Microphysiological platform with embedded electrodes for 3D tissue culture

Abstract

To provide devices, apparatuses, and systems with embedded electrodes for growing, maintaining, and / or using 3D tissues in vitro.SOLUTION: A non-transitory processor-readable medium storing code representing instructions executable by a processor, the instructions causing the processor to: receive, from at least one sensing element configured to grow tissue from cells seeded in a well, a series of measurements, the well comprising a set of electrodes configured to apply electrical stimulation to the tissue according to a set of stimulation parameters; form, based on a comparison between the series of measurements and a set of predetermined criteria associated with the tissue, an updated set of values of the stimulation parameters; and transmit a stimulation signal to the set of electrodes to apply the electrical stimulation to the tissue.SELECTED DRAWING: Figure 2C

Description

Technical Field

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

Background Art

[0002] Due to the increasing cost of drug development and the limitations of cardiotoxicity screening, it is urgent to develop a robust in vitro 3D tissue culture platform that can promote the culture of human heart tissue and provide a non-invasive functional readout for predicting cardiotoxicity in a clinical environment. However, such platforms generally require complex fabrication procedures and are difficult to scale up to high-throughput test platforms. to develop a robust in vitro 3D tissue culture platform that can promote the culture of human heart tissue and provide a non-invasive functional readout for predicting cardiotoxicity in a clinical environment. However, such platforms generally require complex fabrication procedures and are difficult to scale up to high-throughput test platforms. platforms generally require complex fabrication procedures and are difficult to scale up to high-throughput test platforms. platforms generally require complex fabrication procedures and are difficult to scale up to high-throughput test platforms. to high-throughput test platforms.

[0003] Cardiotoxicity is a major cause of drug failure in the late stages of clinical trials and is associated with many post-marketing withdrawals. Verifying the safety of candidate drugs and eliminating unqualified candidates as early as possible is important for reducing the cost of drug development and preventing patient deaths. Conventional monolayer cultures using immortalized cell lines enable high-throughput screening, but they have limited relevance to native myocardium due to the lack of tissue-level function and morphological organization. Artificial heart tissue from human stem cell-derived cardiomyocytes has been shown to more closely mimic the myofibril bundles of native myocardium and exhibits an aligned sarcomere structure and a significant level of contractility. However, producing large quantities of reproducible 3D tissues requires a complex fabrication process that includes constructing hollow or scaffolded microstructures important for promoting functional tissue formation. Furthermore, to detect cardiotoxicity, functional to verify the safety of candidate drugs and eliminate unqualified candidates as early as possible is important for reducing the cost of drug development and preventing patient deaths. Conventional monolayer cultures using immortalized cell lines enable high-throughput screening, but they have limited relevance to native myocardium due to the lack of tissue-level function and morphological organization. Artificial heart tissue from human stem cell-derived cardiomyocytes has been shown to more closely mimic the myofibril bundles of native myocardium and exhibits an aligned sarcomere structure and a significant level of contractility. However, producing large quantities of reproducible 3D tissues requires a complex fabrication process that includes constructing hollow or scaffolded microstructures important for promoting functional tissue formation. Furthermore, to detect cardiotoxicity, functional to eliminate unqualified candidates as early as possible is important for reducing the cost of drug development and preventing patient deaths. Conventional monolayer cultures using immortalized cell lines enable high-throughput screening, but they have limited relevance to native myocardium due to the lack of tissue-level function and morphological organization. Artificial heart tissue from human stem cell-derived cardiomyocytes has been shown to more closely mimic the myofibril bundles of native myocardium and exhibits an aligned sarcomere structure and a significant level of contractility. However, producing large quantities of reproducible 3D tissues requires a complex fabrication process that includes constructing hollow or scaffolded microstructures important for promoting functional tissue formation. Furthermore, to detect cardiotoxicity, functional using immortalized cell lines enable high-throughput screening, but they have limited relevance to native myocardium due to the lack of tissue-level function and morphological organization. Artificial heart tissue from human stem cell-derived cardiomyocytes has been shown to more closely mimic the myofibril bundles of native myocardium and exhibits an aligned sarcomere structure and a significant level of contractility. However, producing large quantities of reproducible 3D tissues requires a complex fabrication process that includes constructing hollow or scaffolded microstructures important for promoting functional tissue formation. Furthermore, to detect cardiotoxicity, functional from native myocardium due to the lack of tissue-level function and morphological organization. Artificial heart tissue from human stem cell-derived cardiomyocytes has been shown to more closely mimic the myofibril bundles of native myocardium and exhibits an aligned sarcomere structure and a significant level of contractility. However, producing large quantities of reproducible 3D tissues requires a complex fabrication process that includes constructing hollow or scaffolded microstructures important for promoting functional tissue formation. Furthermore, to detect cardiotoxicity, functional from human stem cell-derived cardiomyocytes has been shown to more closely mimic the myofibril bundles of native myocardium and exhibits an aligned sarcomere structure and a significant level of contractility. However, producing large quantities of reproducible 3D tissues requires a complex fabrication process that includes constructing hollow or scaffolded microstructures important for promoting functional tissue formation. Furthermore, to detect cardiotoxicity, functional from native myocardium and exhibits an aligned sarcomere structure and a significant level of contractility. However, producing large quantities of reproducible 3D tissues requires a complex fabrication process that includes constructing hollow or scaffolded microstructures important for promoting functional tissue formation. Furthermore, to detect cardiotoxicity, functional However, producing large quantities of reproducible 3D tissues requires a complex fabrication process that includes constructing hollow or scaffolded microstructures important for promoting functional tissue formation. Furthermore, to detect cardiotoxicity, functional from artificial heart tissue is required. from artificial heart tissue is required. No reading is required either.

[0004] Built-in sensors are incorporated into many microphysiological platforms. For example, a thin muscle membrane is fabricated on a thin layer of polydimethylsiloxane (PDMS) or polyurethane membrane. Built-in sensors are incorporated into many microphysiological platforms. For example, a thin muscle membrane is fabricated on a thin layer of polydimethylsiloxane (PDMS) or polyurethane membrane. The deformation of the membrane can be easily modeled and related to the contractile force generated by the overlying cardiomyocytes. Recently, electrical circuits have been embedded in thin polymer membranes using 3D printing. This technology can use modeling techniques to convert the electrical signals of the embedded resistors into contractile movements. However, these systems only allow for the culture of single-cell layers of structures that are a few cell layers thick. The widely used two-post platform employs PDMS posts as force sensors and non-invasively reports the contractile forces from 3D tissues. The widely used two-post platform employs PDMS posts as force sensors and non-invasively reports the contractile forces from 3D tissues. However, the cultured tissue is likely to slip off the posts, leading to device failure. Furthermore, these devices rely on the use of PDMS, which is hydrophobic and thus prone to drug absorption. SUMMARY OF THE INVENTION

