A microbial physiological platform with embedded electrodes for 3D tissue culture

Abstract

The embodiments described herein generally relate to devices, apparatus, and systems with embedded electrodes for growing, maintaining, and / or using 3D tissue in vitro. The devices, apparatus, and systems described herein can provide scalable, automated tissue stimulation.

Description

[Technical field]

[0001] The present disclosure generally relates to a platform with embedded electrodes for growing three-dimensional (3D) tissue in vitro. [Background technology]

[0002] Due to the increasing costs of drug development and limitations in cardiotoxicity screening, there is an urgent need to develop robust in vitro 3D tissue culture platforms that can facilitate the cultivation of human cardiac tissue and provide non-invasive functional readouts that predict cardiotoxicity in clinical settings. However, such platforms generally require complex fabrication procedures and are difficult to scale up into high-throughput testing platforms.

[0003] Cardiotoxicity is the leading cause of drug failure in the late stages of clinical trials and is associated with many post-marketing discontinuations. Validating the safety of drug candidates and eliminating unqualified candidates as early as possible is critical to reduce the cost of drug discovery and prevent patient deaths. Although traditional monolayer cultures with immortalized cell lines enable high-throughput screening, they have limited relevance to native myocardium due to the lack of tissue-level functional and morphological organization. Engineered cardiac tissues from human stem cell-derived cardiomyocytes have been shown to more closely mimic the myofibril bundles of native myocardium, exhibiting aligned sarcomere structures and remarkable levels of contractile force. However, producing reproducible 3D tissues in large quantities requires a complex fabrication process, which includes constructing hollow or suspended microstructures, which are critical to promote functional tissue formation. In addition, functional readouts from engineered cardiac tissues are also needed to detect cardiotoxicity.

[0004] Built-in sensors have been incorporated into many microphysiological platforms. For example, thin muscle membranes have been fabricated on top of a thin layer of polydimethylsiloxane (PDMS) or polyurethane membrane. The deformation of the membrane can be easily modeled and related to the contractile forces generated by superimposed cardiomyocytes. Recently, electrical circuits have been embedded into thin polymer membranes using 3D printing. This technique can convert the electrical signals of embedded resistors into contractile motion using modeling techniques. However, these systems only allow the culture of cell monolayers in structures that are a few cell layers thick. A widely used two-post platform employs PDMS posts as force sensors to noninvasively report contractile forces from 3D tissues. However, cultured tissues are prone to slipping off the posts, leading to failure of the device. Furthermore, these devices rely on the use of PDMS, which is prone to drug absorption due to its hydrophobicity. Summary of the Invention

[0005] One aspect of the present disclosure relates to a device comprising: a substrate; at least one electrode pair at least partially embedded in the substrate, the electrode pair having a first electrode and a second electrode separated by a gap; at least one well on the substrate having a bottom, a first end in contact with the first electrode, and a second end in contact with the second electrode, the well configured to grow tissue from cells seeded therein, the electrode pair configured to apply an electrical stimulus to the tissue; and at least two elastic sensing elements positioned across the well such that a gap exists between the sensing elements and a bottom of the well, the sensing elements configured to (a) allow attachment of tissue to be formed therebetween, thereby suspending the tissue above the bottom of the well, and (b) deform in response to a contractile force exerted by the tissue on the sensing elements, thereby simulating a physiological environment specific to the tissue and / or enabling measurements of contractile forces.

[0006] In some embodiments, the device comprises two or more wells, for example 6 wells, 12 wells, 24 wells, 48 ​​wells, or 96 wells.

[0007] In some embodiments, the electrode pair is completely embedded in the substrate.

[0008] In some embodiments, the electrodes comprise conductive carbon, gold, platinum, palladium, stainless steel, tin, tungsten, titanium, or combinations thereof.

[0009] In some embodiments, the conductive carbon is non-porous.

[0010] In some embodiments, the first electrode and the second electrode are separated by a gap in the range of 1 mm to 5 cm.

[0011] In some embodiments, the first electrode is parallel or substantially parallel to the second electrode.

[0012] In some embodiments, the device comprises two or more electrode pairs, with at least one well disposed between each electrode pair.

[0013] In some embodiments, each of the electrodes are parallel or substantially parallel to one another.

[0014] In some embodiments, the electrode pair is connected to a stimulator, the stimulator configured to apply electrical stimulation between the electrode pair.

[0015] In some embodiments, the device comprises two or more electrode pairs, two or more wells are disposed between each electrode pair, and the stimulator is configured to independently control electrical stimulation between each electrode pair.

[0016] In some embodiments, the device includes between 2 and 25 sensing elements per well.

[0017] In some embodiments, the sensing element comprises a polymer. In some embodiments, the polymer is at least one of polylactic acid, poly(lactic-co-glycolic) acid, poly(caprolactone), polyglycolide, polylactide, polyhydroxobutyrate, polyhydroxyalkanoic acid, chitosan, hyaluronic acid, hydrogel, poly(2-hydroxyethyl methacrylate), poly(ethylene glycol), poly(L-lactide) (PLA), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), poly(glycerol sebacate), poly(octamethylene maleic anhydride citric acid) (POMaC), citric acid free POMaC, poly(ε-caprolactone), polyurethane, silk, nanoengineered materials, copolymers, blended polymers, or combinations thereof. In some embodiments, the polymer is POMaC.

[0018] In some embodiments, the polymers have mechanical properties that are tunable during the polymerization reaction.

[0019] In some embodiments, the sensing element is porous, thereby allowing delivery of nutrients and growth factors to the cardiac tissue.

[0020] In some embodiments, the sensing element has a resilience between about 20 kPa and 0.5 MPa.

[0021] In some embodiments, the sensing element is in the form of a wire.

[0022] In some embodiments, the well is configured to have a longitudinal axis.

[0023] In some embodiments, the sensing element is oriented perpendicular, parallel, or oblique to the longitudinal axis of the well.

[0024] In some embodiments, the substrate comprises a polymer. In some embodiments, the polymer is rigid. In some embodiments, the polymer is polystyrene or polycarbonate.

[0025] In some embodiments, the cells are seeded onto the hydrogel.

[0026] In some embodiments, the cells are selected from cardiomyocytes, fibroblasts, skeletal muscle cells, liver cells, kidney cells, chondrocytes, skin cells, contractile cells, blood cells, immune system cells, germ cells, neural cells, epithelial cells, hormone-secreting cells, bone marrow cells, stem cells, tumor cells, smooth muscle cells, endothelial cells, fibroblasts, adipose-derived stem cells, mesenchymal stem cells, progenitor cells, or combinations thereof.

[0027] One aspect of the disclosure relates to a non-transitory processor-readable medium storing code representing instructions executed by a processor, the instructions including code for causing the processor to: receive a set of measurements from at least one sensing element associated with a well configured to grow tissue from cells seeded in the well, the well comprising a set of electrodes configured to apply electrical stimulation to the tissue according to a set of stimulation parameters; determine an amount by which to change a set of values ​​of the set of stimulation parameters based on a comparison of the set of measurements to a set of predetermined criteria associated with the tissue; adjust the set of values ​​based on the amount to form an updated set of values ​​for the set of stimulation parameters; and transmit a stimulation signal to the set of electrodes to apply electrical stimulation to the tissue according to the updated set of values ​​for the set of stimulation parameters.

[0028] In some embodiments, the set of stimulation parameters includes at least one of a stimulation voltage, a stimulation frequency, or a stimulation duration.

[0029] In some embodiments, the at least one sensing element comprises at least two resilient sensing elements positioned across the well such that a gap exists between the sensing elements and the bottom of the well.

[0030] In some embodiments, the initial set of values ​​for the set of stimulation parameters is selected based on a predetermined stimulation protocol.

[0031] In some embodiments, the code for causing the processor to identify includes code for causing the processor to identify an amount to reduce the stimulation voltage based on a set of measurements that meet a tissue maturity criterion.

[0032] In some embodiments, the code for causing the processor to identify includes code for causing the processor to identify an amount to increase the stimulation voltage based on a set of measurements that do not meet a tissue excitation criterion.

[0033] In some embodiments, the code for causing the processor to receive the sequence of measurements includes code for causing the processor to periodically receive instances of the sequence of measurements and store the instances of the sequence of measurements in a memory.

[0034] One aspect of the disclosure relates to an apparatus comprising: a memory; and a processor operatively connected to the memory, the processor configured to receive an image of tissue growing around a wire in a well from cells seeded in the well; form a first binary image based on comparing each pixel from a plurality of pixels in the image to a pixel threshold; detect first and second edges of the wire in the first binary image to form a second binary image representing the first and second edges; calculate midpoints between the first and second edges of each row from a plurality of rows of the second binary image to generate a set of midpoints; calculate a quadratic equation fitting to the set of midpoints based on a polynomial regression of the set of midpoints; and identify a bending point of the wire based on the quadratic equation.

[0035] In some embodiments, the well comprises a series of electrodes configured to apply electrical stimulation to tissue according to the set of stimulation parameters, and the processor is configured to adjust the set of values ​​of the set of stimulation parameters based on a bending point of the wire meeting a predetermined criterion to form an updated set of values ​​of the set of stimulation parameters, and to transmit stimulation signals to the set of electrodes to apply electrical stimulation to the tissue according to the updated set of values ​​of the set of stimulation parameters.

[0036] In some embodiments, the processor is configured to detect the first edge and the second edge using a Sobel operator.

[0037] In some embodiments, the processor is configured to form the first binary image using Otsu's method.

[0038] In some embodiments, the processor is configured to remove artifact pixels from the first binary image using an opening operator prior to detecting the first edge and the second edge of the wire in the first binary image.

[0039] In some embodiments, the processor is configured to smooth the first binary image using a closing operator prior to detecting the first edge and the second edge of the wire in the first binary image.

[0040] In some embodiments, the processor is configured to complete a portion of the first edge or a portion of the second edge that is not included in the second binary image before calculating the midpoint, and each row from the plurality of rows of the second binary image includes a portion of the first edge and a portion of the second edge.

[0041] In some embodiments, the processor is configured to calculate an amount of contractile force exerted by the tissue on the wire based on the flex point.

[0042] In some embodiments, the processor is configured to calculate an amount of resting tension exerted by the tissue on the wire based on the flex point.

[0043] One aspect of the disclosure relates to a system having two or more devices as described herein and two or more driver boards, where each driver board is configured to apply electrical stimulation between each electrode pair of each device, such that the stimulator can independently control the application of electrical stimulation between each electrode pair.

[0044] In some embodiments, the system further comprises an interface tray having a hinged lid and configured to electrically connect the device to a stimulator.

[0045] In some embodiments, the system further comprises an optical microscope configured to generate images of the sensing elements and / or tissue within each device.

[0046] In some embodiments, the system further comprises a computing device configured to connect to the microscope, analyze the images, and / or control the stimulator.

[0047] In some embodiments, a computing device comprises a non-transitory processor-readable medium as described herein.

[0048] In some embodiments the computing device comprises an apparatus described herein. [Brief description of the drawings]

[0049] [Figure 1] FIG. 1 is a device 100 having embedded electrodes according to some embodiments of the present disclosure.

[0050] [Figure 2A-C] 2A-2C show a schematic diagram of Biowire 96-well plate fabrication.

[0051] [Figure 2A] FIG. 2A shows the incorporation of carbon electrodes into a Biowire plate base.

[0052] [Figure 2B] FIG. 2B is the casting of polymer microwires onto the Biowire plate base.

[0053] [Figure 2C] Figure 2C shows a representative image of the assembled Biowire plate and cardiac tissue cultured in the Biowire chamber and spanned across two parallel polymer wires. Scale bar is 1 mm.

[0054] [Figure 3A-C] 3A-3C show the hot embossing, assembly, and post-processing of the Biowire plate base.

[0055] [Figure 3A] FIG. 3A is a hot embossed polystyrene plate base with embedded carbon electrodes.

[0056] [Figure 3B] Figure 3B is an image of a polystyrene plate base with an embedded carbon electrode, with the top surface of the carbon electrode exposed.

[0057] [Figure 3C] Figure 3C is a fully assembled Biowire plate with both polymer wires and carbon electrodes viewed from the bottom. The arrows indicate the location of the carbon electrodes and tissue chambers.

[0058] [Figure 4A-D] 4A-4D show high speed casting of polymer wires across a 96-well plate format.

[0059] [Figure 4A] FIG. 4A is the polymer injection molding, UV crosslinking, wire casting, and demolding.

[0060] [Figure 4B] FIG. 4B shows polymer injection by capillary forces, where the polymer solution was able to perfuse throughout the microchannels suspended above the tissue chamber.

