiment, the measurement chamber further comprises: a pacing board comprising a plurality of electrode pairs, the pacing board being configured for insertion of the electrode pairs into sample wells in a one-to-one correspondence. In at least one embodiment, the pacing board is operatively coupled to the pacing unit.

[0025] In at least one embodiment, the pacing board comprises conductive tracks arranged in an interdigitated configuration to provide a two-dimensional arrangement of electrode pairs on the pacing board.

[0026] In at least one embodiment, the evaluation unit is configured to capture measurement data from the stimulation of the one or more tissue samples by the pacing unit.

[0027] In at least one embodiment, the measurement chamber is operatively coupled to a computing device configured to provide visualization of the measurement data.

[0028] In at least one embodiment, the measurement data comprises contractile data for the tissue samples.

[0029] In at least one embodiment, the one or more tissue samples comprise engineered heart tissue (EHT) samples.

[0030] In at least one embodiment, the pacing unit is configured to stimulate a plurality of the tissue samples simultaneously.

[0031] A further aspect of the present invention relates to a pacing board configured for use with a measurement chamber to analyze tissue samples, the pacing board comprising a plurality of electrode pairs, the pacing board being configured for insertion of the electrode pairs into sample wells of a multi-well plate in a one-to-one correspondence.

[0032] In at least one embodiment, the pacing board comprises conductive tracks arranged in an interdigitated configuration to provide a two-dimensional arrangement of electrode pairs on the pacing board.

[0033] In at least one embodiment, the pacing board is configured to interface with a pacing unit operatively coupled to a measurement chamber.

[0034] In at least one embodiment, the pacing board is configured to simultaneously apply a stimulation signal to tissue samples in the multi-well plate when the plurality of electrode pairs are inserted thereon in response to a signal received from the pacing unit when operatively coupled thereto.

[0035] A further aspect of the present invention relates to an assembly for characterizing a plurality of tissue samples, the assembly comprising: a multi-well plate comprising a plurality of wells; a sample transport tray disposed on the multi-well plate; and a plurality of sample racks disposed on the sample transport tray. In at least one embodiment, each of the plurality ofsample racks comprises at least one tissue sample that extends through an aperture of the sample transport tray and is inserted into one of the plurality of wells.

[0036] In at least one embodiment, the assembly further comprises: a pacing board disposed above the plurality of sample racks, the pacing board comprising a plurality of electrode pairs. In at least one embodiment, each of the plurality of electrode pairs is inserted into a well of the multi-well plate.

[0037] A further aspect of the present invention relates to a method of characterizing a plurality of engineered heart tissue (EHT) samples, the method comprising: placing a multi-well plate onto a sample stage of a measurement chamber, the multi-well plate comprising, in each well, one of the plurality of EHT samples; and causing a pacing unit of the measurement chamber to deliver a stimulation signal to the plurality of EHT samples.

[0038] In at least one embodiment, the method further comprises: inserting electrodes of a pacing board into each well of the multi-well plate prior to placing the multi-well plate onto the sample stage. In at least one embodiment, the stimulation signal is delivered via the electrodes of the pacing board.

[0039] In at least one embodiment, the method further comprises: computing one or more parameters associated with contractility of the EHT samples based on the stimulation.

[0040] In at least one embodiment, the method further comprises: capturing fluorescence images or video of the EHT samples; and deriving one or more parameters of the EHT samples from the fluorescence images or video.

[0041] In at least one embodiment, the method further comprises: capturing images and / or video of the plurality of EHT samples during the stimulation.

[0042] In at least one embodiment, the method further comprises: displaying, by a computing device, the captured images and / or video.

[0043] A further aspect of the present invention relates to a transduction plate adapted for treating a plurality of tissue samples, the transduction plate comprising: a rigid body; and a plurality of wells formed in the body in a grid configuration. In at least one embodiment, each of the plurality of wells comprises a transduction chamber formed within the well having a noncircular shape.BRIEF DESCRIPTION OF DRAWINGS

[0044] The disclosure described herein is illustrated by way of example and not by way of limitation in the accompanying figures.

[0045] FIG. 1 A illustrates an exemplary measurement chamber in accordance with at least one embodiment.

[0046] FIG. IB illustrates an open view of the exemplary measurement chamber in accordance with at least one embodiment.

[0047] FIG. 1C illustrates a cutaway view of a measurement area within the exemplary measurement chamber in accordance with at least one embodiment.

[0048] FIG. 2 illustrates an exemplary system for tissue measurement in accordance with at least one embodiment.

[0049] FIG. 3 illustrates an automated data pipeline for use in accordance with at least one embodiment.

[0050] FIG. 4 illustrates an exemplary information infrastructure in accordance with at least one embodiment.

[0051] FIG. 5 illustrates multidimensional analytics for the analysis of sample data in accordance with at least one embodiment.

[0052] FIG. 6A shows an upside down view of an illustrative casting mold spacer in accordance with at least one embodiment.

[0053] FIG. 6B shows an upside down side view of a technical drawing of a casting mold spacer in accordance with at least one embodiment.

[0054] FIG. 7A shows an upside down view of an illustrative sample rack in accordance with at least one embodiment.

[0055] FIG. 7B shows a technical drawing of an upside down side view of a sample rack in accordance with at least one embodiment.

[0056] FIG. 8Ais an orthographic view of an exemplary transduction plate in accordance with at least one embodiment.

[0057] FIG. 8B shows a cross-sectional view of the exemplary transduction plate in accordance with at least one embodiment.

[0058] FIG. 9A shows a top-down view of an exemplary sample transport tray in accordance with at least one embodiment.

[0059] FIG. 9B shows a bottom-up view of the exemplary sample transport tray in accordance with at least one embodiment.

[0060] FIG. 10A shows an exemplary transport tray holding six sample racks placed onto a 24-well plate.

[0061] FIG. 10B shows a user removing the transport rack by hand to transport the six sample racks in parallel.

[0062] FIG. 11 A illustrates an exemplary pacing board in accordance with at least one embodiment.

[0063] FIG. 1 IB illustrates engagement of the exemplary pacing board into a multi-well in accordance with at least one embodiment.

[0064] FIG. 12 illustrates a top down schematic view of conductive tracks on an exemplary pacing board in accordance with at least one embodiment.DETAILED DESCRIPTION

[0065] Embodiments of the present disclosure relate to a system, device, and method for the examination, treatment, measurement, and analysis of engineered tissue, as well as components to facilitate the same. Moreover, embodiments of the present disclosure relate to a system comprising components that provide automated and reproducible examinations and evaluation of tissue samples, such as muscle tissue samples (e.g., heart tissue or engineered heart tissue samples). During examination, a series of image recordings of the tissue sample can be captured in a short time and made available for evaluation. The system is configured to generate series of images of a large number of such samples simultaneously. For example, in certain embodiments, a video sequence of the samples moving during the recordings is generated from the series of recorded images.

[0066] Further embodiments of the present disclosure relate to a microtiter plate for the treatment of tissue samples. The microtiter plate can include individual recesses for receiving and holding samples in a manner that provides a small volume adapted to the size of each sample to be treated. The amount of medium required for treatment, which can contain viral vectors therefore be comparatively expensive, can be minimized compared to the use of conventional microtiter plates. In at least one embodiment, this treatment or the use of this microtiter plate takes place before the examination of the samples with image generation.

[0067] Further embodiments of the present disclosure relate to methods and devices for stimulating samples with electrical pulses. For example, in at least one embodiment, a pacing board is utilized for providing electrical pulses to the samples (e.g., tissue samples). In at least one embodiment, the pacing board comprises electrodes, such as graphite electrodes. Electrical connections and tracks of conductive material along the pacing board may comprise gold or another conductor that may be coated with gold. In at least one embodiment, the pacing board can be used in combination with handling frames (e.g., a sample transport tray) and support elements, where the handling frame and / or the pacing board include suitable structures, stops, locking lugs, or the like to ensure a correct arrangement in relation to each other.

[0068] Certain embodiments of the present disclosure may be utilized to facilitate examination of samples via fluorescence imaging. In such embodiments, samples may first be treated with suitable dyes (e.g., for calcium transients) and illuminated within the measurementchamber such that the treated samples are excited and emit radiation, and fluorescence images are recorded and evaluated. The evaluation may take into account the spatial and / or temporal radiation intensity. Temporal radiation intensity is useful, for example, for measuring calcium transients during the contraction of heart tissue within the range of a few milliseconds. In the case of the heart, the embodiments can allow for frequency-controlled measurements to be performed via electrical stimulation of the cells / tissues.

[0069] When characterizing tissue samples, such as EHT samples, for functional performance, several endpoints are typically measured to assess their viability, maturity, and physiological relevance, including voltage (action potentials), calcium transients and contractility. Contractility assessments are often considered as primary endpoint due to their direct relevance to cardiac function and pathophysiology. Voltage and calcium measurements complement contractility analysis by providing mechanistic insights into the electrical and calcium handling properties of EHT samples. The embodiments described herein provide the ability to measure both calcium transients and voltage using fluorescence dyes. The use of such dyes allows for the non-invasive detection of dynamic changes in intracellular calcium and membrane potential (or other targets, like reactive oxygen species) in living cells, including cardiomyocytes within EHT samples and also cells / tissues including those of solid organs stained with fluorophores for respective processes of interest.