[0005] One aspect of the present disclosure includes a substrate and at least one pair of electrodes at least partially embedded in the substrate, the pair of electrodes having a first electrode and a second electrode separated by a gap, at least one pair of electrodes, and 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 being configured to grow tissue from cells seeded therein, the pair of electrodes being configured to apply an electrical stimulus to the tissue, at least one well, and at least two elastic sensors. One aspect of the present disclosure includes a substrate and at least one pair of electrodes at least partially embedded in the substrate, the pair of electrodes having a first electrode and a second electrode separated by a gap, at least one pair of electrodes, and 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 being configured to grow tissue from cells seeded therein, the pair of electrodes being configured to apply an electrical stimulus to the tissue, at least one well, and at least two elastic sensors. One aspect of the present disclosure includes a substrate and at least one pair of electrodes at least partially embedded in the substrate, the pair of electrodes having a first electrode and a second electrode separated by a gap, at least one pair of electrodes, and 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 being configured to grow tissue from cells seeded therein, the pair of electrodes being configured to apply an electrical stimulus to the tissue, at least one well, and at least two elastic sensors. One aspect of the present disclosure includes a substrate and at least one pair of electrodes at least partially embedded in the substrate, the pair of electrodes having a first electrode and a second electrode separated by a gap, at least one pair of electrodes, and 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 being configured to grow tissue from cells seeded therein, the pair of electrodes being configured to apply an electrical stimulus to the tissue, at least one well, and at least two elastic sensors. One aspect of the present disclosure includes a substrate and at least one pair of electrodes at least partially embedded in the substrate, the pair of electrodes having a first electrode and a second electrode separated by a gap, at least one pair of electrodes, and 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 being configured to grow tissue from cells seeded therein, the pair of electrodes being configured to apply an electrical stimulus to the tissue, at least one well, and at least two elastic sensors. One aspect of the present disclosure includes a substrate and at least one pair of electrodes at least partially embedded in the substrate, the pair of electrodes having a first electrode and a second electrode separated by a gap, at least one pair of electrodes, and 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 being configured to grow tissue from cells seeded therein, the pair of electrodes being configured to apply an electrical stimulus to the tissue, at least one well, and at least two elastic sensors. ​A sensing element, wherein the well is arranged horizontally such that there is a gap between the sensing element and the bottom of the well and the sensing element is configured to (a) allow the attachment of tissue formed therebetween, thereby bridging the tissue above the bottom of the well, (b) deform in response to the contractile force exerted on the sensing element by the tissue, thereby simulating the physiological environment specific to the tissue and / or enabling the measurement of the contractile force, and comprises at least two elastic sensing elements relates to a device In some embodiments, the device comprises two or more wells, such as 6-well, 12-well 24-well, 48-well, or 96-well

[0006] In some embodiments, the electrode pair is completely embedded in the substrate In some embodiments, the electrodes comprise conductive carbon, gold, platinum, palladium, stainless steel, tin, tungsten, titanium, or combinations thereof

[0007] In some embodiments, the conductive carbon is non-porous

[0008] In some embodiments, the first electrode and the second electrode are separated by a gap in the range of 1 mm to 5 cm In some embodiments, the first electrode is parallel or substantially parallel to the second electrode

[0009] In some embodiments, the device comprises two or more electrode pairs, with at least one well disposed between each electrode pair

[0010] In some embodiments, the first electrode is parallel or substantially parallel to the second electrode

[0011] In some embodiments, the first electrode is parallel or substantially parallel to the second electrode In some embodiments, the device comprises two or more electrode pairs, with at least one well disposed between each electrode pair

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

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

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

[0016] In some embodiments, the device comprises 2 to 25 sensing elements per well.

[0017] In some embodiments, the sensing element comprises a polymer. In some embodiments, the polymer is polylactic acid, poly(lactic-co-glycolic) acid, poly(caprolactone), polyglycolide, polylactide, polyhydroxybutyrate, polyhydroxyalkanoate, chitosan, hyaluronic acid, hydrogel, poly(2-hydroxyethyl methacrylate) , poly(ethylene glycol), poly(L-lactide) (PLA), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), poly(sebacic acid glycerol) , poly(octamethylene maleic (anhydride) citrate) (POMaC), POMaC without citric acid, poly(ε-caprolactone), polyurethane, silk, nanofabricated materials, copolymers, blend polymers, or at least one of combinations thereof. In some embodiments, the polymer is POMaC.

[0018] ​​​​​In some embodiments, the polymer has adjustable mechanical properties during the polymerization reaction.

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

[0020] In some embodiments, the sensing element has an elasticity of about 20 kPa to 0.5 MPa.

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

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

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

[0024] In some embodiments, the substrate includes 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 in the hydrogel.

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

[0027] ​​​​​One aspect of the present disclosure relates to a non-transitory processor-readable medium storing code representing instructions executable by a processor, the instructions causing the processor to receive a series of measurements from at least one sensing element associated with a well configured to grow tissue from cells seeded therein, the well comprising a series of electrodes configured to apply electrical stimulation to the tissue according to a series of stimulation parameters, to identify an amount by which to vary a series of values of the series of stimulation parameters based on a comparison of the series of measurements with a series of predetermined criteria associated with the tissue, to adjust the series of values based on the amount to form an updated series of values of the series of stimulation parameters, and to transmit stimulation signals 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 some embodiments, the series of stimulation parameters includes at least one of a stimulation voltage, a stimulation frequency, or a stimulation time. In some embodiments, the at least one sensing element comprises at least two elastic sensing elements disposed across the well such that a gap exists between the sensing elements and the bottom of the well. In some embodiments, the initial series of values of the series of stimulation parameters is selected based on a predetermined stimulation protocol. In some embodiments, the code causing the processor to identify causes the processor to identify an amount by which to decrease a stimulation voltage based on a series of measurements meeting a tissue maturity criterion.

[0028]

[0029]

[0030]

[0031] ​​​​​​​​​​​​​​ include.

[0032] In some embodiments, the code that causes the processor to identify meets a tissue excitation criterion. A code that causes the processor to identify the amount to increase the stimulus voltage based on a series of measurements that do not include Includes do.

[0033] In some embodiments, the code for causing the processor to receive the series of measurements comprises: The sensor periodically receives a set of measurement instances, and The method includes code for storing the

[0034] One aspect of the present disclosure relates to an apparatus, the apparatus comprising: a memory; and a processor operatively connected to the memory. the processor detects the amount of the wire in the well from the cells seeded in the well. An image of the surrounding growing tissue is received, and each pixel from a plurality of pixels in the image is thresholded. forming a first binary image based on the comparison; and determining a first edge of the wire in the first binary image. and detecting the first edge and the second edge to generate a second binary value representing the first edge and the second edge. forming an image, and determining a distance between a first edge and a second edge of each of a plurality of rows of the second binary image; Calculate the midpoints of the set of midpoints to generate a set of midpoints, and then add the set of midpoints to the set of midpoints based on a polynomial regression of the set of midpoints. Calculate the fitting quadratic equation and identify the bending points of the wire based on the quadratic equation. and a processor configured to:

[0035] In some embodiments, the wells can be configured to apply electrical stimulation to tissue according to a set of stimulation parameters. a processor for detecting voltages that meet predetermined criteria; A series of stimulation parameters are adjusted based on the deflection point of the ear, and a series of stimulation parameters are adjusted based on the deflection point of the ear. configured to form a series of updated values of the data and to transmit a stimulation signal to a series of electrodes to apply an electrical stimulation to the tissue according to a series of updated values of a series of stimulation parameters.

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

[0037] In some embodiments, the processor is configured to form a 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 before detecting a first edge and a second edge of a wire in the first binary image.

[0039] In some embodiments, the processor is configured to smooth the first binary image using a closing operator before detecting a first edge and a second edge of a 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 a second binary image before calculating a midpoint, and each row from a 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 contraction force exerted on the wire by the tissue based on inflection points. ​​​​​​​​​​​​

[0042] In some embodiments, the processor is configured to calculate the amount of static tension exerted on the wire by the tissue based on the inflection points.

[0043] One aspect of the present disclosure relates to a system having a substrate with two or more devices and two or more drivers, each driver substrate being configured to apply an electrical stimulus between each pair of electrodes of each device, whereby the stimulation device can independently control the application of the electrical stimulus between each pair of electrodes.