[0061] [Figure 4C] FIG. 4C shows the polymer wire successfully draped across the tissue chamber after removal of the mold.

[0062] [Figure 4D] Figure 4D shows the assembled Biowire plate immersed in distilled water after (1) 1 minute, (2) 24 hours, and (3) air drying. Scale bars are 1 mm.

[0063] [Figure 5A-D] 5A-5D show the characterization of polymer wires in a 96-well plate device.

[0064] [Figure 5A] FIG. 5A is a force probe used to deflect a polymer wire and relate force to displacement.

[0065] [Figure 5B] Figures 5B-5D are force-displacement curves of POMaC wires obtained after (Figure 5B) 2 days (n=8), (Figure 5C) 4 days (n=3), and (Figure 5D) 6 days (n=7) of post-treatment baking at 70 °C. The graphs show the raw data traces of the individual force-displacement curves, along with the 95% confidence intervals around the mean values, the polynomial equations of the fitted curves, and the R2 values.

[0066] [Figure 6A-C]6A-6C show the characterization of cardiac tissue cultured in a 96-well plate device.

[0067] [Figure 6A] Figure 6A is a brightfield image of cardiac tissue cultured in a Biowire II 96-well plate. The final image was stitched together from multiple images.

[0068] [Figure 6B] Figure 6B is a fluorescent image of cardiac tissue cultured in a 96-well plate device stained for sarcomeric α-actinin (green) and F-actin (red). Scale bar, 30 μm, (n=3).

[0069] [Figure 6C] Figure 6C is a fluorescent image of cardiac tissue captured automatically on a SpectraMax imaging cytometer. The image clearly shows the autofluorescent polymer wire in green, potentially enabling tracking of wire displacement over time in a high-throughput manner. The final image was stitched from multiple images. Scale bar 1 mm, (n=4).

[0070] [Figure 7A-H]Figures 7A-7H are functional evaluations of human engineered cardiac tissues after 12 days of electrical stimulation with integrated carbon electrodes and their response to thapsigargin. Figure 7A shows stabilized tissue dimensions after 7 days of self-organization, observed in daily representative images (n=3) (Figure 7B). Improved electrical properties were demonstrated by a trend toward decreased excitation threshold (Figure 7C) comparing days 7 (before electrical conditioning) and 19 (endpoint), and a significant increase in maximum capture rate (Student's t-test, n=6) (Figure 7D). The 96-well plate device allows for non-destructive force readout and pulsatile characterization of cultured tissues. Quantification of (Figure 7E) resting tension, active force, (Figure 7F) contraction time, relaxation time, and total duration, (Figure 7G) contraction and relaxation slopes were compared between days 7 and 19 (n=3). Figure 7H is a representative trace showing the response of cultured tissues to thapsigargin at the end of electrical stimulation (day 19) (n=2). Data are presented as mean ± standard deviation, and differences between experimental groups were analyzed by Student's t-test. *p<0.05 was considered significant in all tests.

[0071] [Figure 8A-C] 8A-8C show the outline and dimensions of the Biowire 96 well plate fabrication.

[0072] [Figure 8A] FIG. 8A is a schematic diagram of the Biowire 96-well plate fabrication protocol.

[0073] [Figure 8B] Figure 8B shows the dimensions of the PDMS mold for polymer wire perfusion (in millimeters). The feature height is 200 μm.

[0074] [Figure 8C] Figure 8C shows the dimensions of the polystyrene plate base (in millimeters). The feature height is 200 μm.

[0075] [Figure 9A-B]9A-9B show the Biowire 96-well plate with sputtered gold electrodes.

[0076] [Figure 9A] Figure 9A is an image of the backside of a Biowire 96-well plate with sputtered gold electrodes. The inset image shows the variation in coating thickness at different positions of the electrodes across the entire 120 mm wide plate.

[0077] [Figure 9B] Figure 9B is an image of scratches caused by trying to connect to an electrode with an alligator clamp. Such surface scratches can easily destroy the connection of the entire electrode circuit.

[0078] [Figure 10A-B] 10A-10B show the voltage drop across carbon electrodes.

[0079] [Figure 10A] Figure 10A shows the percentage of voltage drop across the embedded carbon electrodes between wells in different rows of the plate (n=3). Three wells from each row are used for the measurements.

[0080] [Figure 10B] Figure 10B shows the formula used to calculate the voltage drop. Data are presented as mean ± standard deviation, and differences between experimental groups were analyzed by one-way analysis of variance. p < 0.05 was considered significant in all tests.

[0081] [Figure 11A-C] 11A-11C show the consistency of POMaC wire properties across a 96-well plate.

[0082] [Figure 11A]FIG. 11A is the force at 100 μm displacement of the POMaC wire from each well. Both diagrams and heat maps are displayed (n≧4). The displacement force from the wire in each well is shown as a heat map. White boxes indicate wells with missing or missing visually detectable wires. The two outermost rows were intentionally excluded from the measurements because of the non-uniformity found due to uneven pressure at the edges.

[0083] [Figure 11B] FIG. 11B shows the consistency of POMaC wires from all wells at displacements of 50 μm, 100 μm, and 150 μm (n=70).

[0084] [Figure 11C] Figure 11C is a representative tensile and relaxation curve of POMaC wires. Data are presented as mean ± standard deviation, and differences between experimental groups were analyzed by one-way ANOVA. p<0.05 was considered significant in all tests.

[0085] [Figure 12] FIG. 12 is an abstract system configuration diagram according to some embodiments of the present disclosure.

[0086] [Figure 13] FIG. 13 is a schematic diagram of a system for performing 3D tissue culture, according to some embodiments of the present disclosure.

[0087] [Figure 14] FIG. 14 is a flow chart illustrating a method of image processing for 3D tissue culture according to some embodiments of the present disclosure.

[0088] [Fig. 15A-15M] 15A-15M are various stages of a method for tracking a wire according to some embodiments of the present disclosure.

[0089] [Figure 16]FIG. 16 is a flow chart illustrating a method of engineering a tissue culture according to some embodiments of the present disclosure.

[0090] [Figure 17] FIG. 17 is a schematic diagram of the electronic components within the stimulator. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0091] The present disclosure provides devices, apparatus, and systems with embedded electrodes for growing, maintaining, and / or using 3D tissue in vitro. Because the electrodes are physically embedded in the substrate of the device, the electrodes are provided with leads for a stimulator to apply current directly to the embedded electrodes, and the tissue in the device is already in position to receive electrical stimulation as well as medium and nutrients without being moved to a separate dish or device. The devices, apparatus, and systems described herein can be used in a variety of applications, including drug testing. By exposing the tissue to a test agent, changes in tissue contractility and / or resting tension can be used to assess the efficacy, safety, and / or toxicity of the test agent.

[0092] One aspect of the disclosure relates to a device 100 of Figure 1. The device 100 can include a substrate 102, an electrode pair including a first electrode 104 and a second electrode 106, a first sensing element 112, a second sensing element 114, and a well 120.

[0093] The substrate 102 can include a polymer. In some embodiments, the polymer is rigid, optically clear, and / or medically compatible. In some embodiments, the polymer can include polystyrene or polycarbonate.

[0094] The electrode pair is at least partially embedded in the substrate 102. In some embodiments, the electrode pair is completely embedded in the substrate 102. In some embodiments, the first electrode 104 is parallel or substantially parallel to the second electrode 106.

[0095] The first electrode 104 and the second electrode 106 can be separated by a gap. The gap can have a width of at least about 1 mm, at least about 1.5 mm, at least about 2 mm, at least about 2.5 mm, at least about 3 mm, at least about 3.5 mm, at least about 4 mm, at least about 4.5 mm, at least about 5 mm, at least about 5.5 mm, at least about 6 mm, at least about 6.5 mm, or at least about 7 mm. The gap can have a width of about 10 cm or less, about 9.5 cm or less, about 9 cm or less, about 8.5 cm or less, about 8 cm or less, about 7.5 cm or less, about 7 cm or less, about 6.5 cm or less, about 6 cm or less, about 5.5 cm or less, about 5 cm or less, about 4.5 cm or less, about 4 cm or less, about 3.5 cm or less, about 3 cm or less, about 2.5 cm or less, about 2 cm or less, about 1.5 cm or less, or about 1 cm or less.

[0096] Combinations of the above-referenced ranges for gap width are also possible (eg, at least about 1 mm and no more than about 10 cm, at least about 1 mm and no more than about 5 cm, or at least about 5 mm and no more than about 1.5 cm).

[0097] Each electrode can comprise conductive carbon, gold, platinum, palladium, stainless steel, tin, tungsten, titanium, or combinations thereof. Further examples of conductive materials for tissue stimulation are described in Merrill et al., "Electrical Stimulation of Excitable Tissue: Design of Efficacious and Safe Protocols," J. of Neuroscience Methods 2005, 141, 171-198; Tandon et al., "Characterization of Electrical Stimulation Electrodes for Cardiac Tissue Engineering," Proceedings of the 28th Annual Meeting of the American Society of Cardiac Stimulation and Neurosurgery 2006, 117-120; and in Tandon et al., "Characterization of Electrical Stimulation Electrodes for Cardiac Tissue Engineering," Proceedings of the 28th Annual Meeting of the American Society of Cardiac Stimulation and Neurosurgery 2006, 117-120. th IEEE, pages 845-848, the contents of each of which are incorporated herein by reference.

[0098] In some embodiments, the conductive carbon is non-porous, e.g., glassy carbon. Non-porous carbon can prevent the test compound from being absorbed into the carbon. In some embodiments, the carbon electrode is not an epoxy-reinforced carbon electrode.

[0099] The well 120 has a bottom on the substrate 102 and has a first end 122 in contact with the first electrode 104 and a second end 124 in contact with the second electrode 106. The well can be configured to grow tissue 130 from cells seeded therein. The electrode pair can be configured to apply an electrical stimulus to the tissue 130.

[0100] The shape of the well 120 is not particularly limited and can be square, rectangular, circular, oval, rectangular with rounded corners, triangular, or any combination of shapes. Other dimensions of the well 120 may also be varied in any suitable manner. For example, the depth of the well 120, the height of the well 120, and the length of the well 120, as well as the overall volume of the well 120 can be altered in any suitable manner.

[0101] For example, the length, height, or width of well 120 can be about 0.1-1 mm, about 0.2-2 mm, about 0.3-3 mm, about 0.4-4 mm, about 0.5-5 mm, about 0.6-6 mm, about 0.7-7 mm, about 0.8-8 mm, about 0.9-9 mm, about 1-10 mm, about 1-100 mm, or about 10-100 mm.

[0102] The well 120 can be characterized by a longitudinal axis. The longitudinal axis can run along the length of the well 120.

[0103] The surface of the well 120 may be modified with any suitable surface treatment, including chemical modifications (e.g., ligands, charged substances, binding agents, growth factors, antibiotics, antifungals, etc.) or physical modifications (e.g., spikes, curved portions, folds, pores, uneven portions, or various shapes and forms, etc.) that may facilitate the tissue culture process.

[0104] In some embodiments, the well 120 can be disposed within a cell culture well 140. Media can be added to the cell culture well 140 for growing and / or maintaining the tissue 130.

[0105] The first sensing element 112 and the second sensing element 114 are positioned across the well 120 such that a gap exists between the bottom of the well 120 and the sensing elements 112 and 114. The sensing elements 112 and 114 can be configured to (a) allow attachment of the tissue 130 to be formed between them, thereby suspending the tissue 130 above the bottom of the well 120, and (b) deform in response to contractile forces exerted by the tissue 130 on the sensing elements 112 and 114, thereby simulating a physiological environment specific to the tissue 130 and / or allowing measurements of contractile forces.

[0106] The sensing elements 112 and 114 can have an orientation perpendicular or substantially perpendicular to the longitudinal axis of the well 120. The tissue 130 can be aligned in the same or substantially the same direction as the longitudinal axis of the well 120.

[0107] Device 100 can include at least two sensing elements per well, at least three sensing elements per well, at least four sensing elements per well, or at least five sensing elements per well. Device 100 can include no more than 25 sensing elements per well, no more than 20 sensing elements per well, no more than 18 sensing elements per well, no more than 15 sensing elements per well, or no more than 10 sensing elements per well.

[0108] Combinations of the above ranges for the number of sensing elements per well are also possible (e.g., at least 2 and up to 25 per well, at least 2 and up to 20 per well, or at least 2 and up to about 10 per well). Any number of sensing elements per well can be provided, so long as tissue is capable of forming around each sensing element and bonding between them such that the tissue spans above the bottom of the well.

[0109] The sensing element can include a polymer. The polymer can be synthetic or biological. The polymer can also be biodegradable or non-biodegradable.