[0070] Moreover, fluorescence measurements are widely used in various tissues and cell types for studying cell functions, tissue organization, and disease mechanisms. They offer detailed insights into dynamic processes at the cellular and subcellular levels, significantly contributing to the understanding of the biology and pathology of various tissues. It is contemplated that the embodiments described herein may be utilized for evaluation of samples utilizing fluorescent dyes in tumor and cancer research to investigate cell proliferation, apoptosis, cell migration, and tumor metastasis, as well as for the purpose of optogenetics studies.

[0071] Advantages of the present embodiments, which are described in greater detail herein, include, but are not limited to: (1) sterile video-optical recording of tissue samples; (2) userindependent tissue recognition within samples; (3) parallel analysis of multiple samples (e.g., up to 24 or more) within a short time period (e.g., less than about 2 minutes); (4) fully-automated functional characterization of samples, including contraction against an elastic resistance; (5) an integrated pacing unit for simultaneous stimulation of multiple samples; (6) deep data insight capability via processing of multidimensional data arrays for comprehensive sample analysis; (7) improved data integrity via artificial intelligence-driven quality checks and intelligent classification of measurements; (8) improved data integration via a combination of informationfrom disparate data sources; and (9) sample evaluation performed in an automated and reproducible matter independent of the ability and skill of the user of the system.MEASUREMENT CHAMBER EMBODIMENTS

[0072] An exemplary measurement chamber configured for analysis of tissue samples in accordance with various embodiments of the disclosure is now described. FIGS. 1 A and IB illustrate closed and open views, respectively, of the exemplary measurement chamber 100 (which may also be referred to herein as an examination chamber). FIG. 1C shows a cutaway view of the measurement area within the measurement chamber 100. In at least one embodiment, the measurement chamber is equipped with a sample tray 110 that opens upon request (which may be software-based command received from a user) to insert a sample plate 112 containing tissue samples (e.g., a 24-well cell culture plate). In at least one embodiment, the positioning of the plate 112 may be automated via an input shaft and a handling mechanism.

[0073] In at least one embodiment, the measurement chamber 100 includes a code reader 111 (e.g., a QR code reader) configured to capture information about the samples on the plate 112, which can be defined prior to plate insertion and linked by an individual code (e.g., a QR code). In at least one embodiment, after plate insertion into the measurement chamber 100, various environmental conditions for the samples, including gas (CO2, O2, and N2), relative humidity, and temperature can be regulated and maintained within the measurement area. In at least one embodiment, the measurement chamber 100 includes one or more gas inlets to control the environmental conditions for the tissue samples. The gas mixture of CO2, O2, and N2 may depend on the respective assay / project and may be mixed independently and then supplied to the measurement chamber 100. Conditions can measured, for example, via one or more sensors and presented for presentation in a display device via accompanying software.

[0074] In at least one embodiment, the measurement chamber 100 is configured to provide fully automated and user-independent contractility measurements on samples. Software-based initiation of the measurements can provide automatic figure recognition of the individual samples. In at least one embodiment, figure recognition takes place on one or more individual cameras 120A-120B (e.g., high-speed cameras) positioned below the plate 112, and which function as a recording unit. In at least one embodiment, one or more lighting panels 122A- 122B (e.g., light-emitting diode lighting panels) are positioned below the plate 112 at an angle to optimize the lighting conditions for the samples. In at least one embodiment, the one or more lighting panels are configured to provide illumination during the entire measurement period (e.g., on average 90-120 seconds for a plate with 24 EHT samples). In other embodiments, the one or more lighting panels 122A-122B may comprise LEDs combined with controlled shutters.

[0075] In at least one embodiment, the one or more cameras 120A-120B is / are configured to record image and video of the samples through an optically transparent base of the plate 112. In at least one embodiment, the one or more lighting panels 122A-122B is / are arranged so as to not protrude into and obscure the optical axis / axes of the lenses / apertures 121A-121B of the one or more cameras 120A-120B, respectively.

[0076] In at least one embodiment, the one or more cameras 120A-120B is / are used to simultaneously record images of a large number of tissue samples arranged, for example, on the small posts inside the wells of the plate 112 when filled with medium. In such embodiments, individual image recording areas and / or foci can be adjusted by a control unit of the one or more cameras 120A-120B, which may be under the direct control by a user using a software interface. In at least one embodiment, a video-sequence / movie is generated from a series of image recordings.

[0077] FIG. 2 illustrates an exemplary system 200 in accordance with at least one embodiment. The system 200 includes a pacing unit 210, which is communicatively coupled to a computing device 250, for example, via a network (e.g., an ethernet connection, a wireless connection, such as a Wi-Fi network, etc.).

[0078] In at least one embodiment, the pacing unit 210 is included within or operatively coupled to the measurement chamber 100. The pacing unit 210 comprises a processing device 220, an amplifier 230, and a power supply 240. For example, in at least one embodiment, the amplifier 230 is operatively coupled to a pacing board (such as the pacing board 1100 described with respect to FIGS. 11 A, 11B, and 12) to provide electrical stimulation via electrodes to tissue samples contained within a sample plate.

[0079] In at least one embodiment, the processing device 220 may comprises one or more of a main memory (e.g., read-only memory (ROM), flash memory, dynamic random access memory (DRAM) such as synchronous DRAM (SDRAM) or Rambus DRAM (RDRAM), etc ), a static memory (e.g., flash memory, static random access memory (SRAM), etc.), and a data storage device, which communicate with each other via a bus. In at least one embodiment, the processing device 220 represents one or more general-purpose processing devices such as a microprocessor, central processing unit, or the like. More particularly, the processing device 220 may be a complex instruction set computing (CISC) microprocessor, reduced instruction set computing (RISC) microprocessor, very long instruction word (VLIW) microprocessor, or a processor implementing other instruction sets or processors implementing a combination of instruction sets. The processing device 220 may also be one or more special-purpose processing devices such as an ASIC, a field programmable gate array (FPGA), a digital signal processor (DSP), network processor, or the like. The processing device 220 is configured to executeinstructions for performing the operations and steps discussed herein, such as operations associated with operation of the measurement chamber 100 and its components, as well as with data processing and sample stimulation. In at least one embodiment, the processing device 220 is a Raspberry Pi device.

[0080] In at least one embodiment, the processing device 220 may include a computer- readable storage medium on which is stored one or more sets of instructions (e.g., software) embodying any one or more of the methodologies or functions described herein. The instructions may also reside, completely or at least partially, within a main memory of the processing device 220, within a memory of the computing device 250, or within a separate memory. The instructions may further be received over a network from the computing device 250 (e.g., via an ethernet connection). In at least one embodiment, the processing device 220 may be separate from the pacing unit 210, and one or more similar processing devices may be utilized within the measurement chamber 100 (e.g., for recording measurement data, for controlling the cameras 120A-120B, etc.).

[0081] In at least one embodiment, the power supply 240 may be an uninterruptible power supply (UPS) that is part of the pacing unit 210 or separate from the pacing unit 210. In at least one embodiment, the power device may provide information about the power level of the UPS. For example, the power supply 240 may provide one or more interfaces to provide an indication of a power level, a time window remaining prior to shutdown of the pacing unit 210 or one or more of its components, a power consumption rate, an indicator of whether the pacing unit 210 is utilizing an external power source or battery power, and other power related information.

[0082] It is noted that the pacing unit 210, in certain embodiments, may be optional or may not be utilized for stimulating tissue samples. For example, the system 200 may still be used for the measurement, evaluation, and / or visualization of spontaneous movements of tissue samples under various environmental conditions (e.g., conditions of the medium, the presence of various agents or toxins introduced into the medium, gas conditions, etc.) without direct electrical stimulation from the pacing unit 210.

[0083] In at least one embodiment, the computing device 250 is representative of one or more computing devices such as a server, a router computer, a server computer, a personal computer, a mainframe computer, a laptop computer, a tablet computer, a desktop computer, etc. In at least one embodiment, the computing device 250 may be a server utilized by the processing device 220, for example, to process and display sample data. In at least one embodiment, additional servers may be present. In at least one embodiment, the computing device 250 may function as an evaluation unit for the evaluation of images and / or the generation and evaluationof video sequences, which may be performed using evaluation software stored and executed thereon.AUTOMATED DATA PIPELINE AND DATA ANALYTICS EMBODIMENTS

[0084] In at least one embodiment, the system is configured to provide an automated data pipeline and data analytics functionality using software implemented on one or more of the processing device 220 or the computing device 250. The software may comprise back-end and front-end components. In at least one embodiment, the back-end component is implemented by the processing device 220 to control the functioning of the measurement chamber 100 (e.g., data acquisition, handling, and storage). In at least one embodiment, the front-end component is implemented by the computing device 250 to provide user control, and may be web-based (e.g., implemented through a web-based user interface on the computing device 250 with access being provided by a separate server).