[0044] In some embodiments, the system further comprises an interface tray having a hinged lid and configured to electrically connect the device to the stimulation device. [[ID=2l]]

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

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

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

[0048] In some embodiments, the computing device comprises the apparatus described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0049]

Figure 1

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DETAILED DESCRIPTION OF THE INVENTION

[0091] The present disclosure provides devices, apparatuses, and systems having embedded electrodes for growing, maintaining, and / or using 3D tissue in vitro. Since the electrodes are physically embedded in the substrate of the device, the electrodes are provided with leads for applying a direct current to the embedded electrodes by a stimulation device, and the tissue within the device is already in a position to receive electrical stimulation as well as media and nutrients without being transferred to another dish or device. The devices, apparatuses, and systems described herein can be used in a variety of applications including drug testing. By exposing the tissue to a test drug, changes in the contractility and / or resting tension of the tissue can be used to evaluate the efficacy, safety, and / or toxicity of the test drug. The electrodes are physically embedded in the substrate of the device, so the electrodes are provided with leads for applying a direct current to the embedded electrodes by a stimulation device, and the tissue within the device is already in a position to receive electrical stimulation as well as media and nutrients without being transferred to another dish or device. The electrodes are physically embedded in the substrate of the device, so the electrodes are provided with leads for applying a direct current to the embedded electrodes by a stimulation device, and the tissue within the device is already in a position to receive electrical stimulation as well as media and nutrients without being transferred to another dish or device. The electrodes are physically embedded in the substrate of the device, so the electrodes are provided with leads for applying a direct current to the embedded electrodes by a stimulation device, and the tissue within the device is already in a position to receive electrical stimulation as well as media and nutrients without being transferred to another dish or device. The electrodes are physically embedded in the substrate of the device, so the electrodes are provided with leads for applying a direct current to the embedded electrodes by a stimulation device, and the tissue within the device is already in a position to receive electrical stimulation as well as media and nutrients without being transferred to another dish or device. The devices, apparatuses, and systems described herein can be used in a variety of applications including drug testing. By exposing the tissue to a test drug, changes in the contractility and / or resting tension of the tissue can be used to evaluate the efficacy, safety, and / or toxicity of the test drug. By exposing the tissue to a test drug, changes in the contractility and / or resting tension of the tissue can be used to evaluate the efficacy, safety, and / or toxicity of the test drug. By exposing the tissue to a test drug, changes in the contractility and / or resting tension of the tissue can be used to evaluate the efficacy, safety, and / or toxicity of the test drug.

[0092] One aspect of the present disclosure relates to the device 100 of FIG. 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. 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. 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, highly optically transparent, and / or medically compatible. In some embodiments, the polymer can include polystyrene or polycarbonate. In some embodiments, the polymer is rigid, highly optically transparent, and / or medically compatible. In some embodiments, the polymer can include polystyrene or polycarbonate. 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 m m, 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 c m or less. Combinations of the ranges referred to above for the width of the gap are also possible (e.g., at least

[0096] about 1 mm to about 10 cm or less, at least about 1 mm to about 5 cm or less, or at least about 5 mm to about 1.5 cm or less).

[0097] Each electrode can include conductive carbon, gold, platinum, palladium, stainless steel, tin, tungsten, titanium, or combinations thereof. Conductive materials for tissue stimulation Another example is "Electrical Stimulation of Excitable Tissue: Design of Efficacious and Safe Protocols" by Merrill et al., J. of Neuroscience Me thods 2005, 141, 171 - 198, "Characterization of Electrical Stimulation Elect rodes for Cardiac Tissue Engineering" by Tandon et al., Pr oceedings of the 28 IEEE, pages 845 - 848, the contents of each of which are incorporated herein by reference. th and are located on pages 845 - 848 of the 28th IEEE, the contents of each of which are incorporated herein by reference.

[0098] In some embodiments, the conductive carbon is non - porous, such as vitreous carbon. Non - porous carbon can prevent test compounds from being absorbed by the carbon. In some embodiments, the carbon electrode is not an epoxy - reinforced carbon electrode.

[0099] Well 120 has a bottom on 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 seeded cells. The electrode pair can be configured to apply electrical stimulation to tissue 130.

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

[0101] For example, the length, height, or width of well 120 can be about 0.1 to 1 mm, about 0.2 to 2 m m, about 0.3 to 3 mm, about 0.4 to 4 mm, about 0.5 to 5 mm, about 0.6 to 6 mm, about 0 .7 to 7 mm, about 0.8 to 8 mm, about 0.9 to 9 mm, about 1 to 10 mm, about 1 to 100 m m, or about 10 to 100 mm.

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

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

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

[0105] The first sensing element 112 and the second sensing element 114 are disposed across well 120 such that there is a gap between the bottom of well 120 and the sensing elements 112 and 114. It is possible. The sensing elements 112 and 114 are configured to (a) enable the adhesion of the tissue 130 formed therebetween, thereby bridging the tissue 130 above the bottom of the well 120, and (b) deform in response to the contraction force exerted on the sensing elements 112 and 114 by the tissue 130, thereby simulating the physiological environment specific to the tissue 130 and / or enabling the measurement of the contraction force. 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. The 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. The device 100 can include 25 or fewer sensing elements per well, 20 or fewer sensing elements per well, 18 or fewer sensing elements per well, 15 or fewer sensing elements per well, or 10 or fewer sensing elements per well. Combinations within the above ranges for the number of sensing elements per well are also possible (e.g., at least 2 to 25 or fewer per well, at least 2 to 20 or fewer per well, or at least 2 to about 10 or fewer per well). Any number of sensing elements per well can be provided as long as the tissue can be formed around each sensing element and joined between them such that the tissue is bridged above the bottom of the well.

[0106] The sensing elements 112 and 114 can be configured to (a) enable the adhesion of the tissue 130 formed therebetween, thereby bridging the tissue 130 above the bottom of the well 120, and (b) deform in response to the contraction force exerted on the sensing elements 112 and 114 by the tissue 130, thereby simulating the physiological environment specific to the tissue 130 and / or enabling the measurement of the contraction force. 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] The 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. The device 100 can include 25 or fewer sensing elements per well, 20 or fewer sensing elements per well, 18 or fewer sensing elements per well, 15 or fewer sensing elements per well, or 10 or fewer sensing elements per well. Combinations within the above ranges for the number of sensing elements per well are also possible (e.g., at least 2 to 25 or fewer per well, at least 2 to 20 or fewer per well, or at least 2 to about 10 or fewer per well). Any number of sensing elements per well can be provided as long as the tissue can be formed around each sensing element and joined between them such that the tissue is bridged above the bottom of the well. The sensing elements 112 and 114 can be configured to (a) enable the adhesion of the tissue 130 formed therebetween, thereby bridging the tissue 130 above the bottom of the well 120, and (b) deform in response to the contraction force exerted on the sensing elements 112 and 114 by the tissue 130, thereby simulating the physiological environment specific to the tissue 130 and / or enabling the measurement of the contraction force. 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.

[0108] Combinations within the above ranges for the number of sensing elements per well are also possible (e.g., at least 2 to 25 or fewer per well, at least 2 to 20 or fewer per well, or at least 2 to about 10 or fewer per well). Any number of sensing elements per well can be provided as long as the tissue can be formed around each sensing element and joined between them such that the tissue is bridged above the bottom of the well. The sensing elements 112 and 114 can be configured to (a) enable the adhesion of the tissue 130 formed therebetween, thereby bridging the tissue 130 above the bottom of the well 120, and (b) deform in response to the contraction force exerted on the sensing elements 112 and 114 by the tissue 130, thereby simulating the physiological environment specific to the tissue 130 and / or enabling the measurement of the contraction force. 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.

[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 the 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 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 adjusted by controlling polymerization using different crosslinking energies. The adjustment ability can also be controlled by the mixing ratio of polymer units during the polymerization reaction. The adjustment ability can also be controlled by the mixing ratio of polymer units during the polymerization reaction.

[0113] In some embodiments, the polymer is polylactic acid, poly(lactic-co-glycolic) acid , poly(caprolactone), polyglycolide, polylactide, polyhydroxybutyrate , polyhydroxyalkanoic acid, chitosan, hyaluronic acid, hydrogel, poly(2-hydroxy ethyl methacrylate), poly(ethylene glycol), poly(L-lactide) ( PLA), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) ( PMMA), poly(glycerol sebacate), poly(octamethylene maleic (anhydride )) citrate (POMaC), POMaC without citric acid, poly(ε-caprolactone ), polyurethane, silk, nanomachined materials, copolymers, blend polymers, or at least one of combinations thereof.

[0114] The shape, thickness, length, orientation, and surface morphological properties of the sensing element can be deformed, bent, or changed in shape in response to the contraction action or activity of the tissue connected therebetween, and such deformation, bending, or shape change can be reliably measured. In response to the contraction action or activity of the tissue connected therebetween, the sensing element can be deformed, bent, or changed in shape, and such deformation, bending, or shape change can be reliably measured. ​​​Possibly, it can be varied in many suitable ways. In some embodiments, the sensing element The element is in the form of a wire, for example a polymer wire.