[0110] The sensing element can include a polymer having a Young's modulus similar to that of a tissue supported thereon. For example, the Young's modulus of the polymer can be within ±2000% of the Young's modulus of the tissue, the Young's modulus of the polymer can be within ±1000% of the Young's modulus of the tissue, the Young's modulus of the polymer can be within ±500% of the Young's modulus of the tissue, the Young's modulus of the polymer can be within ±250% of the Young's modulus of the tissue, the Young's modulus of the polymer can be within ±100% of the Young's modulus of the tissue, the Young's modulus of the polymer can be within ±50% of the Young's modulus of the tissue, the Young's modulus of the polymer can be within ±30% of the Young's modulus of the tissue, the Young's modulus of the polymer can be within ±25% of the Young's modulus of the tissue, the Young's modulus of the polymer can be within ±20% of the Young's modulus of the tissue, the Young's modulus of the polymer can be within ±15% of the Young's modulus of the tissue, or the Young's modulus of the polymer can be within ±10% of the Young's modulus of the tissue.

[0111] In some embodiments, the sensing element can include a polymer having a Young's modulus in the range of 10 kPa to 800 kPa. For example, the polymer can have a Young's modulus in the range of 20 kPa to 700 kPa, 20 kPa to 600 kPa, 20 kPa to 500 kPa, 50 kPa to 500 kPa, or 100 kPa to 500 kPa. In some embodiments, the polymer can have a Young's modulus of about 150 kPa, about 200 kPa, about 250 kPa, about 300 kPa, about 350 kPa, about 400 kPa, about 450 kPa, about 500 kPa, or about 550 kPa.

[0112] In some embodiments, the sensing element can include a polymer whose mechanical properties can be tuned by controlling the polymerization using different crosslinking energies. The tunability can also be controlled by the mixing ratio of the polymer units during the polymerization reaction.

[0113] In some embodiments, the polymer can be at least one of polylactic acid, poly(lactic-co-glycolic) acid, poly(caprolactone), polyglycolide, polylactide, polyhydroxobutyrate, polyhydroxyalkanoic acid, chitosan, hyaluronic acid, hydrogel, poly(2-hydroxyethyl methacrylate), poly(ethylene glycol), poly(L-lactide) (PLA), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), poly(glycerol sebacate), poly(octamethylene maleic anhydride citric acid) (POMaC), citric acid free POMaC, poly(ε-caprolactone), polyurethane, silk, nanoengineered materials, copolymers, blended polymers, or combinations thereof.

[0114] The shape, thickness, length, orientation, and surface morphology of the sensing elements can be varied in many suitable ways, so long as the sensing elements are capable of deforming, bending, or changing shape in response to the contractile action or activity of the tissue coupled therebetween, and such deformation, bending, or change in shape can be reliably measured, hi some embodiments, the sensing element is in the form of a wire, e.g., a polymer wire.

[0115] In some embodiments, the sensing element is porous, thereby allowing delivery of nutrients and growth factors to the tissue 130.

[0116] Cells can be seeded into the hydrogel to create tissue 130. The cells can be selected from cardiomyocytes, fibroblasts, skeletal muscle cells, liver cells, kidney cells, chondrocytes, skin cells, contractile cells, blood cells, immune system cells, germ cells, neural cells, epithelial cells, hormone-secreting cells, bone marrow cells, stem cells, tumor cells, smooth muscle cells, endothelial cells, fibroblasts, adipose-derived stem cells, mesenchymal stem cells, progenitor cells, or combinations thereof.

[0117] In some embodiments, the hydrogel comprises collagen or a collagen derivative, intestinal submucosa or a derivative thereof, cellulose or a cellulose derivative, proteoglycan, heparin sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, elastin, fibronectin, thrombin, laminin, fibrin, chitosan, alginate, Matrigel®, Geltrex®, agarose, decellularized extracellular matrix, polyethylene glycol or a derivative thereof, silicone or a derivative thereof, or a combination thereof. In some embodiments, the hydrogel comprises Matrigel®.

[0118] The present disclosure also provides a multiwell device having multiple devices 100 on a single substrate (e.g., a single plate). In some embodiments, the multiwell device can include multiple wells, e.g., 6 wells, 12 wells, 24 wells, 48 ​​wells, or 96 wells. The multiwell device can include two or more electrode pairs, with at least one well disposed between each electrode pair. In some embodiments, the multiwell device can include 2-20 electrode pairs (e.g., 2-20 pairs, 2-10 pairs, or 4-8 pairs). In some embodiments, the multiwell device can include 2 pairs, 3 pairs, 4 pairs, 5 pairs, 6 pairs, 7 pairs, 8 pairs, 9 pairs, or more electrode pairs. In some embodiments, 1-20 wells (e.g., 1-15 wells, 2-20 wells, 2-10 wells, or 4-8 wells) can be disposed between each electrode pair. In some embodiments, 1 well, 2 wells, 3 wells, 4 wells, 5 wells, 6 wells, 7 wells, 8 wells, 9 wells, or more wells can be placed between each electrode pair. In some embodiments, the multiwell device can be in the form of a multiwell plate. In some embodiments, the multiwell plate is a 24 well plate in a 4x6 configuration. In the 4x6 configuration, there are 6 electrode pairs, with 4 wells placed between each electrode pair.

[0119] Each electrode pair can be used to apply electrical stimulation to all wells between the electrode pairs. The operation of each electrode pair can be independent of the other electrode pairs, allowing each electrode pair to have different stimulation parameters than the other electrode pairs.

[0120] Figure 2C shows a multiwell device with 96 wells on a single substrate. The multiwell device includes 12 electrode pairs, each of which is parallel or substantially parallel to the other electrodes. The black lines indicate the electrodes. Eight wells are located between each electrode pair.

[0121] Hot embossing and injection molding techniques can be used to fabricate the devices described herein. In some embodiments, a custom multiwell plate with embedded electrodes and channels can be generated first, and sensing elements can be placed in the channels. Then, a bottomless plate with multiple cell culture wells can be attached on top of the multiwell plate. During attachment, the wells on the multiwell plate are aligned with the cell culture wells of the bottomless plate, so that each cell culture well contains one well.

[0122] In one aspect, the present disclosure provides a system comprising two or more devices (e.g., multi-well devices) as described herein, and a stimulator having two or more driver boards, each driver board configured to apply electrical stimulation between each electrode pair of each device, such that the stimulator can independently control the application of electrical stimulation between each electrode pair.

[0123] For in vitro tissue generation, the current state of the art has the following disadvantages: (a) manual programming of electrical stimuli is required at every step of the process; (b) the electromechanical interfaces are hand-made and manually connected; and (c) the hand-made systems are not designed for robustness, stability, and ease of assembly / manufacturability.

[0124] In comparison, the system described herein provides a scalable and automated platform for the generation, manipulation, and maintenance of tissues in vitro. This system allows all steps of the major workflow (e.g., cell seeding, maturation / stimulation, and end-point testing) to occur in the same location, without the need to transfer tissues from one chamber or Petri dish to another. Each tissue can be a self-contained, isolated bioreactor. In some embodiments, when the multi-well bottomless plate is attached to a custom-made polystyrene plate, each microwell is effectively sealed off from the rest of the plate. Media and nutrients can be applied to each tissue during the culture process. Furthermore, since the electrodes are already embedded in the plate, there is no need to move the tissue to apply the electrical stimuli of the maturation protocol. Given that each tissue is isolated, there is no need to separate them after maturation to apply any test compounds to perform tests (e.g., contraction tests). As a result, each tissue can be individually treated independently of the other tissues on the plate (e.g., different doses of test compounds).

[0125] In some embodiments, the stimulator may include at least 2 driver boards, at least 3 driver boards, at least 4 driver boards, at least 5 driver boards, or at least 6 driver boards. In some embodiments, the stimulator may include 25 or fewer driver boards, 20 or fewer driver boards, 19 or fewer driver boards, 18 or fewer driver boards, 17 or fewer driver boards, 16 or fewer driver boards, 15 or fewer driver boards, or 10 or fewer driver boards.

[0126] Combinations of the above ranges for the number of driver boards are also possible (e.g., at least 2 to no more than 25, at least 5 to no more than about 15, or at least 5 to no more than 10). In some embodiments, the stimulator can include about 10 driver boards.

[0127] In some embodiments, each driver board can have up to six channels. Each channel is individually configurable output (e.g., stimulation frequency) from the driver board. For example, a 24-well plate can be stimulated by six channels driven by one driver board. Thus, each driver board has the ability to control the stimulation parameters of each plate relative to the rows of wells, allowing one row of a plate to have different parameters than other rows of the same plate. Commercially available stimulators cannot provide this level of control.

[0128] Each driver board can be used to control one device, so the number of driver boards depends on the number of devices in the system, for example, if there are five multiwell devices in the system, there can be at least five driver boards, and if there are ten multiwell devices in the system, there can be at least ten driver boards.

[0129] In some embodiments, the electronic components of the stimulator are configured as shown in FIG.

[0130] The system allows for an expansion of the number of channels, with each channel being independently configurable. Benefits derived from such independent configurability include, but are not limited to, (1) better temperature regulation and control, (2) the ability to operate within an incubator without separate thermal control, (3) the ability to automate stimulation parameters over the maturation cycle, and (4) recordable stimulation history for each driver board.

[0131] In some embodiments, the system can further include an interface tray (e.g., a base plate) configured to electrically connect the device to a stimulator. The interface tray can include a hinged lid to secure the device and ensure that electrical contacts at the bottom of the device are connected to the leads coming from the stimulator. When a lab operator needs to remove the multi-well plate from the incubator, he or she lifts the hinged lid and removes the plate without unplugging or removing any components, making the process easier and safer for the organization.

[0132] In some embodiments, the system may further comprise an incubator configured to culture cells and tissues grown within the device. During the tissue maturation process, the device needs to be kept within the incubator at a constant temperature of approximately 37° C. while connected to the stimulator. To accomplish this, the incubator may comprise a custom incubator rack that houses multiple multi-well plates within the incubator. In each space within the incubator rack, one of the incubator trays is secured to the bottom of the rack, allowing an operator to secure the multi-well plate in place. The connections between the stimulator, the incubator rack, and the interface tray may be managed by standard ribbon cables and a custom printed circuit board (PCB) secured to the back of the incubator rack.

[0133] The system may further comprise an imaging subsystem configured to generate images of the sensing elements of each device and / or the tissue. The imaging subsystem may comprise an optical microscope.

[0134] The system may further comprise a computing device configured to connect to the imaging subsystem and analyze the images and / or control the stimulator. The computing device may comprise a graphical user interface (GUI) for connecting to the stimulator and controlling its settings. The GUI allows an operator to remotely review and adjust the stimulator settings and configure the stimulator to automatically execute a pre-determined stimulation protocol. Additionally, the GUI may report errors, such as open circuits, over / under voltage, and network disconnections (e.g., between the controller and the stimulator), to the operator. The stimulator GUI also allows an operator to view and track the progress of the maturation of the multi-well plate according to the protocol settings or the project for which the organization is specified.

[0135] The stimulator may be electrically connected to the incubator and / or the imaging subsystem.

[0136] Figure 12 is an abstract system block diagram according to some embodiments of the present disclosure. As shown in Figure 12, the system includes the following subsystems: (a) maturation and incubation subsystem, (b) test and imaging subsystem, (c) system controller, and (d) a computer that runs a GUI to set operational parameters. To accommodate expandability beyond a single stimulator box, maturation incubator, or test / imager, each subsystem can be connected to the system via a wired or wireless network.

[0137] Once mature, the tissue is ready for drug testing. Testing can be done in the testing and imaging subsystem. In some embodiments, this subsystem can include a Nikon imager, an X / Y stage controller to move the wells into the camera's field of view, a liquid handler to inject drugs into each well, and a stimulator box. The goal of the system is to integrate these four components into a single physical platform. Once integrated, drug testing tests can be run row by row, one plate at a time, to automate the orchestration of the tests. Each row represents each pair of embedded electrodes. The testing protocol also requires coordination of the same stimulation patterns.

[0138] In some embodiments, the system can have more than one stimulator. A system controller can be used to manage all stimulators. As the system expands, each stimulator can be assigned a unique identification by the system controller. The system controller is responsible for communicating the stimulation parameters for each row, each plate, and each stimulator, as parameterized in the GUI. It also monitors the status of each stimulator, periodically recording and time-stamping the operating parameters as well as checking the status of the stimulators (checking for overcurrent warning conditions).

[0139] A browser-based GUI running on a network computer can be the interface to the system controller. As long as the computer is on the same network, its physical location is irrelevant. The GUI computer can access the system controller remotely.