[0085] FIGS. 3 and 4 illustrate an automated data pipeline and an information infrastructure, respectively, in accordance with at least one embodiment. In at least one embodiment, the system 200 comprises a fully automatic pipeline for data processing, storage, and analysis. The pipeline may include video-optical recordings and user independent figure recognition implemented via one or more machine learning models, followed by automatic parameter calculation (e.g., analysis of the sample data based on a gradient descent machine learning algorithm). After data processing, the raw data (images) and their derived param eters / metadata can be stored in one or more databases (e.g., PostgreSQL database) and in the cloud (e.g., Amazon S3 cloud). In at least one embodiment, all user activities are stored for later audit trails (e.g., using Elastic Search / Kibana), and further information may be stored based on application monitoring and error tracking (e.g., using sEntry). In at least one embodiment, the automated data pipeline and information infrastructure represent a complete good laboratory practice (GLP) compliant data acquisition process.

[0086] In at least one embodiment, pattern recognition utilized in data analysis may be based on a Halcon script. In at least one embodiment, the pattern recognition may be implemented based on a You-Only-Look-Once (YOLO) algorithm, for example, when fluorescence measurements are performed. YOLO is a real-time object detection algorithm that uses a convolutional neural network to predict the bounding boxes and class probabilities of objects in input images.

[0087] In at least one embodiment, the system 200 is configured to run data analytics software that may allow the user to query and analyze sample data (e.g., via a web interface). The evaluation of the images and / or the generation and evaluation of video sequences, and thusthe examination of the movement of the individual samples, takes place by means of a suitable evaluation software. In at least one embodiment, a series of image recordings of the samples positioned in the measurement chamber 100 are generated simultaneously. Generated data can be collected automatically, transferred into contractile parameters, stored, and displayed. In at least one embodiment, the data analytics software may operate based on the online analytical processing (OLAP) theory by utilizing a multi-dimensional array of each possible column in the database as a “dimension” and then to calculate a “measure” per cube. Multidimensional analysis is depicted visually in FIG. 5, where the dimensions correspond to the well position within a sample plate, well dish plate ID, and recording number. One or more additional dimensions may be used in combination with or in lieu of those shown including, but not limited to, sample age, force measurements, frequency of stimulation, stimulation amplitude, etc. Multidimensional data analysis allows for comprehensive query functions allowing the user to explore and navigate multiple dimensions of data, as well as view and visually compare multiple measurements simultaneously within a single view.SAMPLE PREPARATION

[0088] Described now are devices and methods for preparing tissue samples for evaluation in the measurement chamber 100. While the embodiments are described with respect to engineered tissue samples (such as EHT samples), these embodiments are merely exemplary and provided to illustrate how a sample may be prepared to utilize the tissue sample evaluation functionality of the measurement chamber 100.

[0089] Prior to transduction, EHT samples can be prepared utilizing, for example, the components now described. FIGS. 6A and 6B show an illustrative casting mold spacer 600 and a technical drawing thereof, respectively, in accordance with at least one embodiment. The casting mold spacer 600 can be used for the generation of casting molds in a gel-forming material, and includes a body 610, a plurality of spacer extensions 620 each protruding from a shoulder 622 along the length of the body 610. In at least one embodiment, the casting mold spacer is a unitary structure formed, for example, from PTFE. It is to be understood that other suitable materials may be used, as appreciated by those of ordinary skill in the art.

[0090] The casting mold spacer 600 and its features may have geometries and sizes adapted for fitting into a row of wells within a standard multi-well plate (also referred to herein as a microtiter plate), and may vary depending on the plate size and geometry for which the casting molds are to be formed. For example, each shoulder 622 is sized and spaced in order to fit the diameters and match the spacing of round wells within the multi-well plate within which molds will be cast, while securing the casting mold spacer 600 in place. The height 630 of each spacerextension 620 is such that the combined height of shoulder 622 and spacer extension 620 are about equal to or less than the depth of the corresponding well. The length 640 and perpendicular width of each spacer extension 620 may be selected to correspond to a target EHT sample size.

[0091] In at least one embodiment, the casting mold spacer 600 is utilized in the generation of molds in a gel-forming material (e.g., liquid agarose) within a multi-well plate. In an exemplary embodiment, liquid agarose is filled into wells of the multi-well plate having the spacer extensions 620 of the casting mold spacer 600 inserted therein. Once the agarose hydrogel molds are formed, the casting mold spacer 600 is then removed from the wells, leaving behind cavities within the hydrogel molds.

[0092] After the molds are formed in each well, EHT samples can be formed by using a sample rack 700 as illustrated in FIGS. 7A and 7B. In at least one embodiment, the sample rack 700 is formed from a unitary silicone material, such as polydimethylsiloxane (PDMS). In at least one embodiment, the sample rack 700 is transparent, or may be opaque if pattern recognition or contrast is preferred during sample imaging. The sample rack includes a plurality of solid posts 720 extending from a frame 710. Each post 720 may include a bulged portion at its distal end so as to maintain a tissue sample in place when fixed thereon. As shown, the sample rack 700 includes post pairs 730 each having a spacing sized to fit within the a gel mold formed in a multi-well plate, as previously described. In at least one embodiment, a center-to- center spacing 740 of posts within each post pair 730 is from about 5 mm to about 15 mm.

[0093] In at least one embodiment, the sample rack is placed over a multi-well plate containing gel molds such that each post pair 730 is inserted into a corresponding well within a cavity formed by a spacer extension 620 during the gel molding. To form an EHT sample, a composition comprising a hydrogel-forming liquid (e.g., containing fibrinogen and thrombin) and cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC). Within each cavity, the tissue sample is formed across and fixed to each post pair 730. During culture, the CMs align along force lines and start to beat spontaneously and coherently. Generated forces and contractile parameters can be observed in the EHT samples. The EHT samples can serve as a testbed to evaluate the effect of compounds including viral vectors and their transduction efficacy.

[0094] In accordance with the embodiments described herein, transduction of multiple three- dimensional EHT samples in parallel reduces the required volume of surrounding liquid and brings the transduction agents in close proximity to the cells. Current transduction of three- dimensional tissue samples requires large volumes of, for example, viral vector for the treatment of such samples prior to measurement and analysis. Such approaches are particularly cost-intensive in gene therapy studies, where large amounts of transduction agents (such as viral vectors, lipid nanoparticles, etc.) are needed to achieve reliable transduction in all cells of the tissue within the volume. The approach described herein advantageously reduces the amount of volume that is used to treat the cells by utilizing sample plates with geometries tailored to those of the tissue samples, thus reducing the overall volume needed for submerging and treating the tissue samples and increasing the overall transduction efficiency.

[0095] FIG. 8Ais an orthographic view of an exemplary transduction plate 800, in accordance with at least one embodiment. The transduction plate 800 comprises rigid body 810 having a plurality of wells 820 formed thereon. As illustrated, the transduction plate 800 contains 24 wells 820 for treating tissue samples in parallel, though it is to be understood that the total number of wells may vary (e.g., 48, 96, 384, or any suitable number of wells). In at least one embodiment, the body 810 is formed from a single unitary material such as a plastic material, such as polystyrene or polytetrafluoroethylene (PTFE), or another suitable material having a low binding affinity for transduction agents (e.g., viral vectors), and may be transparent or opaque. Each well 820 of the transduction plate 800 comprises a transduction chamber 822 in the form of a sub-well that extends deeper into the body 810. In at least one embodiment, each transduction chamber 822 has a predetermined size and geometry to match the corresponding size and geometry of each tissue sample to be treated (e.g., the tissue samples formed using the sample rack 700 described above). For example, in at least one embodiment, each transduction chamber 822 within each well 820 is substantially rectangular in shape (e.g., a rounded rectangle). In at least one embodiment, each transduction chamber 822 is sized to contain a total volume of about 100 pL to about 300 pL (e.g., about 200 pL).

[0096] In at least one embodiment, the internal shape of each transduction chamber 822 is adapted to reduce the volume of surrounding liquid during the transduction process to bring transduction agents within close proximity to the tissue sample when inserted therein, thus reducing the amount of vector needed for effective transduction. It is noted that the geometries of the wells 820 and transduction chambers 822 are not necessarily drawn to scale. FIG. 8B shows a cross-sectional view of the transduction plate 800, corresponding to the dotted line shown in FIG. 8 A. In at least one embodiment, the diameter 830 of each well 820 may range from about 7.5 mm to about 25 mm (provided that the diameter is at least about 2 mm or about 3 mm larger than the largest lateral dimension of its corresponding transduction chamber 822). In at least one embodiment, a depth 832 of each well 820 may range from about 5 mm to about 10 mm. In at least one embodiment, a depth 834 of each transduction chamber 822 may range from about 5 mm to about 15 mm. In at least one embodiment, a lateral width 836 of each transduction chamber 822 may range from about 3 mm to about 8 mm. In at least oneembodiment, a lateral length (perpendicular to the width 836) of each transduction chamber 822 may range from about 5 mm to about 15 mm. In at least one embodiment, a transduction plate may have one or more transduction chambers 822 that have dimensions different from other transduction chambers 822 within the transduction plate 800.