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

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

[0117] In some embodiments, the hydrogel comprises collagen or a collagen derivative, intestinal mucus, or the like. Submembrane tissue or its derivatives, cellulose or cellulose derivatives, proteoglycan , heparin sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, elastin, Pronectin, thrombin, laminin, fibrin, chitosan, alginate, Matri gel®, Geltrex®, agarose, decellularized extracellular matrix polyethylene glycol or its derivatives, silicone or its derivatives, or a combination thereof. In some embodiments, the hydrogel comprises Matrig Includes el (registered trademark).

[0118] The present disclosure also provides for the use of multiple devices 100 on a single substrate (e.g., a single plate). To provide a multi-well device. In some embodiments, the multi-well device can comprise a plurality of wells, such as 6 wells, 12 wells, 24 wells, 48 wells, or 96 wells. The multi-well device can comprise two or more electrode pairs, with at least one well disposed between each electrode pair. In some embodiments , the multi-well device can comprise from 2 to 20 electrode pairs (e.g., 2 to 20 pairs, 2 to 10 pairs, or 4 to 8 pairs). In some embodiments, the multi-well device can comprise 2 pairs, 3 pairs, 4 pairs, 5 pairs, 6 pairs, 7 pairs, 8 pairs, 9 pairs, or more electrode pairs. In some embodiments, from 1 to 20 wells (e.g., 1 to 15 wells, 2 to 20 wells, 2 to 10 wells, or 4 to 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 disposed between each electrode pair. In some embodiments, the multi-well device can be in the form of a multi-well plate . In some embodiments, the multi-well plate can be a 24-well plate in a 4x6 configuration . In a 4x6 configuration, there are 6 electrode pairs, with 4 wells disposed between each electrode pair. Using each electrode pair, electrical stimulation can be applied to all wells between the electrode pair. The operation of each

[0119] electrode pair can be independent of other electrode pairs, thereby enabling each electrode pair to have different stimulation parameters from other electrode pairs.

[0120] FIG. 2C shows a multi-well device having 96 wells on a single substrate. The multi- well device comprises 12 electrode pairs, each electrode being parallel or substantially parallel to the other electrodes. The black lines indicate the electrodes. Eight wells are disposed between each electrode pair.

[0121] The devices described herein can be manufactured using hot embossing and injection molding techniques. In some embodiments, a custom multi-well plate having embedded electrodes and channels can be first generated, and sensing elements can be placed in the channels. Then, a bottomless plate having a plurality of cell culture wells can be attached on top of the multi-well plate. During attachment, the wells on the multi-well plate are aligned with the cell culture wells of the bottomless plate, such 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) described herein, and a stimulation device having two or more driver substrates, each driver substrate being configured to apply an electrical stimulation between each electrode pair of each device, such that the stimulation device can independently control the application of the electrical stimulation between each electrode pair.

[0123] In the case of in vitro tissue generation, the state of the art includes the following: (a) manual programming of electrical stimulation is required at all steps of the process, (b) the electromechanical interface is handmade and manually connected, (c) the handmade system lacks robustness, stability, and ease of assembly / ​​​​​​​​​​​​​It has the drawback of not being designed for ease of manufacture.

[0124] In comparison, the system described herein provides a scalable and automated platform for in vitro tissue generation, manipulation, and maintenance. With this system, all steps of the main workflow (e.g., cell seeding, maturation / stimulation, and endpoint testing) can be performed in the same location, eliminating the need to transfer the tissue from one chamber or Petri dish to another. Each tissue can be a self - contained isolation bioreactor. In some embodiments, when a multi - well bottomless plate is attached to a custom - ordered polystyrene plate, each microwell is effectively sealed from the rest of the plate. During the culture process, media and nutrients can be applied to each tissue. Furthermore, since the electrodes are already embedded in the plate, there is no need to move the tissue to apply electrical stimulation for the maturation protocol. Considering that each tissue is isolated, there is no need to separate them after maturation to apply any test compounds for testing (e.g., contraction testing). As a result, each tissue can be processed individually, independent of other tissues on the plate (e.g., test compounds at different doses).

[0125] In some embodiments, the stimulation device can comprise at least 2 driver substrates, at least 3 driver substrates, at least 4 driver substrates, at least 5 driver substrates, or at least 6 driver substrates. In some embodiments, In one state, the stimulation device can include 25 or fewer driver substrates, 20 or fewer driver substrates, 19 or fewer driver substrates, 18 or fewer driver substrates, 17 or fewer driver substrates, 1 6 or fewer driver substrates, 15 or fewer driver substrates, or 10 or fewer driver substrates.

[0126] It is also possible to combine the above ranges for the number of driver substrates (e.g., at least 2 to 25 or fewer, at least 5 to about 15 or fewer, or at least 5 to 10 or fewer). In some embodiments, the stimulation device can include about 10 driver substrates.

[0127] In some embodiments, each driver substrate can include up to 6 channels. Each channel can have an output (e.g., stimulation frequency) that can be configured individually from the driver substrate. For example, a 24-well plate can be stimulated by 6 channels driven by 1 driver substrate. Thus, each driver substrate has the function of controlling the stimulation parameters of each plate for a column of wells, and one column of a plate can have different parameters from other columns of the same plate. Commercially available stimulation devices cannot provide this level of control. Since each driver substrate can be used to control one device, the number of driver substrates depends on the number of devices in the system. For example, if there are 5 multi-well devices in the system, there may be at least 5 driver substrates, and if there are 10 multi-well devices in the system, there may be at least 10 driver substrates.

[0128] ​ is possible.

[0129] In some embodiments, the configuration of the electronic components of the stimulation device is shown in FIG. 17.

[0130] This system allows for expansion of the number of channels, and each channel can be configured independently. Advantages resulting from such independent configurability include: (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 a maturation cycle, and (4) the recordable stimulation history of each driver board, among others.

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

[0132] In some embodiments, the system can further include an incubator configured to culture cells and tissues grown within the device. During the tissue maturation process, the device remains at a constant temperature of approximately 37° C. in an incubator while connected to the stimulation device. To achieve this, the incubator must The incubator is equipped with a custom incubator rack that accommodates multiple multiwell plates. Each space in the incubator rack can accommodate an incubator tray. One of the multi-well plates is fixed to the bottom of the rack, allowing the operator to place the multi-well plate in the desired position. The stimulator, incubator rack, and interface The connection between the trays is via a standard ribbon cable and a cable fixed to the back of the incubator rack. The system can be managed by a custom printed circuit board (PCB).

[0133] The system is configured to generate an image of the sensing element and / or tissue of each device. The imaging subsystem may further include an optical microscope. It can be prepared.

[0134] The system further includes a connection to an imaging subsystem for analyzing the images and / or for stimulating the device. The system may include a computing device configured to control the system. The computing device provides a graphical interface for connecting to the stimulator and controlling its settings. It can be equipped with a user interface (GUI). The GUI allows the operator to Remotely checks and adjusts stimulator settings and configures the stimulator to perform pre-determined The GUI can automatically execute the stimulation protocol you have selected. These include open circuits, overvoltage / undervoltage, and network issues (e.g., between the controller and the stimulator). Network disconnections can be reported to the operator. The stimulator GUI The regulator may also decide to approve the project depending on the protocol settings or the organization specified. The maturation progress of the multiwell plates can be viewed and tracked.

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

[0136] FIG. 12 is an abstract system configuration diagram according to some embodiments of the present disclosure. As shown in Figure 1, the system includes the following subsystems: (a) Maturation and Incubation Subsystem (b) test and imaging subsystem; (c) system controller; and and (d) a computer that runs the GUI to set the operating parameters. Expandable beyond a single stimulator box, maturation incubator, or test / imager To accommodate this, each subsystem must be connected to the system via a wired or wireless network. It is possible.

[0137] Once mature, the tissue is ready for drug testing, which is performed in the testing and imaging subsystem. In some embodiments, this subsystem includes a Nikon imaging device. a stage controller for moving the wells into the camera's field of view; The device may include a fluid handler for injecting drugs into the tube, and a stimulator box. The goal of this system is to integrate these four components into a single physical platform. Integrating this allows drug testing tests to be organized in columns to automate the testing process. , can be performed on one plate at a time. Each column represents one pair of embedded electrodes. The protocol also requires adjustment of the same stimulation pattern.