[0140] The adjustable parameters of the stimulator include, but are not limited to, pulse voltage, pulse frequency, and pulse duration. The stimulator is also responsible for monitoring the voltage and current applied to each pair of implanted electrodes. These measurements can be periodically logged to a database. The electrical stimulation can be user adjustable via a GUI application running on a networked computer.

[0141] FIG. 13 shows a schematic diagram of a system 300 for performing 3D tissue culture, according to one embodiment. The system 300 comprises a tissue growth device 310 further comprising a growth well 312 and a control unit 316. In some embodiments, the growth well 312 can be substantially similar to the above-mentioned device 100 shown in FIG. 1. For example, the growth well 312 can comprise one or more embedded electrodes (not shown in FIG. 13) configured to stimulate tissue growth. Additionally, the growth well 312 also comprises one or more wires (not shown in FIG. 13) configured to sense tissue growth. The control unit 316 is configured to control the operation of the growth well 312. In some embodiments, the control unit 316 can comprise a power source (not shown in FIG. 13) configured to provide power to the embedded electrodes. In some embodiments, the control unit 316 is configured to receive power (e.g., electricity) from an external power source. In some embodiments, the control unit 316 comprises a signal generator (not shown in FIG. 13) configured to provide an electrical signal to the embedded electrodes. Additionally, the control unit 316 may be configured to modify characteristics of one or more electrical signals, such as, for example, the amplitude, frequency, repetition rate, and / or duration.

[0142] The system 300 also includes an imaging device 320 configured to monitor tissue growth within the tissue growth device 310. In some embodiments, the imaging device 320 is configured to monitor a shape of a sensing wire within the tissue growth device 310 to estimate a state of the tissue. The imaging device 320 can include any suitable type of sensor, such as a charge-coupled device (CCD), a complementary metal-oxide semiconductor (CMOS) device, a photodiode, and / or a phototransistor, etc. In some embodiments, the imaging device 320 can include optical elements, such as lenses, collimators, waveplates, polarizers, and / or filters, etc., to facilitate image acquisition. In some embodiments, the imaging device 320 includes a microscope.

[0143] The processing device 330 is operatively connected to the imaging device 320 and configured to analyze images acquired by the imaging device 320. The processing device 330 comprises a memory 332, a processor 334, and a communication interface 336. The memory 332 can be, for example, a random access memory (RAM), a memory buffer, a hard drive, a database, a read only memory (ROM), an erasable programmable read only memory (EPROM), an electrically erasable programmable read only memory (EEPROM), and / or the like. In some cases, the memory 332 includes a set of instructions or code used to perform one or more methods of data collection and / or data processing (including image classification), as described below with reference to FIGS. 14-16. The processor 334 can be any suitable processor, for example, a general purpose processor (GPP), a central processing unit (CPU), an accelerated processing unit (APU), a graphics processing unit (GPU), a network processor, a front-end processor, an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), etc. Thus, the processor 334 may be configured to implement and / or execute a series of instructions, processes, modules, and / or code stored in the memory 332 .

[0144] The communications interface 336 can be any suitable device capable of communicating with and processing other devices, such as a device implementing the user interface 340, the tissue growth device 310, and / or any suitable device. In some embodiments, the communications interface 336 can include one or more wired and / or wireless interfaces, such as a network interface card (NIC), an Ethernet interface, an optical carrier (OC) interface, an asynchronous transfer mode (ATM) interface, and / or a radio interface (e.g., a WiFi® radio, a Bluetooth® radio, a near field communication (NFC) radio, etc.).

[0145] The user interface 340 is configured to allow a user to interact with other parts of the system 300. In some embodiments, the user interface 340 is configured to display available protocols that can be executed by the system 300 and to allow a user to select one or more protocols to be executed. In some embodiments, the user interface 340 is configured to display analysis results generated by the processing device 330. In these embodiments, the analysis results can include, for example, images acquired by the imaging device 320 (with or without processing), the growth status of the tissue in the tissue growth device 310, and potential defects of the tissue. In some embodiments, the user interface 340 includes a graphic user interface (GUI) associated with a web page, a PC application, a mobile application, and the like. In some embodiments, the user interface 340 can include a touch screen configured to receive tactile user input. In some embodiments, the user interface 340 can be implemented by any suitable computing device, for example, a desktop, a laptop, a personal computer, a server, a mainframe computer, a smartphone, a tablet, a wearable device, and the like.

[0146] 14 is a flow chart illustrating a method 400 for image processing, according to one embodiment. In some embodiments, method 400 can be performed by processor 334 (e.g., by executing instructions stored in memory 332). Method 400 includes receiving 410 an image of tissue growing around a wire in a well from cells seeded in the well (also referred to as an input image). In some embodiments, the image is acquired by imaging device 320 of system 300 of FIG. 13.

[0147] The method 400 also includes, at 420, forming a first binary image based on comparing each pixel from the plurality of pixels in the image to a pixel threshold. In some embodiments, to generate the first binary image, pixel values ​​equal to or greater than the pixel threshold are set to 1 and pixel values ​​less than the pixel threshold are set to 0. In some embodiments, this definition can be reversed, i.e., pixel values ​​less than or equal to the pixel threshold are set to 1 and pixel values ​​greater than the pixel threshold are set to 0. In some embodiments, the first binary image is formed using Otsu's method (see details with reference to FIGS. 15A-15B below).

[0148] At 430 of the method 400, a first edge and a second edge of the wire in the first binary image are detected and a second binary image representing the first edge and the second edge is formed. In some embodiments, the first edge and the second edge are detected based on a gradient of pixels in the first binary image. In some embodiments, the first edge and the second edge are detected using a Sobel operator (see details with reference to Figures 15G and 15H below).

[0149] At 440 of the method 400, a midpoint between the first edge and the second edge is calculated for each row of the second binary image, resulting in a series of midpoints. In some embodiments, the midpoint of the mth row of the second binary image is represented by a two-dimensional (2D) coordinate (x m ,y m ), and x m =(x1+x2) / 2, where (x1,y m ) are the coordinates of the pixel of the first edge, and (x2, y m ) are the coordinates of the pixel of the second edge. In some embodiments, the wires in the image (including the first edge and the second edge) are along the x direction. In these embodiments, the midpoint can be calculated for each column of the second binary image.

[0150] The method 400 also includes calculating 450 a quadratic equation that fits the set of midpoints based on a polynomial regression of the set of midpoints. In some embodiments, the quadratic equation represents the contour (or shape) of the wire in the input image. At 460, the method 400 identifies flex points of the wire based on the quadratic equation.

[0151] In some embodiments, the well comprises a series of electrodes configured to apply electrical stimulation to tissue according to the series of stimulation parameters. In these embodiments, the method 400 further includes adjusting the series of values ​​of the series of stimulation parameters based on the bending points of the wire (i.e., identified at 460) that meet the predetermined criteria to form an updated series of values ​​of the series of stimulation parameters. Stimulation signals are then sent to the series of electrodes to apply electrical stimulation to the tissue according to the updated series of values ​​of the series of stimulation parameters. In other words, the bending points identified at 460 can be used (e.g., by the processor 334) to derive growth conditions for the tissue.

[0152] In some embodiments, the method 400 also includes removing artifact pixels from the first binary image using an opening operator before detecting the first and second edges of the wire in the first binary image (see details with reference to Figures 15C and 15D below). In some embodiments, the method 400 also includes smoothing the first binary image using a closing operator before detecting the first and second edges of the wire in the first binary image (see details with reference to Figures 15E and 15F below).

[0153] In some embodiments, the method 400 further includes completing the portion of the first edge or the portion of the second edge that is not included in the second binary image prior to calculating the midpoint at 440. In this manner, each row of the second binary image includes a portion of the first edge and a portion of the second edge (see details with reference to Figures 15K and 15L below).

[0154] In some embodiments, the flex point identified at 460 can be used to calculate the amount of contractile force exerted by the tissue on the wire. In some embodiments, the flex point identified at 460 can be used to calculate the resting tension of the wire. The contractile force and / or resting tension can be indicative of the growth state (e.g., maturity) of the tissue and therefore can be used to form a feedback control loop (see more below with reference to FIG. 16).

[0155] 15A-15M illustrate a method for wire tracking (also referred to as a wire tracking method) according to some embodiments. In general, the wire tracking method identifies the edges of a wire sensor in each input image and calculates a series of 2D coordinates (x i ,y i ), where i=1, 2, 3, ..., N, and N is a positive integer. The 2D coordinates of the skeleton are then fitted to a second order polynomial (e.g., using least squares fitting). Without loss of generality, a second order polynomial (also called a parabola) is written as: TIFF0007654677000001.tif15170

[0156] In some embodiments, the wire tracing method includes a bit conversion step in which the input image is converted to an 8-bit image (i.e., an image having a grayscale ranging from 0 to 255). In some cases, the data values ​​of each pixel of the input image are in the form of a 16-bit integer format. In such cases, it is beneficial to convert these images to 8-bit images to facilitate further processing.

[0157] Direct conversion of 16-bit values ​​to 8-bit values ​​may result in certain data loss and may also affect the threshold if a particular frame has a slight brightness gradient. The effect on the threshold may lead to baseline spikes. To address this issue, a normalization step may be performed when converting the input image from 16-bit to 8-bit. More specifically, in this normalization step, a range minimum (i.e., minimum pixel value) and a range maximum (i.e., maximum pixel value) are identified for each frame. Then, each pixel value in the frame is adjusted according to contrast stretching, e.g., the minimum value of the range is stretched to 0 and the maximum value of the range is stretched to 256. Intermediate values ​​between the minimum and maximum are stretched proportionally to their location in the range from the minimum to the maximum. The 8-bit input image is then converted to a binary image by thresholding. In some embodiments, the pixel value of each pixel of the input image is compared to a threshold. If the pixel value is equal to or greater than the threshold, the pixel value is set to 1, otherwise the pixel value is set to 0. In some embodiments, the definition may be reversed, i.e., the pixel value is set to 0 if it is equal to or greater than the threshold, and set to 1 if it is less than the threshold. In some embodiments, the 8-bit image can be inverted before thresholding, i.e. the wire areas are set to black and the background areas are set to white.

[0158] In some embodiments, the binary image is generated using Otsu's method. Without being bound to a particular theory or mode of operation, Otsu's method is an adaptive thresholding method for binarization in image processing. By exploring possible thresholds (e.g., 0 to 255), Otsu's method allows finding an optimal and / or desirable threshold for an input image. Unlike other thresholding methods that use a fixed threshold, Otsu's method calculates the threshold based on the input image. This means that in Otsu's method, a different threshold is calculated for each image.

[0159] In some embodiments, the optimal and / or desired threshold is the within-class variance V w or between-class variance V bFor a given threshold T, the pixels of the input image can be divided into two classes, including class 1 for pixels with pixel values ​​less than or equal to T, and class 2 for pixels with pixel values ​​greater than T. Sometimes, class 1 and class 2 are referred to as the background and foreground of the input image, respectively. The within-class variance V w can be calculated as follows: TIFF0007654677000002.tif15170 (where μ is the average pixel value of a particular class, N is the number of pixels in a particular class, and Xi is the pixel value of each pixel in a particular image)

[0160] Between-class variance V b characterizes the variance between two classes. In some embodiments, the between-class variance V b V b =V t -V w , where V t is the total variance, i.e. the variance of all pixels in the input image. For a given image, V t is usually a constant. Therefore, V w By minimizing V b becomes maximum.

[0161] Fig. 15A shows an example input image before thresholding. Fig. 15B shows the input image after thresholding using Otsu's method. Additionally, the input image in Fig. 15A is inverted before thresholding. As can be seen in the binary image in Fig. 15B, the wire portion is set as background (i.e., black) and the rest of the image is set as foreground (i.e., white).

[0162] The binary image produced after thresholding is then subjected to one or more morphological operations. Morphological operations can include two operations: erosion and dilation. Both operations can have two inputs: (1) the image to be processed, and (2) a structuring element (also called a kernel). Each of the two inputs is represented by a set of coordinates.

[0163] More specifically, an erosion operation on a binary image can be performed as follows: Let X be the set of Euclidean coordinates corresponding to the input binary image, and K be the set of coordinates corresponding to the structuring element. Furthermore, let Kx be the translation of K such that the origin of the structuring element is at x. Using these concepts, the erosion of X by K is the set of all points x, such that Kx is a subset of X.

[0164] To calculate the erosion of a binary input image by this structuring element A, each of the foreground pixels of the input image can be considered in turn. For each foreground pixel (also called an input pixel), the structuring element is overlaid on top of the input image such that the origin of the structuring element coincides with the input pixel coordinates. For every pixel in the structuring element, if the corresponding pixel in the binary image below it is a foreground pixel, then the input pixel is left as is. However, if any of the corresponding pixels in the image are background, then the input pixel is also set to the background value.