[0097] FIGS. 9A and 9B show top-down bottom-up views, respectively, of an exemplary sample transport tray 900. The sample transport tray 900 may be adapted to hold / secure a plurality of sample racks (e.g., similar to sample rack 700) to facilitate transport and handling in a manual or automated fashion. In at least one embodiment, the body 910 of the sample transport tray 900 is a unitary structure formed from a polymeric material, such as an FDA- approved and / or autoclavable material (e.g., iglidur® A350). In at least one embodiment, the smallest thickness of the sample transport 900 tray may range from 4 mm to about 8 mm (e.g., about 5 mm).

[0098] In at least one embodiment, a single sample transport tray 900 may be adapted to hold a plurality of linear sample racks (e.g., sample rack 700) in an array (e.g., at total of 6 sample racks each having 4 pairs of posts for insertion of the EHT samples into a 24-well transduction plate). In at least one embodiment, the transport tray comprises a plurality of apertures 920 separated by a central support that runs longitudinally along the transport tray and provides structural support. In at least one embodiment, the sample transport tray includes opposing sidewalls 930 that facilitate gripping and transporting of sample racks. FIG. 10A shows an exemplary transport tray holding six sample racks placed onto a 24-well plate. FIG. 10B shows a user removing the transport rack by hand to transport the six sample racks in parallel. The sample transport tray design may be used for both sample treatment applications (e.g., placing EHT samples into a transduction plate, such as the transduction plate 800) and for sample stimulation and evaluation (as discussed below).PACING BOARD EMBODIMENTS

[0099] FIGS. HA and 11B illustrate an exemplary pacing board 1100 and engagement of a multi -well plate in accordance with at least one embodiment. As shown, the pacing board 1100 is configured to stimulate 24 EHT samples in parallel, though the design may be adapted to accommodate other types of plate sizes and layouts as would be appreciated by those of ordinary skill in the art. The pacing board 1100 is configured to operatively couple to a pacing unit (such as the pacing unit 210), which can be controlled by software interface (for example, as described with respect to the system 200). The pacing board 1100 may be used to advantageously stimulate EHT samples without the production of toxic products (e.g., due to electrolysis, reactive oxygen species, etc.).

[0100] As illustrated in FIG. 11 A, the pacing board includes a plurality of electrode pairs 1110, each comprising needle-like electrodes configured for insertion into wells of a plate 1120 in proximity to a tissue sample. For example, the pacing board 1100 may be placed on top sample racks 1130 that are placed above and within their corresponding wells of the plate 1120. The bridges between the ends of the post represent EHT samples that span the posts within each well. For example, electrodes 1112 and 1114 can be inserted into the well 1122 and are near sample 1132, which spans a pair of posts of the corresponding sample rack. In at least one embodiment, the tip of an electrode is less than about 3 mm from its corresponding post. In at least one embodiment, the electrodes 1112 and 1114 may comprise graphite. In at least one embodiment, conductive portions of the electrodes and on the body of the pacing board may be plated with a conductive material, such as gold, while the portions of electrodes that are submerged within the wells of the plate are uncoated graphite.

[0101] FIG. 12 illustrates a top down schematic view of conductive tracks 1212 and 1214 , where the white dots represent electrodes oriented into the plane of the schematic. The square portions of the conductive tracks 1212 and 1214 correspond to electrode contacts for interfacing, for example, with the pacing unit 210. In at least one embodiment, the conductive tracks 1212 and 1214 form an interdigitated pattern that spans the underlying plate 1120 such that each pair of electrodes includes a working electrode and counter electrode. In at least one embodiment, the pacing board 1100 includes at least 2 electrode pairs, at least 4 electrode pairs, at least 8 electrode pairs, at least 16 electrode pairs, at least 24 electrode pairs, at least 48 electrode pairs, at least 96 electrode pairs, or any number of pairs defined and inclusive of these points (e.g., from 16 electrode pairs to 48 electrode pairs). The electrode pairs may be arranged in a two- dimensional grid configuration with the electrodes extending in the normal direction from the pacing board 1100.ILLUSTRATIVE EMBODIMENTS

[0102] The following examples are set forth to assist in understanding the disclosed embodiments and should not be construed as specifically limiting the embodiments described and claimed herein. Such variations of the embodiments, including the substitution of all equivalents now known or later developed, which would be within the purview of those skilled in the art, and changes in formulation or minor changes in experimental design, are to be considered to fall within the scope of the embodiments incorporated herein.

[0103] Exemplary methods of producing EHT samples are described, for example, in U.S. Patent No. 7,618,452 B2, the disclosure of which is hereby incorporated by reference hereinin its entirety. Examples 2-4 describe methods for producing EHT samples for analysis in accordance with the various embodiments described herein.Example 1: Exemplary measurement chamber components

[0104] Exemplary components for use in the measurement chamber 100, system 200, and pacing unit 210 and are now described. It is to be understood that other components may be utilized in addition to or in lieu of the components described below, as would be appreciated by those of ordinary skill in the art.

[0105] Camera(s): two cameras (Baumer VLXT 240M); heat sink (Heat Sink Type B); I / O cable (Cable Power Adapter M12 / DC Connector); Ethernet (Cable GigE M12x / RJ45); lens (EdmundOptics 75mm / F2.8); camera tube (Tube M62 hard anodized); network card (10-GigE PCIe interface board).

[0106] Illumination: lamps (Lumimax LQHP80 (32° lenses)); power I / O cable (Lumimax connection cable).

[0107] Computing / processing devices: Power-PC (Thinkstation P620; 128 GB RAM).

[0108] Sensors / actuators: Modbus TCP gateway (Advantech EKI-1221-CE); CO2 sensor (GMP251); temperature / humidity sensor (HMP110); Modbus TCP IO device (Advantech ADAM 605); Modbus switch (Moxa EDS-205 / 2005); power supply unit (ATM300T-A240 300W / 100-240VAC / 24VDC / 12.5A desktop power supply unit).

[0109] Motorized components: linear guide (Igus SLNV-27); spindle stepper motor (Igus MOT-ST-28-L-A-A); motor control (Igus DI Motor control).

[0110] Pacing unit: trigger cable coax (Coaxial cable 5m); BNC connector (BNC plug to terminals); spring contact (UEBK-3026 l-UEBK-30262 UWEelectronic, UEBK-30262 contact plate, UEBK seal); level converter Pacer (LEG SF1); processing device (Raspberry Pi).

[0111] Heating: power supply (ATM300T-P240); silicone heating foil (Thermo Technologies 3326115); heat sink (Aluminum Components 122AB); heater controller (Heater controller TR-46 (24V)); temperature sensor (Temp sensor PT 100); heating relay (SS relay + DIN rail adapter (24VDC / 10A) = 240W); fan (WINSINN 40 mm Blower Fan 24 V, alternative DC radial fan, 24 V, 51 x 51 x 15 mm, ebm-papst, RLF 35-8 / 14 N); holder for heating controller (DI Y DB- Eyssen).

[0112] Scanner: MB006 (N4680, 2D scanner Honeywell); KSP (Adapter-PCB (Phytec) PL2600); FFC cable 12x0.5mm, l=50mm; USB-A cable (self-made).

[0113] Power supply: BACO NB02AGQ switch-disconnector 16A, Bachmann device supply cable halogen-free 5m, Bachmann device extension cable, Kopp 226320016 socket strip, assembly Baco by Jager Elektrotechnik.

[0114] Other components: flange Socket (computer housing) Degson 15CDGV-3.81-02P-14- 00AH; plug (computer housing) Degson 15EDGKD-3.81-02P-14-00AH-1; hook-and-loop cable ties (Voltcraft Velcro cable ties); Brennenstuhl 6-way socket strip; enclosure base (Essentra 466197 d48 125).Example 2: Culture ofhiPSCs for use inEHT samples

[0115] Human induced pluripotent stem cells (hiPSC) are frozen in aliquots and thawed upon request for expansion on Geltrex®-coated 6-well-dishes (Nunc) and subsequent differentiation into cardiomyocytes (Example 3 below). During the culture, hiPSCs are monitored closely (e.g., daily) to avoid and detect spontaneous differentiation or contamination. Screening with FACS for pluripotency markers (e.g., SSEA3) is recommended, if feasible.