[0138] In some embodiments, the system can comprise two or more stimulation devices. The system controller can be used to manage all the stimulation devices. As the system expands, each stimulation device can be assigned a unique identification by the system controller. The system controller has the role of communicating the stimulation parameters of each column, each plate, and each stimulation device, parameterized in the GUI. Also, it monitors the state of each stimulation device and not only checks the state of the stimulation device (checks for overcurrent warning conditions), but also periodically

[0139] records the operating parameters and stamps them with a timestamp. A browser-based GUI executed on a network computer can be an interface to the system controller. As long as the computer is on the same network, the physical location does not matter. The GUI computer can remotely access the system controller.

[0140] Adjustable parameters of the stimulation device include, but are not limited to, pulse voltage, pulse frequency, and pulse duration. The stimulation device also has the role of monitoring the voltage and current applied to each pair of embedded electrodes. These measurements can be periodically recorded in a database. The electrical stimulation can be adjustable by the user via a GUI application

[0141] Figure 13 is a schematic diagram of a system 300 for performing 3D tissue culture is shown. The system 300 further includes a growth well 312 and a control unit 316. comprises a tissue growth device 310. In some embodiments, the growth well 312 can be substantially similar to the 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. Furthermore, the growth well 312 also comprises one or more wires (not shown in FIG. 13) configured to detect 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 supply 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 electrical signals to the embedded electrodes. Furthermore, the control unit 316 can be configured to change the characteristics of one or more electrical signals, such as amplitude, frequency, repetition rate, and / or duration.

[0142] The system 300 also comprises an imaging device 320 configured to monitor the growth of tissue within the tissue growth device 310. In some embodiments, the imaging device 320 is configured to monitor the shape of the sensing wires within the tissue growth device 310 to estimate the state of the tissue. The imaging device 320 can be any suitable type of sensor, such as a charge-coupled ​​​​​​It can include elements such as a charge-coupled device (CCD), a complementary metal-oxide semiconductor (CMOS) device, a photodiode, and / or a phototransistor. In some embodiments, the imaging device 320 can include optical elements, such as a lens, a collimator, a wavelength plate, a polarizer, and / or a filter, etc., to facilitate image acquisition. In some embodiments, the imaging device 320 includes a microscope.

[0143] The processing device 330 is operably connected to the imaging device 320 and is configured to analyze the image acquired by the imaging device 320. The processing device 330 includes 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 others. In some cases, the memory 332 includes a series of instructions or code for performing 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, such as 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 is configured to implement and / or execute a series of instructions, processes, modules, and / or code stored in the memory 332.

[0144] The communication interface 336 can be any suitable device capable of communicating with other devices to perform processing, for example, a device implementing the user interface 340, the tissue growth device 310, and / or any suitable device. In some embodiments, the communication interface 336 can be 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 wireless interface (e.g., WiFi ( registered trademark) wireless, Bluetooth (registered trademark) wireless, near field communication (NFC) wireless, etc.).

[0145] The user interface 340 is configured such that a user can interact with other parts of the system 300. In some embodiments, the user interface 340 is configured to display available protocols executable by the system 300, and in some embodiments, the user interface 340 is configured such that a user can 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 be, for example, images (with or without processing) acquired by the imaging device 320, tissue growth data It can include the growth state of the tissue within the vice 310, and potential defects of the tissue, etc. In some embodiments, the user interface 340 is a graphical user interface (GUI) associated with a web page, a PC application lication, a mobile application, etc. In some embodiments, the user interface 3 40 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, such as a desktop, a laptop, a personal computer , a server, a mainframe computer, a smartphone, a tablet, a wearable device, etc.

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

[0147] The method 400 also includes, at 420, forming a first binary image based on comparing each pixel from a plurality of pixels in the image with a pixel threshold. In some embodiments, to generate the first binary image, a pixel value greater than or equal to the pixel threshold is set to 1, and a pixel value less than the pixel threshold is set to 0. The elemental value is set to 0. In some embodiments, this definition can be reversed, i.e., pixel values below 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] In 430 of method 400, the first edge and the 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 the gradient of the pixels in the first binary image. In some embodiments, the first edge and the second edge are detected using the Sobel operator (see details with reference to FIGS. 15G and 15H below).

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

[0150] ​​​​​​​​​ Method 400 also includes, at 450, calculating a quadratic equation that fits a series of midpoints based on a polynomial regression of the series of midpoints. In some embodiments, the quadratic equation represents the contour (or shape) of the wire in the input image. At 460 of method 400, the inflection points of the wire are identified based on the quadratic equation. In some embodiments, the well comprises a series of electrodes configured to apply electrical stimulation to tissue according to a series of stimulation parameters. In these embodiments, method 400 further includes adjusting a series of values of a series of stimulation parameters based on the inflection points of the wire (i.e., identified at 460) that meet a predetermined criterion to form an updated series of values of the series of stimulation parameters. Then, a stimulation signal is 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 inflection points identified at 460 can be used (e.g., by processor 334) to derive the growth conditions of the tissue. In some embodiments, 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 FIGS. 15C and 15D below for details). In some embodiments, 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 FIGS. 15E and 15F below for details).

[0151] In some embodiments, the well comprises a series of electrodes configured to apply electrical stimulation to tissue according to a series of stimulation parameters. In these embodiments, method 400 further includes adjusting a series of values of a series of stimulation parameters based on the inflection points of the wire (i.e., identified at 460) that meet a predetermined criterion to form an updated series of values of the series of stimulation parameters. Then, a stimulation signal is 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 inflection points identified at 460 can be used (e.g., by processor 334) to derive the growth conditions of the tissue. In some embodiments, the well comprises a series of electrodes configured to apply electrical stimulation to tissue according to a series of stimulation parameters. In these embodiments, method 400 further includes adjusting a series of values of a series of stimulation parameters based on the inflection points of the wire (i.e., identified at 460) that meet a predetermined criterion to form an updated series of values of the series of stimulation parameters. Then, a stimulation signal is 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 inflection points identified at 460 can be used (e.g., by processor 334) to derive the growth conditions of the tissue. In some embodiments, the well comprises a series of electrodes configured to apply electrical stimulation to tissue according to a series of stimulation parameters. In these embodiments, method 400 further includes adjusting a series of values of a series of stimulation parameters based on the inflection points of the wire (i.e., identified at 460) that meet a predetermined criterion to form an updated series of values of the series of stimulation parameters. Then, a stimulation signal is 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 inflection points identified at 460 can be used (e.g., by processor 334) to derive the growth conditions of the tissue. In some embodiments, the well comprises a series of electrodes configured to apply electrical stimulation to tissue according to a series of stimulation parameters. In these embodiments, method 400 further includes adjusting a series of values of a series of stimulation parameters based on the inflection points of the wire (i.e., identified at 460) that meet a predetermined criterion to form an updated series of values of the series of stimulation parameters. Then, a stimulation signal is 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 inflection points identified at 460 can be used (e.g., by processor 334) to derive the growth conditions of the tissue.

[0152] In some embodiments, 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 FIGS. 15C and 15D below for details). In some embodiments, 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 FIGS. 15E and 15F below for details). In some embodiments, 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 FIGS. 15C and 15D below for details). In some embodiments, 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 FIGS. 15E and 15F below for details). In some embodiments, 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 FIGS. 15C and 15D below for details). In some embodiments, 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 FIGS. 15E and 15F below for details). ​​​​​​​​​

[0153] In some embodiments, before calculating the midpoint at 440, method 400 further completes a portion of the first edge or a portion of the second edge that is not included within the second binary image. In this way, each row of the second binary image includes a portion of the first edge and a portion of the second edge (see FIGS. 15K and 15L below for details). In some embodiments, the inflection points identified at 460 can be used to calculate the amount of contraction force applied to the wire by the tissue. In some embodiments, the inflection points identified at 460 can be used to calculate the resting tension of the wire. Since the contraction force and / or the resting tension can indicate the growth state (e.g., maturity) of the tissue, they can be used to form a feedback control loop (see FIG. 16 for details below). See FIGS. 15K and 15L below for details.