[0165] In some embodiments, the structuring element is 3x3, and the effect of an erosion process using this structuring element is to remove any foreground pixels that are not completely surrounded by other white pixels (assuming 8-connected). Because these pixels may be on the edges of white regions, the net effect of the erosion is to shrink the foreground region and make the holes in it larger.

[0166] The dilation process for a binary image can be performed as follows. First, let X be the set of Euclidean coordinates corresponding to the input binary image, K be the set of coordinates of the structuring element, and Kx be the translation of K such that the origin of K is at x. Then, the dilation of X by K is the set of all points x such that the intersection of Kx and X is nonempty. More specifically, to compute the dilation of a binary input image by a structuring element K, one can consider each of the background pixels of the input image in turn. For each background pixel (also called an input pixel), a structuring element is overlaid on top of the input image such that the origin of the structuring element coincides with the input pixel location. If at least one pixel in the structuring element coincides with a foreground pixel in the image below, the input pixel is set to a foreground value. However, if all corresponding pixels in the image are background, the input pixel remains at its background value.

[0167] In some embodiments, the structuring element is 3x3, and the effect of a dilation operation using this structuring element is to set to the foreground color all background pixels that have a neighboring foreground pixel (assuming 8-connected). Because these pixels may be on the edge of a white region, the net effect of the dilation operation is to enlarge the foreground region and shrink any holes in it.

[0168] In the wire tracing method described herein, the binary image produced by thresholding is processed with opening and closing operations. Morphological opening and closing are essentially erosion and dilation operators applied in different orders. Without being bound to a particular theory or mode of operation, an opening is defined as an erosion followed by a dilation using the same structuring element in both operations, and a closing operation is defined as a dilation followed by an erosion using the same structuring element in both operations.

[0169] The basic effect of opening is to remove some of the foreground (i.e., bright) pixels from the edges of regions of foreground pixels. However, opening is usually less destructive than erosion. Although the closing operation tends to expand the boundaries of foreground (i.e., bright) regions in an image (and shrink background-colored holes in such regions), it is generally less destructive than dilation.

[0170] FIG. 15C shows a binary image generated after thresholding, and FIG. 15D shows a binary image generated after performing an opening operation on the binary image shown in FIG. 15C. FIG. 15E shows a binary image before a closing operation, and FIG. 15F shows a binary image generated after performing a closing operation on the binary image shown in FIG. 15E. In FIGS. 15C-15F, the structuring element is 5×5. In some embodiments, the closing operation is performed after the opening operation. In some embodiments, the order can be reversed, i.e., the opening operation is performed after the closing operation.

[0171] As can be seen in Figures 15C-15F, the opening and closing morphological operations help to remove and / or reduce artifacts that may interfere with the edges of the wires. The opening operator is applied to remove unwanted stray pixels and clean up the edges of the wires, while the closing operator can fill empty spaces and remove unwanted lines (e.g., as seen on the left side of the input image for the output images in Figures 15E-15F).

[0172] The wire tracking method also includes an edge detection step in which edges of the wire in an input image are identified. The input image in this step can be, for example, an image after the morphological operations described above. In some embodiments, edge detection is performed using a Sobel operator configured to calculate gradients of pixel values ​​in both the x and y directions. The Sobel operator convolves a kernel with the input image and calculates an approximation of the derivative to detect edges.

[0173] More specifically, the first kernel G x We can convolve with the input image from left to right to obtain the gradient in the first direction (say, the x direction). Without loss of generality, the first kernel can be written as: TIFF0007654677000003.tif17170

[0174] The second kernel G y We can convolve with the input image from top to bottom to obtain the gradient in the second direction (say, the y direction). Without loss of generality, the second kernel can be written as: TIFF0007654677000004.tif18170

[0175] The above two convolution steps produce two vectors representing the gradient of the image in the x and y directions. Therefore, the gradient magnitude of each pixel of the input image can be calculated as follows: TIFF0007654677000005.tif18170

[0176] Fig. 15G shows the binary image after the above opening and closing operations, and Fig. 15H shows the binary image after edge detection using the Sobel operator. The image after edge detection (Fig. 15H) is also called the edge detection image. As can be seen in Fig. 15H, two edges of the wire (i.e., the right edge and the left edge) are detected and shown as white lines.

[0177] In some embodiments, the edge-detected image (e.g., the image shown in FIG. 15H) is processed with another thresholding step in the wire-tracing method to generate another binary image. In some embodiments, the thresholding at this step can be performed using Otsu's method as described above. FIG. 15I shows the edge-detected image, and FIG. 15J shows the binary image generated after thresholding using Otsu's method.

[0178] In some embodiments, the edge-detected image is further processed with edge filling (e.g., after thresholding). In some cases, wire edges are trimmed in the edge-detected image, and edge filling is configured to fill empty edges (e.g., at the top and bottom of the detected edges). In some cases, the trimming of wire edges in the edge-detected image may be due to the kernel used in the edge detection process, and edges that are trimmed out of the image frame are not detected. Thus, the left and right edges of each row are not fully calculated.

[0179] The edge filling technique described herein traverses up and down from the center row of the edge detected image until it finds a row where either the right edge point or the left edge point is the last or first column of the image (i.e., the wire edge point is also at the edge of the image). From there, the rows above are filled with the right or left edge as the last or first column.

[0180] FIG. 15K shows the edge detection image after thresholding, and FIG. 15L shows the image obtained by performing edge filling on the image shown in FIG. 15K. The image after edge filling is also called an edge-filled image. As can be seen in FIG. 15K, the bottom of the right edge of the wire is filled in along the right edge of the image.

[0181] The next step in the wire tracking method involves calculating the midpoints. In this step, the midpoint of the wire is calculated for each row of the edge fill image. The midpoint can be calculated by taking the average of a first coordinate representing the left edge and a second coordinate representing the right edge. Figure 15M shows an example image showing a series of calculated midpoints.

[0182] Once the midpoints are calculated, they can be used as inputs for polynomial regression to generate a quadratic equation that best fits the midpoints. In statistics, polynomial regression is a form of regression analysis in which the relationship between an independent variable x and a dependent variable y is modeled as an nth order polynomial in x. More specifically, the regression method described herein uses a least squares method to fit a quadratic polynomial. The midpoints calculated above are represented by corresponding x and y coordinates. The coordinates are essentially row and column numbers. These sets of x and y values ​​are used to calculate an equation using the polynomial regression method.

[0183] Without loss of generality, the regression method can be expressed, for example, as follows: TIFF0007654677000006.tif12170 (In the formula, x i and y i is the midpoint coordinate calculated for each image frame)

[0184] Equation (6) can be written in matrix form as follows: TIFF0007654677000007.tif24170 or more succinctly can be written as: TIFF0007654677000008.tif10170 (where Y is the element (y1, y2, ..., y n ), X is the calculated midpoint x i , A is a vector containing the fitting parameters a, b, and c)

[0185] Solving equation (8), we obtain the following equation. TIFF0007654677000009.tif10170 Equation (9) is executed for each input image frame to generate a quadratic equation for each frame, and thus fitting parameters (also called fitting coefficients) are calculated for each image frame.

[0186] The quadratic equation fitting coefficients from the previous step are used to find the maximum point of deflection or displacement of the parabola. In some embodiments, the deflection point is found by utilizing the derivative of the equation x M Then, for that value of x in equation (6), add x M Substituting, the y coordinate, i.e., y M This y M The value is used in a plot to represent the displacement of the wire.

[0187] Using the above deflection points, the force on the sensing wire can be calculated. In general, quantification of force transients involves precise identification of the location of each transient within the force recording. In some embodiments, peak analysis methods can be used to identify force transients by matching theoretical force trajectories (also called simulated force trajectories) with actual force trajectories. An actual force trajectory is a graph resulting from plotting the change in measured force over time. Contraction of tissue appears as peaks and valleys in the resulting trajectory as the force repeatedly increases and decreases over time. Theoretical force trajectories are generated with artificial transients at known intervals or known frequencies. Peak analysis methods have the advantage of being able to identify transient peaks in recordings with low signal-to-noise ratios (SNR) or in recordings where the amplitude of force transients has been reduced in response to compounds that result in very small force transient recordings.

[0188] The actual force trajectories are then fitted to a polynomial function to reduce noise in the raw force transient trajectories, and contraction parameters are calculated from the polynomial function, including, for example, transient amplitude, transient duration, time to transient peak, time from transient peak, maximum slope of force production, maximum apparent slope, etc.

[0189] Figure 16 is a flow chart illustrating a method 500 of manipulating tissue culture, according to one embodiment. In some embodiments, the method 500 can be performed by the processor 334 of the system 300 illustrated in Figure 13. In these embodiments, the memory 332 in the system 300 comprises a non-transitory processor-readable medium configured to store processor-executable code. The processor 334 performs the method 500 upon execution of the processor-executable code.

[0190] The method 500 includes, at 510, receiving a series of measurements from at least one sensing element associated with a well (e.g., well 312 of FIG. 13 ) configured to grow tissue from cells seeded in the well. The well includes a series of electrodes configured to apply electrical stimulation to the tissue according to the series of stimulation parameters. The method 500 also includes, at 520, identifying an amount to modify a series of values ​​of the series of stimulation parameters based on a comparison of the series of measurements to a series of predetermined criteria associated with the tissue. The series of values ​​is then adjusted using the amount to modify the series of stimulation parameters to form, at 530, an updated series of values ​​for the series of stimulation parameters. A stimulation signal is then transmitted, at 540, to the series of electrodes to apply electrical stimulation to the tissue according to the updated series of values ​​for the series of stimulation parameters.

[0191] In some embodiments, the set of stimulation parameters includes at least one of a stimulation voltage, a stimulation frequency, and / or a stimulation time. In some embodiments, the stimulation signal can be transmitted by the processor 334 to the control unit 316 of the tissue growth device 310 via the communication interface 336. The control unit 316 is then configured to execute the stimulation signal by sending electrical signals to the electrodes characterized by the stimulation parameters specified in the stimulation signal. In other words, the stimulation signal transmitted by the processor 334 serves as an instruction to the control unit 316, which controls the operation of the electrodes based on the instruction.

[0192] In some embodiments, the sensing element comprises at least two elastic sensing elements (e.g., sensing wires) positioned across the well such that a gap exists between the sensing elements and the bottom of the well. In these embodiments, tissue growth within the well can exert a force on the elastic sensing elements, thereby causing the elastic sensing elements to change shape. In some embodiments, a processor 334 within the system 300 is configured to analyze the shape (or change in shape) of the elastic sensing elements to determine a state of tissue growth within the well. In response to the determined growth state, the processor 334 is configured to determine an amount to modify a set of stimulation parameters.

[0193] In some embodiments, the initial set of values ​​for the set of stimulation parameters is selected based on a predefined stimulation protocol. In some embodiments, the user interface 340 is configured to present the predefined stimulation protocol to a user from among a plurality of available stimulation protocols, and the user may select one stimulation protocol as the predefined stimulation protocol.

[0194] In some embodiments, the predetermined criteria associated with the tissue include a maturity criterion and / or an excitation criterion of the tissue. In response to a series of measurements that meet the maturity criterion and / or the excitation criterion, the stimulation voltage can be decreased accordingly (e.g., by the processor 334). In response to a series of measurements that do not meet the maturity criterion and / or the excitation criterion, the stimulation voltage can be increased accordingly (e.g., by the processor 334). In some embodiments, the series of measurements includes a maturity state and / or an excitation criterion of the tissue obtained from an image of the sensing element. For example, processing of data from a sensing wire may indicate that the deflection of the wire is not large enough (e.g., below a threshold amount of deflection), thereby indicating that the tissue is not as mature as expected. In this case, the stimulation voltage can be increased.

[0195] In some embodiments, the series of measurements can be obtained by another sensor, for example a voltage sensor configured to measure the voltage applied to the implanted electrodes. In response to a measured voltage below a threshold voltage (e.g., specified in the protocol), the processor can send an instruction to the control unit 316 to increase the voltage. Any other operational parameters of the tissue growth device 310 can be adjusted similarly.

[0196] In some embodiments, the method 500 further includes periodically receiving (e.g., by the processor 334) an instance of the series of measurements and storing the instance of the series of measurements in memory (e.g., memory 332). In these embodiments, a history of tissue growth in the well can be recorded. In some embodiments, the series of measurements also includes operating parameters of the tissue growth device 310, such as the frequency and voltage applied to the electrodes. In some embodiments, the series of measurements is stored in memory, for example in a comma separated value (CSV) format or any other suitable format, for later analysis and troubleshooting if a problem with the tissue is detected.