[0116] Materials:• Geltrex® (Gibco, cat# A1413202)• Y-27632 (Biorbyt, cat# orbl54626 or Tocris cat# 1254 / 10; 10 mM in H2O use 10 pM). Store aliquots at -20 °C, use within 7 days when thawed and stored at 4 °C.• RPMI 1640 (PanBiotech, cat# P04- 16500)• PBS (PanBiotech, cat# P04-36500)• Accutase (Stemcell Technologies, cat# 07922), store frozen aliquots at -20 °C, after thawing for up to 7 days at 4 °C• Trypan blue (Gibco, cat# 15250-061)• DMSO (Sigma- Aldrich, cat# D8418• Fetal calf serum, FCS (Gibco, cat# 26140079) Heat inactivation (h.i.): Thaw 500 ml of FCS at 4 °C over night. Warm water bath to 56 °C. Wrap parafilm around cap of FCS bottle and place FCS in 56 °C for 1 h. Aliquot immediately afterwards and store aliquots at -20 °C.• Isopropanol (Chemsolute, cat# 1157)

[0117] Media:• mTeSR™ Plus (Stemcell Technologies, cat# 100-0276)• Freezing medium: 90% FCS h.i. with 10 % DMSO

[0118] An exemplary protocol is now described.

[0119] Passaging is performed when hiPSCs have reached approximately 70-80% confluence. hiPSCs are plated at a defined cell density of 0.4 x 106 / l 0 cm2.

[0120] Preparation of Geltrex® stocks and coating of cell culture plates: Passaging is performed when hiPSCs have reached approximately 70-80% confluence. hiPSCs are plated at a defined cell density of 0.4 x 106 / l 0 cm2. Upon receipt, Geltrex® is stored at 80 °C. ThawGeltrex® stock solution at 4 °C over night or on ice, mix 5 ml Geltrex® stock solution with 5 ml ice-cold RPMI (1 :2 pre-dilution). Make 500 pl aliquots of pre-diluted Geltrex® and store those working stocks at -80 °C.1. Thaw working Geltrex® aliquot on ice.2. Dilute Geltrex® working stock 1 :50 in ice-cold RPMI (final 1 : 100).3. Add diluted Geltrex® to cell culture vessel (1 ml / 10 cm2).4. Incubate at room temperature overnight or 1 hour at 37 °C. Coated cell culture vessels can be stored at 4 °C for up to two weeks. Wrap dishes in parafilm to avoid exsiccation.5. If cell culture plate was stored at 4 °C, warm plate at room temperature or 37 °C prior to use.6. Before use, remove diluted Geltrex® by aspiration and either plate cell suspension directly (thawing process, see 4.2) or add mTeSR™ Plus + Y-27632 (1 ml / 10 cm2) prior to hiPSC seeding.

[0121] Thawing of hiPSCs:1. Prepare coated wells, culture medium, water bath.2. Thaw cryotube(s) in water bath at 37 °C for 2-3 min. Carefully spray cryotubes with 70% ethanol, to avoid contaminations.3. Draw up 5 ml of RPMI with Y-27632 (10 pM, 1 :1000) in a pipette. Insert the full pipette into the cryotube and draw up the cell suspension in addition to the medium already in the pipette. The freezing medium will mix only with a slight gradient with the wash medium in the pipette.4. Slowly let cells and wash medium run down the wall of a 50 ml falcon tube and centrifuge for 5 min at 200xg.5. Aspirate supernatant (carefully, avoid hitting the pellet), add 4 ml mTeSR™ Plus + Y27632, scratch falcon tube to loosen pellet, resuspend carefully in with a 10 ml serological pipette and plate in 2 wells of a Geltrex®-coated 6-well plate (2 ml / well for cryotubes with -IxlO6cells). Aspirate Geltrex® shortly before hiPSC seeding to prevent drying of the surface.6. Next day: feed with 2 ml mTeSR™ Plus medium without Y-27632 and cultivate until hiPSC are confluent enough for passaging.

[0122] Passaging of hiPSCs (single cell passaging):1. Thaw Accutase at 4 °C overnight or quickly at 37°C.2. Add Y-27632 (10 pM, 1 : 1000) to required volume of Accutase solution (1 ml / 10 cm2) and mTeSR™ Plus medium (2 ml / 10 cm2) and RPMI medium (1 ml / 10 cm2). Warm up the media and PBS (1.5 ml / 10 cm2) at 37 °C.3. Remove Geltrex® from prewarmed cell culture plate by aspiration.4. Add mTeSR™ Plus + Y-27632 (1 ml / 10 cm2) and incubate cell culture plate at 37 °C, 5% CO2, to prepare the cell culture vessels for hiPSC seeding.5. Aspirate old medium from hiPSC cell culture vessel.6. Wash hiPSC cell culture vessel with PBS (1.5 ml / 10cm2).7. Add Accutase + Y-27632 to hiPSC cell culture vessel (1.0 ml / 10cm2).8. Incubate at 37 °C for 3-4 minutes until hiPSC detach from the surface.9. Use a serological pipette (e.g., 10 ml) to transfer 1 ml of RPMI + Y-27632 to the first vessel (10 cm2) and flush hiPSC by pipetting Accutase solution up and down onto the vessel surface 3-5 times. Take the whole amount to the next vessel and repeat for up to 3 vessels (10 cm2).10. Transfer to 50 ml tube.11. Rinse cell culture vessels with RPMI+ Y-27632 (1.0 ml / 10cm2).12. Transfer to the same tube used ins step 10.13. Centrifuge at 200xg for 5 minutes.14. Aspirate supernatant and add an appropriate volume of mTeSR™ Plus + Y-27632, approximately 2.0 ml / dissociated 6-well plate (max. 50 ml).15. Scratch falcon tube to loosen hiPSC pellet and carefully resuspend pellet by pipetting up and down for 1-2 times using a 10 ml pipette.

[0123] Counting of hiPSCs:1. Add prediluted hiPSC suspension to Trypan Blue (1 :2). Standard dilution works as follows: 450 pl RPMI + 500 pl Trypan Blue + 50 pl hiPSC suspension. Pellets should be resuspended in an appropriate amount of medium to avoid extremely high cell numbers during counting. If cell number is inappropriate during counting (either too high or too low), adjust the volume of the hiPSC suspension (either add medium or centrifuge again, resuspend in less volume).2. Add cell suspension after thorough resuspension to Neubauer counting chamber.3. Count at least 4 quarters and calculate mean.4. Calculate cell concentration per ml: Mean cell count x 10,000 (multiplier Neubauer counting chamber) x 20 (dilution factor) = Number of cells per ml.5. Multiply by volume of suspension for total number.

[0124] Seeding of hiPSCs:1. HiPSCs are plated at a defined cell density of 0.4 x 106 / l 0 cm2.2. Calculate the needed total volume of cell suspension to seed 0.4 x 106cells / 1 ml.3. Fill up the cell suspension with required volume of mTeSR™ Plus + Y-27632 and plate 1 ml directly to pre-filled Geltrex® coated cell culture well (4.3.4). Final volume: 2 ml / well. In case of more than one 6 well plate: pipette the needed amount for the required cell density from the cell suspension into a 15 ml tube (max. for two 6 well plates). Add to the cells the appropriate amount of mTeSR™ Plus supplemented with Y-27632 (e.g., 12 ml for two 6 well plates) with a 10 ml serological pipette and directly distribute the suspension to the vessels.4. Place plates in the incubator (CO2: 5%)

[0125] Feeding of hiPSCs (exemplary schedule):1. Day 0 passaging and seeding of cells.2. On day 1 after seeding feed with mTeSR™ Plus (2 ml / 10 cm2).3. On day 2 after seeding feed with mTeSR™ Plus (2 ml / 10 cm2).4. On day 3 after seeding feed with mTeSR™ Plus (2 ml / 10 cm2) or dissociate and reseed / freeze hiPSCs when cells have reached 70 - 80% confluence.

[0126] Freezing of hiPSCs:1. Prepare freezing medium (heat inactivated FCS+10% DMSO) and label cryotubes.2. Proceed as described under dissociation and counting (Step 0).3. Re-centrifuge desired amount of cells and resuspend in freezing medium.4. Aliquot IxlO6cells per cryotube.5. Quickly transfer cryotubes to freezing container or cell camper, store at -80 °C for the first 24 h, then move the cryotubes to -150 °C for long-term storage.Example 3: Cardiac differentiation of hiPSCs

[0127] The scope of this technique is the differentiation of iPSCs into cardiomyocytes for further experiments. The cardiomyocytes obtained with this protocol can then be used for e.g., EHTs, 2D culture, imaging. HiPSC-cardiomyocytes are dissociated into single cells using a defined collagenase solution.