[0154] In some embodiments, the inflection points identified at 460 can be used to calculate the amount of contraction force applied to the wire by the tissue. In some embodiments, the inflection points identified at 460 can be used to calculate the resting tension of the wire. Since the contraction force and / or the resting tension can indicate the growth state (e.g., maturity) of the tissue, they can be used to form a feedback control loop (see FIG. 16 for details below). See FIG. 16 for details below. See FIG. 16 for details below. See FIG. 16 for details below.

[0155] FIGS. 15A-15M illustrate a method of wire tracking (also referred to as a wire tracking method) according to some embodiments. Generally, the wire tracking method is configured to identify the edges of the wire sensor in each input image and calculate a skeletal structure represented by a series of 2D coordinates (x , y )(where i = 1, 2, 3,..., i , y i )(where i = 1, 2, 3,..., N, and N is a positive integer). Next, the 2D coordinates of the skeleton are fit to a quadratic polynomial (e.g., using least squares fitting). Without loss of generality, the quadratic polynomial (also referred to as a parabola) is written as follows. Next, the 2D coordinates of the skeleton are fit to a quadratic polynomial (e.g., using least squares fitting). Without loss of generality, the quadratic polynomial (also referred to as a parabola) is written as follows. Without loss of generality, the quadratic polynomial (also referred to as a parabola) is written as follows. TIFF2025111430000002.tif15170

[0156] In some embodiments, the wire tracking method includes a bit conversion step, and the input image is converted into an 8-bit image (i.e., an image having a gray scale from 0 to 255). In some cases, the data value of each pixel of the input image is in the form of a 16-bit integer format . In such cases, it is beneficial to convert these images into 8-bit images to facilitate subsequent processing .

[0157] Directly converting 16-bit values to 8-bit values may result in the loss of certain data, and may also affect the threshold when there is a slight brightness gradient in a specific frame. Thus, the influence on the threshold may lead to spikes in the baseline. To address this issue, a normalization step can be performed when converting the input image from 16 bits to 8 bits . More specifically, in this normalization step, the minimum value of the range (i.e., the minimum pixel value) and the maximum value of the range (i.e., the maximum pixel value) are identified for each frame. Then , each pixel value within the frame is adjusted according to contrast stretching. For example, the minimum value of the range is expanded to 0, and the maximum value of the range is expanded to 256. Intermediate values between the minimum and maximum values are expanded proportionally to their positions within the range from the minimum to the maximum value. Next , the 8-bit input image is converted into a binary image by thresholding. In some embodiments , the pixel value of each pixel of the input image is compared with the threshold. If the pixel value is greater than or equal to the threshold, the pixel value is set to 1, and otherwise, the pixel value is set to 0. In some embodiments, the definition can be reversed. That is, the pixel value is set to 0 when it is greater than or equal to the threshold, and set to 1 when it is less than the threshold . In some embodiments, the 8-bit image is threshold processed . ​​​​It can be inverted before. That is, the wire part is set to black and the background part is set to white.

[0158] In some embodiments, the binary image is generated using Otsu's method. Without being bound by any particular theory nor the operating mode, Otsu's method is a suitable adaptive thresholding method for binarization in image processing. By examining possible thresholds (e.g., 0 to 255), Otsu's method can find the optimal and / or desirable threshold of the 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 desirable threshold can be determined by minimizing the within-class variance V w or maximizing the between-class variance V b . For a given threshold T, the pixels of the input image can be divided into two classes: class 1 of pixels having a pixel value less than or equal to T, and class 2 of pixels having a pixel value greater than T. In some cases, class 1 and class 2 are respectively referred to as the background and foreground of the input image. The within-class variance V of each class can be calculated as follows. w TIFF2025111430000003.tif15170 (where μ is the average value of the pixel values of a specific class, N is the number of pixels in a specific class, and Xi is the pixel value of each pixel of a specific image)

[0160] The between-class variance V b characterizes the variance between two classes. In some embodiments, Between-class variance V b is calculated as V b = V t - V w and can be calculated as such, where V t is the combined variance, that is, the variance of all pixels in the input image. For a given image, V t is usually a constant. Therefore, usually, minimizing V w will simultaneously maximize V b .

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

[0162] The binary image generated after thresholding is subject to one or more morphological processes . Morphological processes can include two processes: shrinking and dilation. Both processes can have two inputs: (1) the image to be processed, and (2) a structuring element (also called a kernel ). The two inputs can each be represented by a series of coordinates

[0163] More specifically, the shrinking process for a binary image can be performed as follows. Let X be a series of Euclidean coordinates corresponding to the input binary image, and K be a series of coordinates corresponding to the structuring element. Furthermore, let Kx be K translated so that the origin of the structuring element is at x . Using these concepts, the shrinking of X by K is all points x in the series where Kx is a subset of X

[0164] ​To calculate the erosion of a binary input image by this structural 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 structural element is overlaid on the input image such that the origin of the structural element coincides with the input pixel coordinates. For all pixels within the structural element, if the corresponding pixel in the binary image below is a foreground pixel, the input pixel is left as is. However, if any of the corresponding pixels in the image are background, the input pixel is also set to the background value. In some embodiments, the structural element is 3x3, and the effect of the erosion process using this structural element is to remove any foreground pixel that is not completely surrounded by other white pixels (assuming 8-connectivity). Since these pixels may be at the edge of the white region, the actual effect of the erosion is that the foreground region shrinks and the holes within the region grow. The dilation of a binary image can be performed as follows. First, let X be a series of Euclidean coordinates corresponding to the input binary image, K be a series of coordinates of the structural element, and Kx be K translated so that the origin of K becomes x. Then, the dilation of X by K is all points x such that the intersection of Kx and X is non-empty. More specifically, to calculate the dilation of a binary input image by the structural element K, each of the background pixels of the input image can be considered in turn. For each background pixel (also called an input pixel), the structural element is overlaid on the input image such that the origin of the structural element coincides with the input pixel position. If at least one pixel within the structural element coincides with a foreground pixel of the image below, the input pixel is set to the foreground value. For all pixels within the structural element, if the corresponding pixel in the binary image below is a foreground pixel, the input pixel is left as is. However, if any of the corresponding pixels in the image are background, the input pixel is also set to the background value. In some embodiments, the structural element is 3x3, and the effect of the erosion process using this structural element is to remove any foreground pixel that is not completely surrounded by other white pixels (assuming 8-connectivity). Since these pixels may be at the edge of the white region, the actual effect of the erosion is that the foreground region shrinks and the holes within the region grow. To calculate the erosion of a binary input image by this structural 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 structural element is overlaid on the input image such that the origin of the structural element coincides with the input pixel coordinates.

[0165] For all pixels within the structural element, if the corresponding pixel in the binary image below is a foreground pixel, the input pixel is left as is. However, if any of the corresponding pixels in the image are background, the input pixel is also set to the background value. In some embodiments, the structural element is 3x3, and the effect of the erosion process using this structural element is to remove any foreground pixel that is not completely surrounded by other white pixels (assuming 8-connectivity). Since these pixels may be at the edge of the white region, the actual effect of the erosion is that the foreground region shrinks and the holes within the region grow. For all pixels within the structural element, if the corresponding pixel in the binary image below is a foreground pixel, the input pixel is left as is. However, if any of the corresponding pixels in the image are background, the input pixel is also set to the background value. In some embodiments, the structural element is 3x3, and the effect of the erosion process using this structural element is to remove any foreground pixel that is not completely surrounded by other white pixels (assuming 8-connectivity). Since these pixels may be at the edge of the white region, the actual effect of the erosion is that the foreground region shrinks and the holes within the region grow.