[0197] In some embodiments, method 500 can be executed by a processor without human intervention for a preset period of time (e.g., on the order of days to weeks). In these embodiments, user interface 340 can present a user with several available protocols, and the user selects one to execute. Once the user selects a protocol, system 300 can be configured to execute the protocol (and update any stimulation parameters based on feedback as described in method 500) without further human intervention. In some embodiments, user interface 340 can be configured to display growth results upon completion of execution of the protocol. Growth results can include, for example, images of the tissue, images of the sensing wire, maturation and / or excitability state of the tissue obtained from analysis of the sensing wire, etc.

[0198] In some embodiments, the series of measurements can be used to assess the quality of the tissue growth. Such assessment can include, for example, detecting one or more problems in the tissue growth. In some embodiments, the processor 334 can be configured to detect one or more problems in the tissue growth (e.g., tissue contamination, debris, holes, etc.) based on the series of measurements by machine learning. In these embodiments, the processor 334 can be configured to implement, for example, an artificial neural network configured for image classification. In image classification, an input image can be classified into one of several categories, with each category representing a common problem in the tissue growth. The input image can be an image of tissue, an image of tissue encapsulating the sensing element, or any combination thereof.

[0199] In some embodiments, in response to detecting a problem in tissue growth (e.g., by a machine learning model), processor 334 is further configured to take remedial action, e.g., modifying stimulation parameters. In some embodiments, in response to detecting a problem in tissue growth, processor 334 is further configured to generate an alert signal. In some embodiments, processor 334 is configured to send the alert signal to a user (e.g., via communication interface 336). The alert signal may include, for example, a text message, an email, a voicemail, or a real-time call. In some embodiments, each category of general problem is assigned an error code, and the alert signal sent to the user includes the error code.

[0200] Referring again to FIG. 13, in some embodiments, the system 300 (or processor 334) can be configured to perform end-to-end data analysis. In these embodiments, raw data (e.g., raw images of the sensing wire) is first sent to the processor 334 for wire tracking, which generates force values ​​over time based on the movement of the wire. For example, the processor 334 can be configured to perform the methods described with reference to FIG. 14 and FIGS. 15A-15M. The force values ​​are then used for peak analysis, which is configured to generate a quantitative readout, e.g., peak amplitude, time to peak, etc. The quantitative readout is then used to plot a graphical display. The graphical display can be force trajectories, change in maximum force over time, comparison of time to peak before and after drug treatment, etc. In an end-to-end analysis, the above processes can be performed by the processor 334 (or other processors) without human intervention. For example, a user can select a protocol to be performed by the system 300 and receive a graphical display upon completion of the protocol. In some embodiments, the graphical display can be displayed on the user interface 340. In some embodiments, the graphical representation can be transmitted to the user over a network (wired or wireless), for example, the graphical representation can be transmitted to a smart phone or computer associated with the user.

[0201] In some embodiments, the system 300 (or the processor 334) can be configured to implement a scheduler for operating the tissue growth device 310. In some embodiments, the processor 334 comprises an FPGA and can execute the scheduler via Verilog or other suitable language. In some embodiments, an operator can set the pulse intervals of the system 300 using the user interface 340. The user interface 340 is configured to send the pulse interval information to the scheduler, and the scheduler is configured to generate a timetable to precisely determine when a particular well in the tissue growth device 310 will receive a pulse (e.g., for stimulation). Thus, the scheduler can be used to provide accurate stimulation timing on average with short-term deviations for successive well stimulations.

[0202] Some embodiments described herein relate to methods. Such methods are of course computer-implemented methods (e.g., instructions stored in a memory and executed on a processor). Where the above methods indicate that certain events occur in a certain order, the order of certain events can be changed. Furthermore, certain events can also be repeated, possibly simultaneously in parallel processes, and sequentially as described above. Furthermore, certain embodiments can omit one or more described events.

[0203] Some embodiments described herein relate to computer-readable media. Computer-readable media (or processor-readable media) are non-transient in the sense that they do not include the transitory propagating signal itself (transmission media, e.g., propagating electromagnetic waves that carry information in space or cable). The medium and computer code (sometimes also called code) may be designed and configured for a specific purpose. Examples of non-transitory computer-readable media include, but are not limited to, magnetic storage media, e.g., hard disks, floppy disks, and magnetic tapes, optical storage media, e.g., compact disks / digital video disks (CD / DVDs), compact disk read-only memories (CD-ROMs), and holographic devices, magneto-optical storage media, e.g., optical disks, carrier wave signal processing modules, and hardware devices, e.g., ASICs, PLDs, ROMs, and RAM devices, that are specially configured to store and execute program code. Other embodiments described herein relate to computer program products that may, for example, include the instructions and / or computer code described herein.

[0204] Examples of computer code include, but are not limited to, microcode or microinstructions, such as those generated by a compiler, machine instructions, code used to generate web services, and files containing high-level instructions executed by a computer using an interpreter. For example, embodiments can be implemented using Python, R, Java, JavaScript, C++, or other programming languages ​​and development tools. Further examples of computer code include, but are not limited to, control signals, encryption code, and compression code.

[0205] While the present teachings have been described in conjunction with various embodiments and examples, it is not intended that the present teachings be limited to such embodiments or examples. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0206] Although various inventive embodiments have been described and illustrated herein, those skilled in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application for which the teachings of the present invention are used. Those skilled in the art will recognize many equivalents to the specific inventive embodiments described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that, within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0207] All definitions and those used herein should be understood to control for any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0208] The indefinite articles "a" and "an," as used in the specification and claims, unless expressly indicated to the contrary, should be understood to mean "at least one." Ranges cited herein are inclusive.

[0209] As used throughout this specification and claims, the terms "substantially," "approximately," and "about" generally mean plus or minus 10% of the stated value, e.g., about 100 includes 90-110.

[0210] As used herein, the term "POMaC" refers to poly(octamethylene maleate (anhydride) citrate) (POMaC), or a POMaC prepolymer comprising a mixture of 1,8-octanediol, citric acid, and maleic anhydride. See Tran et al., "Synthesis and characterization of a biodegradable elastomer featuring a dual crosslinking mechanism," Soft Matter, Jan. 1, 2010, Vol. 6, No. 11, pp. 2449-2461, which is incorporated herein by reference in its entirety.

[0211] The phrase "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements described with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" phrase can be present as needed, whether or not related to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising", can refer to only A in one embodiment (possibly including elements other than B), only B in another embodiment (possibly including elements other than A), both A and B in yet another embodiment (possibly including other elements), etc.

[0212] "Or" as used herein and in the claims should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is interpreted as being inclusive, i.e., including at least one of the elements or elements of the list, and possibly other items not listed, but also including two or more of the elements. To the contrary, only terms expressly indicated, such as "only one of" or "exactly one of," or "consisting of," when used in the claims, mean including exactly one element of the elements or elements of the list. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0213] As used herein and in the claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, and not necessarily including at least one of each and every element specifically listed in the list of elements, and not necessarily excluding any combination of elements in the list of elements. By this definition, there may optionally be elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to the specifically identified element. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer to, in one embodiment, the inclusion of at least one A, and possibly more than one A, where B is absent (and possibly including elements other than B); in another embodiment, the inclusion of at least one B, and possibly more than one B; in yet another embodiment, the inclusion of at least one A, and possibly more than one A, and at least one B, and possibly more than one B (and possibly including other elements); etc.

[0214] As in the specification above, in the claims, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," and the like, are to be understood to be open-ended, i.e., meaning inclusive but not limited. As set forth in the U.S. Patent Office Manual of Patent Examining Procedure, Section 2111.03, only the transitional phrases "consisting of" and "consisting essentially of" shall be exclusive or quasi-exclusive transitional phrases, respectively.

[0215] The claims should not be read as limited to the described order or elements unless that intent is stated. It should be understood that various changes in form and detail can be made by those skilled in the art without departing from the spirit and scope of the appended claims. All embodiments that come within the spirit and scope of the following claims and equivalents thereto are claimed. EXAMPLES

[0216] Example 1 Here, an innovative multi-material processing into a scalable and functional platform is proposed in the format of a 96-well plate. Three classes of materials are integrated into the platform. Arrays of soft elastic microwires are used not only as anchors for tissue formation but also as sensors to record tissue contraction. Conductive carbon electrodes are embedded in the plate to drive electrical stimulation for tissue maturation and to modulate tissue contraction during drug testing. The majority of the device is made of rigid polystyrene plastic to eliminate drug-absorbent polydimethylsiloxane (PDMS). The platform allows for a higher throughput than current state-of-the-art devices, significantly reducing the manufacturing and tissue production costs.

[0217] Our tissue culture platform (herein referred to as Biowire II plate) is made of tissue culture plastic (polystyrene), resulting in a PDMS-free final product, minimizing drug absorption. The platform is based on the footprint of a standard 96-well plate and is compatible with widely used plate readers. The device was assembled from four components: 1) a plate cap, 2) a bottomless 96-well plate, 3) a polymer wire array, and 4) a patterned polystyrene base (Figure 2A-2C). The plate cap and bottomless 96-well plate are commercially available components. The polystyrene plate base is customized to fit the 96-well plate format, with one microwell (LxWxH: 5x1x0.2mm) aligned to the center of each bottomless well (Figure 2A). Human pluripotent stem cell-derived cardiomyocytes (hCM) and human cardiac fibroblasts (ratio 10:1) were cultured in a hydrogel matrix (3.0 mg mL -1 Collagen and 15% (v / v) Matrigel) into the microwells. To provide an anchoring point for tissue remodeling and compression, two parallel-positioned elastic microwires (diameter: 0.1 mm) were cast across the microwells (Figure 2B). Over time, the hCM / fibroblast / hydrogel mixture seeded in the microwells self-assembles into a 3D tissue that is spanned and anchored across the two parallel wires (Figure 2C). Within a week, the tissue began to contract and the elastic polymer wires bent. Using a calibration curve generated by a standard force sensor (MicroSquishier, CellScale), the displacement of the polymer wires could be related to the contractile force of the tissue.

[0218] Although the entire device fabrication process can take up to 6 days, most steps, including manual operations, are short and multiple plates can be fabricated in parallel (Figure 8A). The fabrication of the polymer wires and base plate are simultaneous. For these components, a SU-8 master mold was designed and fabricated using standard soft lithography techniques according to the specifications shown in Figure 8B and Figure 8C. A PDMS mold was then fabricated by placing PDMS into the SU-8 master and crosslinking the PDMS at room temperature for 48 hours. The PDMS mold was then used to fabricate the polymer wires so that the polystyrene base plate could be hot embossed. To minimize the effects of thermal expansion, we chose to crosslink the PDMS mold at room temperature rather than by heating. Thus, the resulting PDMS mold retains the designed dimensions over a large surface area, e.g., the footprint of a 96-well plate. However, thermal expansion still occurs during hot embossing of the base plate. Initially, it proved difficult to maintain the pre-determined plate dimensions without shrinkage across this large footprint. Even a shrinkage of 0.1 mm over a 100 mm length can result in significant misalignment between the wires and the microwells. Therefore, the degree of shrinkage after hot embossing was empirically determined and the dimensions of the wire array were readjusted to match the final dimensions of the plate base (Figure 8B).

[0219] PDMS-based master mold for hot embossing

[0220] To fabricate the polystyrene base plate, the back side of the PDMS mold of the base plate was attached to a 6-inch silicon wafer by plasma bonding or corona etching methods. After bonding to the silicon wafer, the mechanical properties of the PDMS master were further enhanced by exposure to 150 °C for 30 min. A polystyrene sheet was pressed and molded against the PDMS master in a hot embosser to obtain a customized polystyrene plate base. Fabrication of a PDMS master for hot embossing is much cheaper than traditional metal etching methods to create a master. As PDMS is softer than metals, feature distortion can occur during hot embossing, but it was found that this issue could be avoided by using high temperatures and low compression forces during embossing. One important advantage of PDMS over metal-based masters is its ability to withstand temporary distortion, allowing the embossed polystyrene sheet to be easily released from the master. This poses a major challenge in that when a hard material (polystyrene) is being embossed against another hard material (a metal master), the microscopic elements must possess strictly vertical walls to prevent the two hard materials from interlocking.