[0128] Materials for hiPSC culture:• Geltrex® (Gibco, cat# A1413202)• Y-27632 (Biorbyt, cat# orbl54626 or Tocris cat# 1254 / 10; 10 mM in H2O; use 10 pM). Store aliquots at -20 °C, use within 7 days when thawed and stored at 4 °C.• mTeSR™ Plus (Stemcell Technologies, cat# 100-0276)• RPMI 1640 (PanBiotech, cat# P04- 16500)• PBS (PanBiotech, cat# P04-36500)• Accutase (Stemcell Technologies, cat# 07922), store frozen aliquots at -20 °C, after thawing for up to 7 days at 4 °C• Trypan blue (Gibco, cat# 15250-061)

[0129] Materials for cardiac differentiation:• STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit (Stemcell Technologies, cat# 05010)• Kit includes the following components: o Basal Medium o STEMdiff™ Ventricular Cardiomyocyte Diff Supplement A ( 1 OX) o STEMdiff™ Ventricular Cardiomyocyte Diff Supplement B (10X) o STEMdiff™ Ventricular Cardiomyocyte Diff Supplement C (10X) o STEMdiff™ Cardiomyocyte Maintenance Basal Medium o STEMdiff™ Cardiomyocyte Maintenance Supplement (50X)• Matrigel (Coming, cat# 356234; MG) is stored at -80 °C and is re-frozen once after initial thawing and aliquoting. Always check that Matrigel LOT has low endotoxin levels.

[0130] Materials for dissociation of cardiomyocytes:• Collagenase solution: o HBSS without Ca2+(Pan Biotech, P04-34500) o + Collagenase II (200 units / ml, changes for each lot, around 1 mg / ml; ThermoFisher, 17101015; stock: powder) o + 10 mM HEPES (Stock 1 M), o Sterile filtration, aliquot preparation (20 and 40x ml), storage at -20°C o Thaw ideally over night at 4°C, add before using. o Before use, add:■ 10 pM Y-27632 ( Biorbyt, orbl54626 or Tocris cat# 1254 / 10); Stock: 10 mM in H2O - after resolving sterile filtered, frozen at -20°C, °C. Thawed aliquots should be stored at 4°C and used within two weeks, if kept cold during procedure.■ 30 pM BTS (N-Benzyl-p-Toluenesulfonamide, Ambeed, A320082-25G); Stock: 30 mM in DMSO, -20°C, Thawed aliquots should be stored at 4°C and used within two weeks, if kept cold during procedure.• Washing solution: HBSS without Ca2+(Pan Biotech, P04-34500)• Blocking buffer: o DMEM (Sigma, D5546) or RPMI (ThermoFisher, 21875091)o + Penicillin / streptomycin 1% (ThermoFisher, 15140122) o + DNAse solution (6 pl DNase / ml) o Dissolve 100 mg DNase II Type V (Sigma, D8764-150KU) in 50 ml PBS and make aliquots of 2 ml. Store at -20°C for up to 1 year.• Basic medium: o DMEM (Pan Biotech, P04-01548) or RPMI (Pan Biotech, P04- 16500) o + Penicillin / streptomycin 1% (ThermoFisher, 15140122)• Freezing medium: FCS heat inactivated (h. i.) with 10 % DMSO

[0131] An exemplary protocol is now described:

[0132] HiPSCs expansion and preparation: Thaw and expand hiPSC as described in Example 1 on Geltrex® for two passages. The number of thawed cryovials depends on the desired number of cells to be differentiated. Avoid more than two splits on Geltrex® as single cell passing can lead to genetic abnormalities.

[0133] For differentiation, seed hiPSC on High Growth Factor (HGF) Matrigel®-coated 6- wells during the third split.Day -2: Seeding hiPSCs onto Matrigel-coated 6-well plates Matrigel-coating of 6-well cell culture plates• Thaw Matrigel® aliquot on ice.• Dilute Matrigel® 1 :60 in RPMI (ice-cold).• Add diluted Matrigel® to cell culture vessel (1 ml / well).• Incubate at room temperature overnight or 1 h at 37 °C. Coated cell culture vessels can be stored at 4 °C for up to two weeks. Wrap dishes in parafilm to avoid exsiccation.• If cell culture plate was stored at 4 °C, warm plate at room temperature prior to use (min. 1 h).• Before use, remove diluted Matrigel and add mTeSR™ Plus + Y-27632 (1 ml / well).Dissociating and seeding of hiPSCs for differentiation• Dissociate hiPSCs from Geltrex®-coated cell culture vessels using Accutase as described in Example 1• For differentiation, seed cells on prepared Matrigel® coated 6-well plates at a density of 0.9xl06cells / well in mTeSR™ Plus + Y-27632. Seed cell in the afternoon at 2 pm. The number of plated cells is critical since hiPSCs must reach 90-95% confluency at day 0 when differentiation is initiated. The seeding density must be determined for each hiPSC line.Day -1 : Medium change with mTeSR™ Plus medium1. Assess cell density and cell morphology under the microscope.2. Gently remove mTeSR™ Plus medium + Y-27632 by aspiration and replace with 2 ml of mTeSR™ Plus medium (without Y-27632).3. Incubate at 37°C for 24 hours. Do not disturb cells.

[0134] Cardiac differentiation of hiPSCs: Cardiac differentiation is performed according to STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit protocol. The media volumes are adjusted accordingly. Every media change is done with 4 ml / well.

[0135] Preparation of media A, B, C• Thaw Differentiation Supplement A / B / C at room temperature (15 - 25°C). Mix thoroughly. If not used immediately, aliquot Supplement and store at -20°C. Once aliquots are thawed, do not re-freeze.• Dilute Differentiation Supplement A / B / C 1 : 10 with Differentiation Basal Medium, (max. total volume per Kit is 100 ml: 10 ml Supplement A / B / C + 90 ml Differentiation Basal Medium). If not used immediately, store Medium A at 2 - 8°C for up to 2 weeks.• Warm media at 37°C shortly before use (warm Medium A only after Matrigel was added)

[0136] Preparation of Maintenance Medium:• Thaw Maintenance Supplement at room temperature (15 - 25°C). Mix thoroughly. If not used immediately, aliquot Supplement and store at -20°C. Once aliquots are thawed, do not re-freeze.• Dilute Maintenance Supplement 1 :50 with Maintenance Basal Medium, (max. total volume per Kit is 500 ml: 10 ml Supplement + 490 ml Maintenance Basal Medium). If not used immediately, store Medium A at 2 - 8°C for up to 2 weeks. Warm media at 37°C shortly before use.Day 0: Initiation of cardiac differentiation1. Assess cell density and cell morphology under the microscope. Cells must reach 90- 95% confluency before starting the differentiation protocol. If cells are < 90% confluent, do not continue incubation. Instead, detach cells and seed cells at a higher density than previously used.2. Prepare Medium A as described above.3. Thaw Matrigel® on ice and dilute 1 : 100 with cold Medium A.4. Warm Medium A + Matrigel® at 37°C shortly before use.5. Gently remove mTeSR™ Plus medium by aspiration.6. Add 4 ml of Medium A + Matrigel® per well.7. Incubate at 37°C for 48 hours.Days 2 - 15: Full-medium change1. Assess cells daily under the microscope.2. Perform a full-medium change on Day 2 and every 2 days until dissociation (Day 10 - Day 14) as follows:.• Gently remove medium from the wells using a pipettor and a sterile 10 ml pipette (do not aspirate).• Gently add 4 ml of medium B / C or maintenance medium per well as indicated in the protocol diagram (page 6) and in Table 1. Incubate at 37°C.Table 1 : Full-Medium Changes with STEMdiff™ Ventricular Cardiomyocite Differentiation and Maintenance Media*Do not feed differentiating CMs with STEMdiff™ Cardiomyocyte Maintenance Medium before Day 8 of differentiationDay 8: Small areas of beating cardiomyocytes may be visible. Over the course of time, beating areas become larger and more intense.

[0137] Monitor beating pattern daily and perform at least two full- medium changes with Maintenance medium before dissociation of cardiomyocytes (earliest timepoint for dissociation is day 12).

[0138] Harvest cardiomyocytes on the day with the highest contractility. Of note, maintenance medium should be changed the day before dissociation, even though that would include an extra medium change.

[0139] To maintain hiPSC-derived ventricular cardiomyocytes in 2D for 1 month or longer, perform a full-medium change every 2 days with 4 mL of STEMdifll™ Cardiomyocyte Maintenance Medium per well.

[0140] Dissociation of hiPSC-CM:1. Thaw Collagenase (200 U / ml) at 4°C overnight and prepare required volume of collagenase solution, washing solution, blocking buffer and basic medium as described in 3.3. Pre-warm at 37°C.2. Aspirate medium from cell culture vessel with glass pipette.3. Wash cell culture vessel twice with washing solution (1.5 ml / well).4. Add Collagenase solution to cell culture vessel (1.0 ml / well).5. Incubate at 37 °C for 45 min - 2 h until cells round up and detach from their clusters.6. Gently flush cells by pipetting Collagenase solution up and down onto the vessel surface 3-5 times.7. Transfer cells to 50 ml Falcon tube.8. Rinse cell culture vessel with blocking buffer (1.0 ml / well) and immediately transfer to the same 50 ml tube used above.9. Centrifuge at lOOxg for 10 min.10. Resuspend hiPSC-CM pellet in pre-warmed basic medium (5 ml for a small pellet size, 10 ml for intermediate pellet size, and 20 ml for a large pellet size) and gently triturate the cells by pipetting 5-times.11. Count cells and calculate number of hiPSC-CM required for further steps (e.g., generation of EHTs, FACS analyses)12. Perform a second washing step: Centrifuge at lOOxg for 10 min and resuspend pellet in corresponding volume of specific medium required for further analyses (e.g., basic medium for subsequent EHT casting).