[0166] The dilation of a binary image can be performed as follows. First, let X be a series of Euclidean coordinates corresponding to the input binary image, K be a series of coordinates of the structural element, and Kx be K translated so that the origin of K becomes x. Then, the dilation of X by K is all points x such that the intersection of Kx and X is non-empty. More specifically, to calculate the dilation of a binary input image by the structural element K, each of the background pixels of the input image can be considered in turn. For each background pixel (also called an input pixel), the structural element is overlaid on the input image such that the origin of the structural element coincides with the input pixel position. If at least one pixel within the structural element coincides with a foreground pixel of the image below, the input pixel is set to the foreground value. For all pixels within the structural element, if the corresponding pixel in the binary image below is a foreground pixel, the input pixel is left as is. However, if any of the corresponding pixels in the image are background, the input pixel is also set to the background value. More specifically, to calculate the dilation of a binary input image by the structural element K, each of the background pixels of the input image can be considered in turn. For each background pixel (also called an input pixel), the structural element is overlaid on the input image such that the origin of the structural element coincides with the input pixel position. If at least one pixel within the structural element coincides with a foreground pixel of the image below, the input pixel is set to the foreground value. For all pixels within the structural element, if the corresponding pixel in the binary image below is a foreground pixel, the input pixel is left as is. However, if any of the corresponding pixels in the image are background, the input pixel is also set to the background value. If at least one pixel within the structural element coincides with a foreground pixel of the image below, the input pixel is set to the foreground value. If all corresponding pixels in the image are background, the input pixel remains at the background value.

[0167] In some embodiments, the structuring element is 3x3, and the dilation operation using this structuring element has the effect of setting all background pixels having adjacent foreground pixels to the foreground color (assuming 8 - connectivity). Since these pixels may be at the edge of the white region, the actual effect of the dilation operation is that the foreground region expands and the holes within the region shrink.

[0168] In the wire - tracking method described herein, the binary image generated by thresholding is processed by opening and closing operations. Morphological opening and closing are essentially erosion and dilation operators applied in various orders. Without being bound by a particular theory or operation mode, opening is defined as erosion followed by dilation using the same structuring element for both operations, and the closing operation is defined as dilation followed by erosion using the same structuring element for both operations.

[0169] The basic effect of opening is to remove some of the foreground (i.e., bright) pixels from the edge of the foreground pixel region. However, opening is usually less destructive than erosion. The closing operation has a tendency to expand the boundaries of the foreground (i.e., bright) regions in the image (and shrink the holes of the background color in such regions), but is generally less destructive than dilation.

[0170] Figure 15C shows the binary image generated after thresholding, and Figure 15D shows the binary image generated after performing an opening operation on the binary image shown in Figure 15C. Figure 15 ​​​​​​​​​ E represents the binary image before the closing operation, and FIG. 15F shows the binary image generated after performing the 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, this order can be reversed, that is, the opening operation is performed after the closing operation.

[0171] As can be seen from FIGS. 15C - 15F, the morphological operations of opening and closing help to remove and / or reduce artifacts that may interfere with the edges of the wire. The opening operator is applied to remove unwanted floating pixels and clean up the edges of the wire. On the other hand, the closing operator fills in empty spaces and can remove unwanted lines (such as those seen on the left side of the input image relative to the output images of FIGS. 15E - 15F).

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

[0173] More specifically, the first kernel G x is convolved with the input image from left to right to obtain the gradient in the first direction ( for example, the x - direction). Without loss of generality, the first kernel is It can be described as follows. TIFF2025111430000004.tif17170

[0174] The second kernel G y is convolved with the input image from top to bottom to obtain the gradient in the second direction (e.g., the y direction ). Without loss of generality, the second kernel can be described as follows and can be written as such. TIFF2025111430000005.tif18170

[0175] By the above two convolution steps, two vectors representing the gradients of the image in the x and y directions are generated. Therefore, the magnitude of the gradient of each pixel of the input image can be calculated as follows and can be computed. TIFF2025111430000006.tif18170

[0176] FIG. 15G shows the binary image after the above opening and closing operations, and FIG. 15 H 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 detection image (e.g., the image shown in FIG. 15H) is processed by another thresholding process in the wire tracking method to generate another binary image. In some embodiments, the thresholding process in this step can be performed using the above Otsu's method if possible. FIG. 15I shows the edge detection image, and FIG. 15J shows the binary image generated after thresholding using the Otsu's method . after thresholding.

[0178] In some embodiments, the image in which edges are detected is further processed by edge filling (e.g., after thresholding). In some cases, wire edges are trimmed in the edge detection image, and the 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 detection image may be due to the kernel used in the edge detection process, and the edges trimmed from the image frame are not detected. Thus, the left and right edges of each row are not fully calculated. In some embodiments, the image in which edges are detected is further processed by edge filling (e.g., after thresholding). In some cases, wire edges are trimmed in the edge detection image, and the 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 detection image may be due to the kernel used in the edge detection process, and the edges trimmed from the image frame are not detected. Thus, the left and right edges of each row are not fully calculated. In some embodiments, the image in which edges are detected is further processed by edge filling (e.g., after thresholding). In some cases, wire edges are trimmed in the edge detection image, and the 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 detection image may be due to the kernel used in the edge detection process, and the edges trimmed from the image frame are not detected. Thus, the left and right edges of each row are not fully calculated. In some embodiments, the image in which edges are detected is further processed by edge filling (e.g., after thresholding). In some cases, wire edges are trimmed in the edge detection image, and the 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 detection image may be due to the kernel used in the edge detection process, and the edges trimmed from the image frame are not detected. Thus, the left and right edges of each row are not fully calculated. In some embodiments, the image in which edges are detected is further processed by edge filling (e.g., after thresholding). In some cases, wire edges are trimmed in the edge detection image, and the 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 detection image may be due to the kernel used in the edge detection process, and the edges trimmed from the image frame are not detected. Thus, the left and right edges of each row are not fully calculated. In some embodiments, the image in which edges are detected is further processed by edge filling (e.g., after thresholding). In some cases, wire edges are trimmed in the edge detection image, and the 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 detection image may be due to the kernel used in the edge detection process, and the edges trimmed from the image frame are not detected. Thus, the left and right edges of each row are not fully calculated. In some embodiments, the image in which edges are detected is further processed by edge filling (e.g., after thresholding). In some cases, wire edges are trimmed in the edge detection image, and the 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 detection image may be due to the kernel used in the edge detection process, and the edges trimmed from 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 middle row of the edge detection image until it detects a row in which 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). The rows above that are filled with the right or left edge as the last or first column. The edge filling technique described herein traverses up and down from the middle row of the edge detection image until it detects a row in which 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). The rows above that are filled with the right or left edge as the last or first column. The edge filling technique described herein traverses up and down from the middle row of the edge detection image until it detects a row in which 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). The rows above that are filled with the right or left edge as the last or first column. The edge filling technique described herein traverses up and down from the middle row of the edge detection image until it detects a row in which 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). The rows above that are filled with the right or left edge as the last or first column.

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

[0181] The next step of the wire tracking method includes calculating the midpoints. In this step, the midpoints of the wire are calculated for each row of the edge-filled 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. FIG. 15M The next step of the wire tracking method includes calculating the midpoints. In this step, the midpoints of the wire are calculated for each row of the edge-filled 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. FIG. 15M The next step of the wire tracking method includes calculating the midpoints. In this step, the midpoints of the wire are calculated for each row of the edge-filled 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. FIG. 15M shows an example of an image showing a series of calculated midpoints.

[0182] Once the midpoints are calculated, these midpoints can be used as the input 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-degree polynomial in x. More specifically, the regression method described herein uses the 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. The set of these x and y values is used to calculate an equation using the polynomial regression method. 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-degree polynomial in x. More specifically, the regression method described herein uses the 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. The set of these x and y values is used to calculate an equation using the polynomial regression method. More specifically, the regression method described herein uses the 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. The set of these x and y values is used to calculate an equation using the polynomial regression method. The midpoints calculated above are represented by corresponding x and y coordinates. The coordinates are essentially row and column numbers. The set of these x and y values is used to calculate an equation using the polynomial regression method. The midpoints calculated above are represented by corresponding x and y coordinates. The coordinates are essentially row and column numbers. The set of these x and y values is used to calculate an equation using the polynomial regression method. The set of these x and y values is used to calculate an equation using the polynomial regression method.

[0183] Without loss of generality, the regression method can be represented, for example, by the following equation. TIFF2025111430000007.tif12170 (where x i and y i are the midpoint coordinates calculated for each image frame)

[0184] Equation (6) can be written in matrix form as follows. TIFF2025111430000008.tif24170 Or more concisely, it can be written as follows. TIFF2025111430000009.tif10170 (where Y is a vector with elements (y1, y2,..., y n ), X is a matrix containing the calculated midpoint x i and A is a vector containing the fitting parameters a, b, and c)

[0185] Solving Equation (8) gives the following equation. TIFF2025111430000010.tif10170 Equation (9) is executed for each input image frame, generating a quadratic equation for each frame. Therefore, the fitting parameters (also called fitting coefficients) are calculated for each image frame.