[0221] The plate incorporates a pair of built-in carbon electrodes around every microwell to generate a localized electric field throughout each tissue. Pacing the cardiac tissue with external electrodes has been shown to ensure consistency in drug testing, both by maturing the tissue over time and by standardizing the beating frequency. For each row of wells, two long carbon electrodes were placed along the vertical border of the well. Figure 8C shows the design of the base plate. Rectangular posts around the microwells were designed to hold the carbon electrodes to the PDMS master mold before hot embossing (Figure 3A). The carbon electrodes were embedded within the polystyrene sheet during hot embossing, exposed on the plate surface and side walls that make up the microwells (Figure 3B, Figure 3C). These exposed surfaces were strategically placed to be as close as possible to the tissue, sufficient to generate an electric field along the length of the tissue. Embedding the 400 μm thick electrodes within the polystyrene base allowed current to flow with minimal resistance. This is in contrast to other methods, including our initial tests using sputtering (e.g., sputtered gold electrodes), which resulted in thin conductive surfaces with nanometer-scale thickness (Figure 9A). Such conductive surfaces proved difficult to coat uniformly across large surface areas, were easily damaged by scratching, had high electrical resistance, and lacked long-term stability due to delamination (Figure 9B). Compared to the voltage applied from the power supply, the percentage voltage drop across the embedded carbon electrodes was negligible and consistent across the well plate (Figure 10A).

[0222] Rapid wire casting on microwells

[0223] Casting soft elastic polymer microwires that can span across an array of microstructures in a scalable manner can be challenging. To circumvent this, we developed a method to mold and crosslink polymer wires directly onto polystyrene-based microwell structures. To do this, a PDMS mold of the wire array was placed over a base plate, with the microchannels on the mold facing the plate (Figure 4A). Poly(octamethylene maleic (anhydride) citrate) (POMaC) prepolymer, a citric acid-based polyester that can be crosslinked with UV light or thermal energy, was deposited at the channel inlet. The prepolymer solution was drawn into the microchannel by capillary action. Notably, the prepolymer solution continuously filled the channel above the microwells, even though the cross-section of the microchannel was not completely closed (Figure 4B). Following complete perfusion, the POMaC prepolymer was exposed to UV light to generate an array of crosslinked elastomeric wires, after which the PDMS mold was removed. To mitigate the effect of batch-to-batch variations in the bulk polymer solution on the mechanical stiffness of the UV-crosslinked polymer wires, the UV crosslinking energy was adjusted for each batch of polymer such that the Young's modulus of the bulk polymer after crosslinking (measured with a myograph as described) was 33 ± 3 kPa. The UV-crosslinked POMaC wires consistently attached to the polystyrene base, allowing multiple microwires to be cast across the entire plate and spanned across 96 microwells (Figure 4C). This method allowed the attachment of 24 wires spanned across 96 microwells in a single step. The plate base cast with microwires was fully assembled in a thermal bonding step in a hot embosser, fusing a bottomless 96-well plate onto the polystyrene plate base cast with polymer microwires. The assembled plate then underwent several post-processing steps, including washing overnight in distilled water, air drying, packaging, and gamma sterilization. The elasticity of the microwires can be further fine-tuned by another thermal crosslinking by baking the entire plate at 70 °C for several days.Plates were stored at 4°C prior to use and protected from light to minimize unnecessary heat and light exposure prior to use.

[0224] Plate characterization and use

[0225] The cast polymer wires in the plate were used as both an anchoring point for the tissue and as a force sensor to monitor changes in contraction. To do so, we characterized the correlation between wire displacement and force generation. We used a commercially available microscale mechanical testing system, MicroSquisher (CellScale), to generate force-displacement calibration curves (Figures 5A-5D). The probe tip was customized with a curvature similar to the attachment of tissue to the polymer wire. At the center point of the polymer wire, the probe tip was moved relative to the polymer wire to record both force and displacement with the MicroSquisher, generating a force-displacement curve (Figure 5A). The mechanical properties of the polymer wire can be further fine-tuned by post-treatment with thermal crosslinking. The force-displacement curves and fitted polynomials after 2, 4, and 6 days of thermal crosslinking are shown in Figures 5B-5D. The mechanical consistency of the POMaC wires was demonstrated across the 96-well plate. There was no significant difference when comparing the force at 100 μm wire displacement between wells in different rows (Figure 11C). The force required to displace the wire by 50, 100, and 150 μm was also reproducible across all wells (Figure 11B). Furthermore, tensile and relaxation curves of a representative polymer wire showed minimal hysteresis (Figure 11C). Microtissues cultured in each 96-well plate were able to grasp the wire (Figure 6A). Cardiac tissue also exhibited well-aligned sarcomere structures when stained for sarcomeric α-actinin (green) and F-actin (red) (Figure 6B). As the 96-well plate is a widely adopted tissue culture format, we have shown that the Biowire plate is also compatible with commercially available instruments, such as the SpectraMax image cytometer for automated imaging and analysis (Figure 6C).

[0226] Long-term tissue culture and drug testing with electrical stimulation

[0227] We next characterized the utility of the platform for in situ measurements (e.g., force-displacement) and conducted proof-of-principle studies to show that the platform can support long-term culture of cardiac tissue in a manner similar to Biowire I, tracking the functional response of cardiac tissue cultured on the platform under electrical stimulation and treatment with clinically relevant drugs. To generate cardiac tissue, human iPSC-derived cardiomyocytes co-cultured with human cardiac fibroblasts were seeded in a 96-well plate device with hydrogel. Compaction of the tissue was observed over the first 7 days (Figure 7A, Figure 7B), with dramatic changes over the first 2 days, followed by stabilization after 3 days. Electrical field stimulation of the cardiac tissue was initiated on day 7 and continued for an additional 12 days. During this period, the electrical function of the tissue improved as observed by a significantly increased maximum capture rate (Figure 7C, Figure 7D). The plate facilitated non-destructive assessment of tissue contractile motion, and assessments showed that resting tension, active force, contraction and relaxation times remained stable after 12 days of electrical stimulation (Figure 7E-7G). Thapsigargin (50 × 10 -6 When administered in the presence of electrical pacing (1.5 Hz), the tissue contraction and relaxation times were prolonged (Figure 7H). Furthermore, the tissue pulsation frequency was halved upon drug application, despite a constant stimulation frequency (Figure 7H).

[0228] Consideration

[0229] In this study, we developed a novel method for rapid casting of suspended elastic microstructures (e.g., wires) onto an array of microfabricated features (e.g., microwells). Thermal bonding with a hot embosser allowed us to fuse multiple parts of the plate and embed both carbon electrodes and polymer wires into the platform to create a controlled microenvironment around the tissues. Tissue function and response can be manipulated with electrical stimulation and tracked with built-in force sensors. In this multi-well plate format, each tissue was cultured and analyzed individually. The entire manufacturing process takes six days from production to storage, but the entire process is scalable because the manufacturing workload does not increase with the number of wells in the plate. This means that the same process can be applied to create 6-well, 24-well, 96-well, and even 386-well plates. Moreover, multiple plates can be fabricated in parallel to achieve a higher production throughput, compatible with industrial manufacturing processes. Up to 96 tissues can be cultured in a single plate, and the experimental throughput is significantly higher than many conventional organism-on-a-chip platforms. Furthermore, only 100,000 cells are required to form cardiac tissue. Currently, cell seeding remains the rate-limiting step for high-throughput tissue production. However, the open-well design allows for the scale-up of cell seeding and manipulation of this multi-well plate platform using automated cell dispensers that are widely utilized in the pharmaceutical industry.

[0230] Post-treatment of the materials after plate assembly also allowed us to tune the deflection-force profile of this system. Characterization of the mechanical properties of the elastic microwires allows us to infer a functional readout of the contractile motion of the tissue. The mechanical properties of the POMaC wires were reproducible between all wells of a 96-well plate, indicating the reliability of this casting method. Because the polymer wires are always positioned at the same height, the tissue height is always constant and can be easily monitored, in contrast to post-based platforms where the vertical position of the tissue is non-uniform. A non-destructive functional readout is important for long-term chronic drug studies. In our application, we demonstrated the rapid casting of soft elastic microwires for cardiac tissue culture purposes. However, more complex suspended microstructures can be cast to allow the formation of more complex tissue structures with the same method. The platform also incorporates built-in electrodes that can stimulate the tissue continuously for at least 12 days to aid in tissue maturation. Highly conductive carbon electrodes minimize the voltage drop from the power source to the electrodes, thereby facilitating precise control of the culture and testing environment. The presence of electrodes plays a key role in rigorous investigation of drug responses. For example, when testing drugs in an acute or chronic format, stimulating the tissue at a physiologically relevant frequency allows the chronotropic effect of the drug to be separated from the inotropic effect. Thapsigargin treatment slowed the contraction and relaxation of the tissue, decreasing the frequency despite a fixed pacing frequency. This means that the tissue relaxes too slowly to initiate the next depolarization until the relaxation of the previous contraction cycle is finished, and therefore the frequency is reduced by half. Thus, our platform is able to reproduce the known in vivo effects of this compound, suggesting its application as a relevant in vivo screening platform.

[0231] Here, we described a simple method to fabricate arrays of elastic microwires for scalable 3D cardiac tissue culture and contractile force readout in a 96-well plate format. This arrayed tissue culture plate facilitates microscale tissue culture with minimal numbers of cells, promotes cardiomyocyte maturation with built-in carbon electrodes, and enables noninvasive functional assessment for cardiotoxicity prediction. Based on the ease and high throughput of fabrication, as well as the availability of high-fidelity cardiac tissue, the characterized platform provides a valuable tool for evaluating tissue responses to pharmacological compounds.

[0232] Experimental procedure

[0233] Preparation of POMaC prepolymer solution POMaC was used to prepare the self-contained polymer sensor. POMaC was synthesized by first preparing a prepolymer gel via a polycondensation reaction. Citric acid (Caledon, A00019), maleic anhydride (Sigma, 63200), and 1,8-octanediol (Sigma, 03303) were combined in a three-necked 250 mL round-bottom flask with equimolar amounts of carboxylic acid (citric acid + maleic anhydride) relative to the alcohol (1,8-octanediol) functional groups, and a 1:4 ratio of citric acid to maleic anhydride. The reaction solution was heated to produce a molten polymerization solution, which was carried out at 150 °C under nitrogen flow with stirring at 200 rpm for 5 h. The resulting gel was dissolved in 1,4-dioxane (Sigma, D201863) followed by dropwise precipitation in deionized water to remove any unreacted monomers or short-chain oligomers. The aqueous phase was then poured off and the undissolved prepolymer was collected and concentrated. The purified prepolymer was mixed with poly(ethylene glycol) dimethyl ether (PEGDM Mw~500) (Sigma, 445886) in a ratio of 6:4 (w:w) and with 5 (w / w)% of the photoinitiator, 2-hydroxy-1-[4(hydroxyethoxy)phenyl]-2-methyl-1-propane (Sigma, 410896). The resulting solution was stored at 4 °C until use in sensor casting.

[0234] Fabrication of baseplates with built-in carbon electrodes

[0235] A repeating pattern consisting of rectangular microwells (5x1mm, LxW) and rectangular notches (1x0.5mm, LxW) was designed in AutoCAD. The spacing (in all directions) from adjacent microwells was 9mm to achieve compatibility with conventional 96-well tissue culture plates. Using soft lithography, a SU-8 photoresist master mold was generated using a photomask derived from the computer model and used to fabricate a negative PDMS (Sylgard 184 Silicone Elastomer Kit, Dow Coming, 01064291) master mold with feature heights of 200μm. Briefly, the silicone elastomer base and curing agent were mixed vigorously in a cup in a 5:1 (w:w) ratio, and the mixture was poured onto the SU-8 master mold and cured at room temperature. The PDMS master mold was then fixed onto a silicon wafer by plasma bonding or corona etching. To embed the electrodes into the polystyrene base plate, the carbon electrodes were first placed on the PDMS mold and fixed between the rectangular notches. The carbon electrodes were custom computer numerically controlled (CNC) machined at Ohio Carbon Blank. The electrodes were isostatically pressed carbon / graphite (catalog number AR-14, Ohio Carbon Blank). A blank polystyrene sheet and silicon wafer were then placed on top and placed into an EVG 520 Hot Embosser. The temperature and pressure were gradually increased to 190°C and 3000N, and the internal pressure was increased by 5x10 -3 After the temperature and pressure were returned to ambient conditions, the resulting patterned polystyrene base with embedded carbon electrodes was removed from the PDMS mold. The edges were trimmed and it was ready for use.

[0236] 2. Bulk PDMS Mold Fabrication and Wire Attachment

[0237] To fabricate POMaC wires, a PDMS mold with 24 microchannels (cross section: 100x100 μm) arranged in parallel was made from a SU-8 master mold as described above. The silicone elastomer base and curing agent were mixed vigorously in a cup in a ratio of 15:1 (w:w), and 15 g of the mixture was poured into the SU-8 master mold and cured at room temperature. The ends of the channels were opened vertically by edge trimming, and the channel side of the PDMS mold was cleaned with transparent tape to remove dust particles. The PDMS mold was gently pressed onto an embossed polystyrene sheet with adjustments for the position and orientation of the channels as well as the position of the polymer wires to align with the microwells. The POMaC prepolymer solution was dispensed with a syringe onto the top edge of the PDMS mold, and the prepolymer was perfused by capillary action in the dark at room temperature for 48 h. After perfusion, the polymer was irradiated with UV light (1500 mJ cm -2 ) to generate an elastomeric material network. Excess non-perfused POMaC polymer was removed and the PDMS mold was slowly peeled away from the plate base, leaving the polymer wires still attached to the polystyrene sheet.