[0141] Freezing of hiPSC-CM1. Prepare freezing medium and label cryotubes (cell line, condition, amount, date, initials) Preparation of freezing medium is slightly exothermic. Do not add DMSO directly onto cells suspended in FCS. Keep freezing medium at 4 °C for up to 2 weeks.2. Resuspend cells in corresponding amount of cold freezing medium.3. Aliquot 10-20xl06hiPSC-CM per cryotube.4. Quickly transfer cryotubes to freezing container or cell camper (preferred), store at -80 °C for the first 24 h, then move the cryotubes to -150 °C for long-term storage.Example 4: Generation and culture of EHT samples

[0142] Cardiac tissue engineering may provide advanced in vitro models for drug testing and, in combination with induced pluripotent stem cell technology, disease modeling and personalized medicine.

[0143] Materials:• Y-27632 (Biorbyt, cat# orbl54626 or Tocris cat# 1254 / 10; 10 mM in H2O use 10 pM). Store aliquots at -20 °C, use within 7 days when thawed and stored at 4 °C• Agarose (Invitrogen, cat# 1153277): 2%. Add appropriate volume of PBS, autoclave, store at 60 °C• Aprotinin (Genaxxon bioscience, cat# M6361.1010 10 g, or Sgima cat# Al 153-10MG): 33 mg / ml. Solve 1 g aprotinin in 30.3 ml aqua ad injectabilia, filter sterile (0.22 pm), aliquot and store at 4 °C up to one week or for long-term at -20°C.• Fibrinogen (Sigma, cat# F8630-5 g): 200 mg / ml. 0.9%-NaCl solution, solve sterile fibrinogen in appropriate volume of sterile NaCl-solution and store at -20 °C, (25 ml for 5 g). Chop up all big clumps of the powder. Add 72.1 pl aprotinin (33 mg / ml stock) to 25 ml pre-warmed (37 °C) NaCl and mix. Solve the powder in this mixture. Make sure that all big clumps are gone before the stock solution is aliquoted (ca 500 pl each). The dissolution process might take 30-60 minutes and requires repetitive shaking. Aliquote approx. 500 pl / tube. Store up to 3 tubes at -20 °C and all others at -80 °C for long-term storage.• Thrombin (Sigma, cat# 605157): 100 U / ml. For 1000 U: solve thrombin in 6 ml PBS and 4 ml aqua ad injectabilia (all sterile). Mix thoroughly; make aliquots of 450 pl (stock) or 3 pl for EHT-generation (sterile 200 pl tubes), store at -20 °C.• Tranexamic acid (Sigma, cat# SIAL857653-10G): 80 mM in water. With some cell lines, the EHTs tend to thin quickly. For matrix stabilization, add in addition to aprotinin tranexamic acid (200 pM). Aliquots are stored at -20 °C and can be kept for up to one week at 4 °C.

[0144] Materials for media and EHT Mastermix:• lOx DMEM o DMEM powder (Gibco, cat# 52100021), 1.34 g o Add 10 ml auqua ad injectabilia, filter sterile (0.22 pm), aliquot (1 ml) and store at -20 °C. Make sure the DMEM powder is properly closed (hygroscopic) before storage. For smaller volumes than 10 ml, adjust accordingly.• 2x DMEM o lOx DMEM, 2 ml o Horse serum (Thermo Fisher Scientific, 26050088), 2 mlo P / S (Gibco, cat# 15140122), 0.2 ml o Aqua ad injectabilia, 5.8 ml o Filter sterile (0.22 pm), aliquot (200 pl) and store at -20 °C. For smaller volumes than 10 ml, adjust accordingly.• EHT medium o DMEM (Biochrom, cat# 041), containing: o Horse serum (current tested lot, Gibco, cat# 10368902), 10% o P / S (Gibco, cat# 15140122), 1 % o Insulin (Sigma, cat# 19278), 0.1% (10 pg / ml) o Aprotinin (Sigma, cat# A1153-10MG), 0.1% (33 pg / ml)

[0145] An exemplary protocol is now described:

[0146] Preparation of the casting molds:1. Fill wells of transduction tray with 1.6 ml agarose and place spacers on top of the wells.2. Place spacer immediately after pipetting of max. 8 wells - agarose solidifies quickly. Do not leave the agarose at room temperature for longer than 10 minutes. Do not prepare the agarose too much in advance, but rather immediately prior to use, as the agarose molds will lose water over time and then perish. Wait until full solidification of the agarose before removal of the spacer (depending on surrounding temperature, approx. 10 min). Use the time during solidification to prepare enough 3 pl thrombin aliquots in sterile 200 pl tubes, in case no thrombin aliquots were prepared in advance (keep frozen at -20 °C).

[0147] Preparation of the mastermix:1. Take a round bottom 15 ml tube and prepare the master mix as indicated below.Master mix (prepare on 4 °C prechilled lab armor beads): hiPS-CM: 8-10xl062x DMEM 56 plY-27632 1 plFibrinogen 25 plThrombin 30 pl (Just for calculation, do not add it to the master mix) — Fill up with DMEM to 1000 plFibrinogen is the last component to be added to the master mix. It must be lukewarm for pipetting. Slowly fill the tip of the pipet and add it as a drop to the master mix. Do not touch the surface of the cold master mix with the fibrinogen-containing pipet - the viscosity of the fibrinogen would increase immediately.2. Resuspend the master mix thoroughly (e.g., 15 times) and make sure that all fibrinogen clots are gone. If something else needs to be added to the master mix (e.g., AAV), the volume of 2x DMEM (increase) and DMEM (decrease) needs to be adjusted. Of note: the number of cardiomyocytes / EHT might be adjusted to individual cell lines and source of cardiomyocytes (fresh vs. frozen).

[0148] Casting of the EHT samples:1. When the agarose is solid (no longer translucent, but opaque, approx, after 10 min), the spacer can be gently removed, and the casting mold racks are placed with one pair of posts reaching into the mold. Close the lid and lift the plate to check correct positioning of all posts.2. Triturate (serological pipette is recommended according to total volume) the master mix 1015 times (check the master mix in the pipette for homogeneity).3. For each EHT pipet 100 pl master mix in one thrombin aliquot (3 pl in 200 pl tube), mix and pipet the mixture into the agarose-slots quickly.4. Use a new filter tip for each EHT, otherwise the master mix will already start clotting. Resuspend the master mix (trituration with serological pipette ~5 times) after 8 EHTs.5. Incubate the plate at 37 °C, 7% CO2, 40% 02 and 98% relative humidity for 1.5-2 h.

[0149] Transfer of the EHT samples:1. Remove the plate from the incubator and place it under a sterile hood.2. Cover each well with a small amount of a sterile liquid (approx.: 200-500 pl; e.g. PBS, DMEM, old complete medium), shake the plate gently and incubate again at 37 °C for at least 10 min.3. In between, prepare a new 24w-culture plate with 1.5 ml fresh EHT medium per well.4. After incubation, take the plate, go to a sterile hood, gently shake it again and then carefully remove the casting mold racks from the agarose casting molds. Place the removed racks into the prepared medium-filled culture plates. Continue with one rack after the other.

[0150] EHT culture, QC and analysis:1. EHTs should be maintained at 37 °C, 7% CO2, 40% O2 and 98% relative humidity. Medium should be changed every other day (Mo, Wed, Fr). For the first medium change, a fresh culture plate should be used. The old medium is to be kept in the incubator containing the used medium. For all other feedings, remove the used medium from a separate plate without EHTs, refill fresh medium and then transfer the EHTs to this plate. Check that the order of racks is kept (e.g., EHT in Al remains in Al). Carefully check the copy plate for contaminations (e.g., fungi), before processing it. Cells in the EHT should showmicroscopic contractions at day 2-5. Macroscopically coherent beating of the entire EHT should start around day 7-10.2. As soon as macroscopical contractions are visible, EHTs can be measured with an EHT analysis instrument (e.g., the exemplary measurement chamber described herein).

[0151] Cleaning up:1. After generating EHTs, boil the used spacers in aqua dest. twice. Place six spacers , wrap in aluminum foil and autoclave.2. After discarding old EHTs, make sure you clean the silicone rack properly with no EHT residues left. Boil twice in aqua dest. and place three racks each on a layer of tissue paper in an old pipette tip box, wrap in aluminum foil and autoclave.Example 4: Transduction of EHT samples

[0152] This example is an optimized AAV9 Transduction Protocol for hiPSC-derived cardiomyocytes within transduction chambers in accordance with the embodiments described herein and per-treatment with Neuraminidase.

[0153] Materials:• Culture / Transduction medium: DMEM (Biochrom cat. No 0415) containing 1% penicillin / streptomycin (10,000 U / mL) (Gibco cat. No 15140), 0.1% insulin solution human (Sigma, cat. No 19278), 0. 1 % aprotinin (Genaxxon bioscience cat. No M6361.0010)• Complete: Culture medium plus 10% Horse serum heat inactivated (Life Technologies, cat No 26050088)• Nunc 24 well plate Thermo fisher # 142475• Neuraminidase from Vibrio cholerae Type III (Sigma, #7885)

[0154] An exemplary protocol is now described:14 days prior to transduction1. Cast 1 Mio human iPSC-derived cardiomyocytes containing EHTs2. Culture, randomize according to force development into non transduced and transduced Day of Transduction:1. Prepare extra changing plate 24 well 1.5 ml culture medium (no serum), pre-warm transduction chamber and medium.2. Prepare 200 pl culture medium per EHT supplemented with 500 mU / ml of neuraminidase (NA) in each well of the transduction chamber3. Incubate cells with NA for 2 hours at 37° C in 5% CO2 , 40% O2 and 90% humidity, use lid from another 24 well plate for transduction chamber4. Transfer EHTs to changing plate5. Remove medium containing NA and replace with 0.2 ml of cell culture medium (no serum) containing AAV9 (300k MOI).6. Incubate cardiomyocytes with AAV9-containing media in incubator at 37 °C in 5% CO2 and 90% humidity for 4 hours.7. Transfer EHTs back into their culture plate with complete medium-8. Remove Vector from transduction plate, wash transduction chambers, UV hood with transduction chamber, seal and autoclave transduction chamber, cook in water transduction chamber, autoclave again.2 to 14 days after transduction1. Change media as normal, measure baseline regularly.2. Functional effects are earliest expected 4 days after transduction, paced analysis recommended 7 and 14 days post-infection.3. Analyze transgene expression 14 days post-infection: For protein, wash in PBS, remove with forceps and snap freeze in 2 ml Eppis. For IF pre-treat with 1 : 1000 BTS for 1-2 hours (optional), wash with PBS, fix according to antibody (standard: 4% Histofix over night at 4°C).Example 5: Measurement Protocol

[0155] An exemplary measurement protocol utilizing the measurement chamber 100 is now described. Modifications to the protocol may be readily made depending on the sample type, materials, and other factors as would be appreciated by those of ordinary skill in the art.(1) All information related to the 24-well plate measurement (user-, system-, project- and EHT- specific information) is generated or loaded via the system software of the measurement chamber if it already exists. Alternatively, this information can be inputted by scanning the QR code on the plate when it is inserted.(2) The tray is opened to allow for insertion of a plate containing EHT samples, and the plate is taken from a separate incubator by an individual or a robotic arm and placed in the tray.(3) The tray is closed and the measurement can be started, during which EHT samples can be electrically stimulated by an internal pacing unit (e.g., 24 EHT samples simultaneously). The corresponding parameters can be set before the respective measurement (e.g., pulse duration in milliseconds (default 20 milliseconds); frequency in Hertz (default 2 Hertz); voltage potential in V (3.2V); current in A (default 1.2 A); recording time in seconds (default 10 to 20 s)). The measurement process may be indicated by a light on top of the measurement chamber.(4) When the measurement is completed, as may be indicated by the light on top of the measurement chamber, the user can access the measured data via a web interface of the system software (e.g., a report is displayed), and the data can be accessed interactively (e.g., images, videos generated from a sequence of images, and / or plots). All data are already saved accordingly at this time point.

[0156] In the foregoing description, numerous specific details are set forth, such as specific materials, dimensions, processes parameters, etc., to provide a thorough understanding of the embodiments of the present disclosure. The particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. The words “example” or “exemplary” are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “example” or “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the words “example” or “exemplary” is intended to present concepts in a concrete fashion.

[0157] As used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is, unless specified otherwise, or clear from context, “X includes A or B” is intended to mean any of the natural inclusive permutations. That is, if X includes A; X includes B; or X includes both A and B, then “X includes A or B” is satisfied under any of the foregoing instances. In addition, the articles “a” and “an” as used in this application and the appended claims should generally be construed to mean “one or more” unless specified otherwise or clear from context to be directed to a singular form.

[0158] Reference throughout this specification to “an embodiment,” “certain embodiments,” or “one embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase “an embodiment,” “certain embodiments,” or “one embodiment,” in various places throughout this specification are not necessarily all referring to the same embodiment, and such references mean “at least one.”

[0159] It is to be understood that the above description is intended to be illustrative, and not restrictive. Many other embodiments will be apparent to those of skill in the art upon reading and understanding the above description. The scope of the disclosure should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

What is claimed is:

1. A measurement chamber comprising: a sample tray; one or more light sources; and one or more cameras configured to capture images and / or video of a plurality of tissue samples when loaded onto the sample tray, wherein the measurement chamber is operatively coupled to an evaluation unit configured to evaluate the plurality of tissue samples in parallel.

2. The measurement chamber of claim 1, wherein the evaluation unit is configured to evaluate one or more parameters of the plurality of tissue samples, wherein the one or more parameters are selected from action potential, calcium transients, and contractility.

3. The measurement chamber of either claim 1 or claim 2, further comprising: a pacing unit operatively coupled to the one or more light sources and the one or more cameras, wherein the pacing unit is configured to generate a stimulation signal to stimulate one or more tissue samples when loaded onto the sample tray.

4. The measurement chamber of claim 3, further comprising: a pacing board comprising a plurality of electrode pairs, the pacing board being configured for insertion of the electrode pairs into sample wells in a one-to-one correspondence, wherein the pacing board is operatively coupled to the pacing unit.

5. The measurement chamber of claim 4, wherein the pacing board comprises conductive tracks arranged in an interdigitated configuration to provide a two-dimensional arrangement of electrode pairs on the pacing board.

6. The measurement chamber of any one of the preceding claims, wherein the evaluation unit is configured to capture measurement data from the stimulation of the one or more tissue samples by the pacing unit.

7. The measurement chamber of claim 6, wherein the measurement chamber is operatively coupled to a computing device configured to provide visualization of the measurement data.

8. The measurement chamber of claim 6, wherein the measurement data comprises contractile data for the tissue samples.

9. The measurement chamber of any one of the preceding claims, wherein the one or more tissue samples comprise engineered heart tissue (EHT) samples.

10. The measurement chamber of any one of claims 3-7, wherein the pacing unit is configured to stimulate a plurality of the tissue samples simultaneously.

11. A pacing board configured for use with a measurement chamber to analyze tissue samples, the pacing board comprising a plurality of electrode pairs, the pacing board being configured for insertion of the electrode pairs into sample wells of a multi-well plate in a one-to- one correspondence.

12. The pacing board of claim 11, wherein the pacing board comprises conductive tracks arranged in an interdigitated configuration to provide a two-dimensional arrangement of electrode pairs on the pacing board.

13. The pacing board of claim 12, wherein the pacing board is configured to interface with a pacing unit operatively coupled to a measurement chamber.

14. The pacing board of claim 13, wherein the pacing board is configured to simultaneously apply a stimulation signal to tissue samples in the multi-well plate when the plurality of electrode pairs are inserted thereon in response to a signal received from the pacing unit when operatively coupled thereto.

15. An assembly for characterizing a plurality of tissue samples, the assembly comprising: a multi-well plate comprising a plurality of wells; a sample transport tray disposed on the multi-well plate; and a plurality of sample racks disposed on the sample transport tray, wherein each of the plurality of sample racks comprises at least one tissue sample that extends through an aperture of the sample transport tray and is inserted into one of the plurality of wells.

16. The assembly of claim 15, further comprising:a pacing board disposed above the plurality of sample racks, the pacing board comprising a plurality of electrode pairs, wherein each of the plurality of electrode pairs is inserted into a well of the multi-well plate.

17. A method of characterizing a plurality of engineered heart tissue (EHT) samples, the method comprising: placing a multi-well plate onto a sample stage of a measurement chamber, the multi-well plate comprising, in each well, one of the plurality of EHT samples; and causing a pacing unit of the measurement chamber to deliver a stimulation signal to the plurality of EHT samples.

18. The method of claim 17, further comprising: inserting electrodes of a pacing board into each well of the multi-well plate prior to placing the multi-well plate onto the sample stage, wherein the stimulation signal is delivered via the electrodes of the pacing board.

19. The method of claim 18, further comprising: computing one or more parameters associated with contractility of the EHT samples based on the stimulation.

20. The method of any one of claims 17-19, further comprising: capturing fluorescence images or video of the EHT samples; and deriving one or more parameters of the EHT samples from the fluorescence images or video.

21. The method of any one of claims 17-20, further comprising: capturing images and / or video of the plurality of EHT samples during the stimulation.

22. The method of claim 20, further comprising: displaying, by a computing device, the captured images and / or video.

23. A transduction plate adapted for treating a plurality of tissue samples, the transduction plate comprising: a rigid body; anda plurality of wells formed in the body in a grid configuration, wherein each of the plurality of wells comprises a transduction chamber formed within the well having a non-circular shape.

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