[0238] Thermal bonding of the base to a bottomless 96-well plate

[0239] A bottomless 96-well plate was placed bottom-up on a flat surface, the base plate with the wires and electrodes was placed facing it, and pairs of polymer wires were placed in the center of each well. A drop of acetone was placed on the corner of the plate between the base and plate to temporarily bond the plastic layers, and then the combination was permanently bonded by hot embossing. The bottom was pressed with a pressure of 700 N and a 5x10 -3 It was gradually heated to 184°C at an internal pressure of 100 mbar, with the top heated surface maintained at 96°C.

[0240] Post-assembly processing

[0241] After embossing, the wells of the mating plate were filled with distilled water and incubated overnight (37°C, 5% CO2). The water was then removed and the plate was air-dried under sterile conditions. The plate was then baked (70°C) for 6 days and sterilized by gamma radiation. After sterilization, the plate was stored at 4°C protected from light.

[0242] Plate characterization

[0243] To measure the Young's modulus of the bulk polymer, a Myograph apparatus (Kent Scientific), consisting of a mechanical stretcher and a force transducer, was used to measure the Young's modulus of the bulk polymer using UV light (1500 mJ cm) in PBS solution. -2 Strips (0.3x1x10 mm) of POMaC polymer cross-linked with 1000 ng / cm2 were stretched. The slope of the resulting stress-strain curve was used to calculate the Young's modulus. The polymer wire force / displacement curves in the wells were characterized using a commercial micromechanical tester, MicroSquisher (CellScale). Test probes (diameter = 0.1524 mm) were modified with customized tips (semi-ellipse, 0.5, 0.7, and 0.8 mm long axis to diameter ratio 4:1) fabricated from a SU-8 master by soft lithography and attached to a provided tungsten probe using adhesive (T-GSG-01 Titan Gel). The polymer wire was immersed in culture medium for 7 days before testing. The test tip was placed in the center of the microwell, and then the polymer wire was displaced in the center using the probe tip moving perpendicular to the long axis of the wire, and the corresponding force output (n ≥ 12) was recorded. The experimental data for each custom tip were fitted to a third-order polynomial to generate a standard force curve for each custom probe. To ensure consistency across the plate, the forces measured at 100 mm of displacement were compared between the correct wires in each well. The outermost two rows were intentionally excluded from the measurements because it was found that edge pressure caused non-uniformity in those two rows.

[0244] Voltage drop along the electrode

[0245] After assembling the plate, the carbon electrodes along the columns were connected to a voltage source (9 V battery). A portable multiscope (Extech, 381275) was used to measure the voltage difference between each pair of carbon electrodes in different wells (n = 3 wells per row). The voltage drop was calculated by dividing the voltage difference between the carbon electrodes and the voltage source by the voltage from the voltage source.

[0246] Preparation of hydrogels

[0247] Collagen hydrogel (500 μL) was prepared using a high concentration of rat tail collagen (9.82 mg mL -1 Prepare a collagen buffer containing 1xM199 (153 µL at 2.3 mM, Corning) by combining it with 15% (v / v) Matrigel (75 µL, BD Biosciences), NaHCCb (50 µL at 2.3 mM, Sigma), NaOH (5 µL at 10 mM, Sigma), deionized sterile HO (167 µL), and 1xM199 (50 µL, Sigma) to a final collagen concentration of 3.0 mg mL -1 obtained.

[0248] Preparation of cardiomyocytes and generation of engineered cardiac tissue

[0249] Primarily ventricular cardiomyocytes (CMs) were derived from the hiPSC line BJ1D using a monolayer differentiation protocol previously described. On day 21 of differentiation, cardiomyocytes were dissociated into single cells using a previously established method. Briefly, CMs were cultured using 1 mg / mL collagenase type II (Worthington, LS004205) in Hanks' balanced salt solution. -1 was added to the monolayer cardiomyocyte culture and incubated at room temperature overnight. The isolated cells were mixed with cardiac fibroblasts (Lonza, NHCF-V) at a cell number ratio of 10:1. The mixed cells were pelleted and 5.5x10 7 cells mL -1The cells were remixed into a collagen hydrogel at 20°C for 1 h. The cell-gel mixture was seeded at 2 mL per microwell. A drop of medium was placed on the wall of the well to keep it hydrated until gelation was complete. The plate was incubated at 37°C and 5% CO2 for 10 min before medium was added. After seeding (day 0), the tissues were cultured for 7 days to remodel around the POMaC wires. Brightfield images of the tissues were taken daily to monitor tissue morphology. By day 7, the tissues contracted synchronously, bending the POMaC wire with each contraction.

[0250] Batch imaging of tissue morphology

[0251] The tissues in the well plates were imaged using a SpectraMax MiniMax 300 Imaging Cytometer to monitor daily morphological changes in the tissues. Nine pictures were taken for each well of a 96-well plate and stitched together automatically to show a full view of the well. Spontaneous and paced (1 Hz) contractions were imaged using 4',6-diaminodino-2-phenylindole (DAPI) channel imaging and brightfield microscopy (4x objective). The blue channel (10x objective, lambda ex = 350 nm, λ em =470nm, 100 frames -1 A 100-ms exposure (5 ms exposure) was used to record and analyze the contractile behavior of the tissue, and data was output using custom MATLAB code to track the displacement of the wire. The average tissue width and the width of the tissue (Tw), attachment site to the polymer sensor were measured using bright field images in the relaxed state. The passive displacement of the polymer sensor was evaluated in the blue channel. The total and passive deflections (mm) of the polymer sensor were converted to force (mN) using a calibration curve generated as described in the plate characterization section. The final readings of total and passive forces were interpolated according to Tw and custom tip size. The active force was the difference between the total and passive forces. Passive force, active force, contraction, and relaxation times were calculated using custom MATLAB code.

[0252] Immunostaining and confocal microscopy

[0253] Tissues were fixed with 4% paraformaldehyde, permeabilized with 0.2% Tween 20, and blocked with 10% fetal bovine serum. Immunostaining was performed with the following antibodies: mouse anti-α-actinin (Abcam; 1:200) and donkey anti-mouse-Alexa Fluor 488 (Abcam; 1:400). Phalloidin-Alexa Fluor 660 (Invitrogen; 1:200) was used to stain F-actin fibers. Confocal microscopy images were acquired using an Olympus FluoView 1000 laser scanning confocal microscope (Olympus Corporation).

[0254] Electrical Stimulation for Tissue Maintenance and Drug Testing

[0255] Each well of the 96-well plate contained 300 mL of medium, which was replaced every 24 h. Built-in force sensors and electrodes allowed the tissue to be assessed in situ for its contractile motion and drug response. Prior to testing, bright-field videos of the tissue were taken to obtain the measurements required for force calculations. During testing, the pacing voltage was increased to 110% of the excitation threshold. One polymer wire (blue channel, 10x magnification) was imaged throughout for comparison. Once the baseline video was taken, 0.3 μL of thapsigargin (50x10 in DMSO) was added to the tissue. -3 m) was added to the wells using a gel-filled pipette tip. After 15 min, the video was taken again after thapsigargin had taken effect. The video was analyzed using custom MATLAB software.

[0256] statistical analysis

[0257] Statistical analyses were performed using Sigma Plot 12.0 or Prism 6.0. Differences between experimental groups were analyzed by Student's t-test or one-way analysis of variance. For one-way analysis of variance, normality tests (Shapiro-Wilk) and pairwise multiple comparison procedures (Tukey or Holm-Sidak method) were used. p<0.05 was considered significant for all statistical tests. All data were presented as mean ± standard deviation. Sample sizes (n) for each statistical analysis were stated in the figure legends.

[0258] Prior art documents

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Claims

1. A system, comprising: Two or more devices, each device comprising: A substrate; at least one electrode pair at least partially embedded within the substrate, the electrode pair having a first electrode and a second electrode separated by a gap; at least one well on the substrate having a bottom, a first end in contact with the first electrode, and a second end in contact with the second electrode, the well configured to grow tissue from cells seeded therein, and the electrode pair configured to apply an electrical stimulus to the tissue; two or more devices comprising at least two elastic sensing elements positioned across the well such that a gap exists between the sensing elements and the bottom of the well, the sensing elements configured to: (a) allow an attachment of the tissue to be formed between them, thereby suspending the tissue above the bottom of the well; and (b) deform in response to the contractile force exerted by the tissue on the sensing elements, thereby simulating a physiological environment specific to the tissue and / or allowing measurement of the contractile force; a stimulator having two or more driver boards, each driver board applying electrical stimulation between each electrode pair of each device, thereby enabling the stimulator to independently control the application of the electrical stimulation between each electrode pair; an interface tray having a hinged lid and configured to electrically couple the device to the stimulator; an optical microscope configured to generate an image of the sensing element and / or tissue of each device; A system comprising:

2. The system described in claim 1, wherein each device has two or more wells.

3. The system of claim 2, wherein each device has 6 wells, 12 wells, 24 wells, 48 ​​wells, or 96 wells.

4. The system of any one of claims 1 to 3, wherein the electrode pair is completely embedded in the substrate.

5. The system of any one of claims 1 to 4, wherein the electrodes comprise conductive carbon, gold, platinum, palladium, stainless steel, tin, tungsten, titanium, or combinations thereof.

6. The system of claim 5 , wherein the conductive carbon is non-porous.

7. The system of any one of claims 1 to 6, wherein the first electrode and the second electrode are separated by a gap in the range of 1 mm to 5 cm.

8. The system of any one of claims 1 to 7, wherein the first electrode is parallel or substantially parallel to the second electrode.

9. A system described in any one of claims 1 to 8, wherein each device has two or more electrode pairs and at least one well is positioned between each electrode pair.

10. The system of claim 9 , wherein each of the electrodes are parallel or substantially parallel to one another.

11. A system described in any one of claims 1 to 10, wherein each device has 2 to 25 sensing elements per well.

12. The system of any one of claims 1 to 11, wherein the sensing element comprises a polymer.

13. 13. The system of claim 12, wherein the polymer is at least one of polylactic acid, poly(lactic-co-glycolic) acid, poly(caprolactone), polyglycolide, polylactide, polyhydroxobutyrate, polyhydroxyalkanoic acid, chitosan, hyaluronic acid, hydrogel, poly(2-hydroxyethyl methacrylate), poly(ethylene glycol), poly(L-lactide) (PLA), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), poly(glycerol sebacate), poly(octamethylene maleic (anhydride) citric acid) (POMaC), citric acid free POMaC, poly(ε-caprolactone), polyurethane, silk, nanoengineered materials, copolymers, blended polymers, or combinations thereof.

14. The system of claim 13 , wherein the polymer is POMaC.

15. The system of claim 12 , wherein the polymer has tunable mechanical properties during the polymerization reaction.

16. The system of any one of claims 1 to 15, wherein the sensing element is porous, thereby enabling delivery of nutrients and growth factors to the cardiac tissue.

17. The system of any one of claims 1 to 16, wherein the sensing element has an elasticity of from about 20 kPa to 0.5 MPa.

18. The system of any one of claims 1 to 17, wherein the sensing element is in the form of a wire.

19. The system of any one of claims 1 to 18, wherein the well is configured to have a longitudinal axis.

20. 20. The system of claim 19, wherein the sensing element is oriented perpendicular, parallel, or oblique to the longitudinal axis of the well.

21. The system of any one of claims 1 to 20, wherein the substrate comprises a polymer.

22. The system of claim 21 , wherein the polymer is rigid.

23. 23. The system of claim 22, wherein the polymer is polystyrene or polycarbonate.

24. The device of any one of claims 1 to 23, wherein the cells are seeded onto a hydrogel.

25. 25. The system of any one of claims 1 to 24, wherein the cells are selected from cardiomyocytes, fibroblasts, skeletal muscle cells, liver cells, kidney cells, chondrocytes, skin cells, contractile cells, blood cells, immune system cells, germ cells, neural cells, epithelial cells, hormone-secreting cells, bone marrow cells, stem cells, tumor cells, smooth muscle cells, endothelial cells, fibroblasts, adipose-derived stem cells, mesenchymal stem cells, progenitor cells, or combinations thereof.

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