[0186] Using the fitting coefficients of the quadratic equation from the previous step, the maximum point of the parabola's deflection or displacement is detected. In some embodiments, the inflection point can be detected by finding the maximum value of x using the derivative of the equation. Next, substituting that value of x into the equation (6) for x M at to obtain the y coordinate, i.e., y x M This y M value is used for plotting to represent the displacement of the wire M

[0187] Using the above inflection point, the force applied to the sensing wire can be calculated. Generally, the quantification of the force transient state involves the accurate identification of the position of each transient state within the force record. In some embodiments, the peak analysis method is used to identify the force transient state by matching the theoretical force trajectory (also called the simulated force trajectory) with the actual force trajectory. The actual force trajectory is a graph of the result of plotting the change in the measured force over time. The fabric shrinkage appears as peaks and valleys in the resulting trajectory as the force repeatedly increases and decreases over time. The theoretical force trajectory is generated with artificial transient states at known intervals or known frequencies. The peak analysis method is applicable to records with a low signal-to-noise ratio (SNR), or records where the amplitude of the force transient state has decreased in response to a compound that results in a very small force transient state record. ​​​​​​​​, has the advantage of being able to identify transient peaks.

[0188] The actual force trajectory is then fitted to a polynomial function to obtain the The noise is reduced and the shrinkage parameters are calculated from a polynomial function. The parameters include, for example, transient amplitude, transient duration, time to transient peak, transient These include time from the transient peak, maximum slope of force production, and maximum slope of apparent force.

[0189] FIG. 16 is a flow chart illustrating a method 500 for engineering tissue culture, according to one embodiment. In some embodiments, the method 500 is implemented using the system 300 illustrated in FIG. In these embodiments, the system Memory 332 within system 300 is configured to store processor-executable code. The processor 334 includes a non-transitory processor-readable medium. The method 500 is executed when the function code is executed.

[0190] The method 500 includes, at 510, growing tissue from cells seeded in the well. At least one detection signal associated with the well to be detected (e.g., well 312 in FIG. 13) The method includes receiving a series of measurements from the well according to a series of stimulus parameters. The method 500 also includes: 520, a set of measurements based on a comparison of the set of measurements with a set of predetermined criteria associated with the tissue. This involves specifying the amount by which to change the set of values of the stimulation parameters. Adjust the set of values using the amount to change the parameter, and set the set of stimulation parameters in 530. Form a new series of values. Then, a stimulation signal is sent to a series of electrodes at 540, and an electrical stimulation is applied to the tissue according to the new series of values of the series of stimulation parameters.

[0191] In some embodiments, the series of stimulation parameters includes at least one of stimulation voltage, stimulation frequency, and / or stimulation time. In some embodiments, the stimulation signal can be sent by the processor 334 to the control unit 316 of the tissue growth device 3 10 via the communication interface 336. Next, the control unit 316 is configured to send an electrical signal to the electrodes characterized by the stimulation parameters specified by the stimulation signal to execute the stimulation signal. In other words, the stimulation signal sent by the processor 334 functions as an instruction to the control unit 316, and the control unit 3 16 controls the operation of the electrodes based on the instruction.

[0192] In some embodiments, the sensing element includes at least two elastic sensing elements (e.g., sensing wires) arranged across the well such that there is a gap between the sensing element and the bottom of the well. In these embodiments, the growth of the tissue in the well can apply a force to the elastic sensing element, thereby changing the shape of the elastic sensing element. In some embodiments, the processor 334 in the system 300 is configured to analyze the shape (or change in shape) of the elastic sensing element to determine the growth state of the tissue in the well. In response to the determined growth state, the processor 334 is configured to determine the amount by which to change the series of stimulation parameters.

[0193] ​​​​​​In some embodiments, an initial set of values of a series of stimulation parameters is a predetermined stimulation protocol. In some embodiments, the user interface 3 40 is configured to present a user with a predetermined stimulation protocol from among a plurality of available stimulation protocols, and the user can select one stimulation protocol as the predetermined stimulation protocol.

[0194] In some embodiments, a predetermined criterion related to the tissue includes a tissue maturity criterion and / or an excitation criterion. In response to a set of measurements that meet the maturity criterion and / or the excitation criterion, the stimulation voltage can be reduced accordingly (e.g., by the processor 334). In response to a set 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 set of measurements includes the maturity state of the tissue and / or the excitation criterion obtained from an image of the sensing element. For example, processing of data from the sensing wire may indicate that the deflection of the wire is not large enough (e.g., less than 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 set of measurements can be obtained by another sensor, such as a voltage sensor configured to measure the voltage applied to an implanted electrode. In response to a measured voltage less than a threshold voltage (e.g., specified in the protocol), the processor can send a command to the control unit 316 to increase the voltage. Tissue growth data Any other operating parameters of the vice 310 can be adjusted in the same manner.

[0196] In some embodiments, method 500 periodically (e.g., by processor 334) receives instances of a series of measurements and further includes storing the instances of the series of measurements in a memory (e.g., memory 332). In these embodiments, the growth history of the tissue within 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 are stored in the memory in, for example, comma-separated value (CSV) format or any other suitable format for later analysis and troubleshooting in the event that a problem is detected in the tissue.

[0197] In some embodiments, method 500 can be executed by a processor without human intervention for a preset period (e.g., from about several days to several weeks). In these embodiments, the user interface 340 can present several available protocols to the user, and the user selects one to execute. When the user selects a protocol, the 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, the user interface 340 can be configured to display the growth results when the execution of the protocol is complete. The growth results can be, for example, an image of the tissue, an image of the sensing wire, the solution of the sensing wire It can include the maturity state and / or the excitation state, etc. of the tissue obtained from the analysis.

[0198] In some embodiments, a series of measurements can be used to evaluate the quality of tissue growth. This kind of evaluation can include, for example, detecting one or more problems in tissue growth. In some embodiments, the processor 334 can be configured to detect one or more problems in tissue growth (such as tissue contamination, debris, holes, etc.) based on a 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, and each category represents a common problem in tissue growth. The input image can be an image of the tissue, an image of the tissue encapsulating the sensing element, or any combination thereof.

[0199] In some embodiments, in response to the detection of a problem in tissue growth (e.g., by a machine learning model), the processor 334 is further configured to perform a repair operation, such as changing the stimulation parameters. In some embodiments, in response to the detection of a problem in tissue growth, the processor 334 is further configured to generate an alarm signal. In some embodiments, the processor 334 is further configured to transmit the alarm signal to the user (e.g., via the communication interface 336). The alarm signal can include, for example, a text message, an email, a voicemail, or a real-time call. ​​​​​​​In some embodiments, an error code is assigned to each category of common problems, and the warning signal sent to the user includes the error code.

[0200] Referring again to FIG. 13, in some embodiments, the system 300 (or the process sor 334) can be configured to perform end-to-end data analysis. In these embodiments, raw data (e.g., a raw image of the sensing wire) is first sent to the processor 334 for wire tracking, and force values over time are generated based on the movement of the wire. For example, the processor 334 can be configured to perform the methods described with reference to FIGS. 14 and 15A-15M. Then, the force values are quantitatively read out and used for peak analysis configured to generate, for example, peak amplitude, time to peak, etc. And the quantitative readout is used for plotting in a graph display. The graph display can be, for example, a force trajectory, a change in maximum force over time, a comparison of time to peak before and after drug treatment, etc. In end-to-end analysis, the above processing can be performed by the processor -334 (or other processors) without human intervention. For example, the user can select a protocol to be executed by the system 300 and receive a graph display at the end of the protocol. In some embodiments, the graph display can be displayed on the user interface 340. In some embodiments, the graph display can be sent to the user via a network (wired or wireless). For example, the graph display can be sent to a smartphone or computer associated with the user. ​

[0201] In some embodiments, system 300 (or processor 334) can be configured to implement a scheduler for operating tissue growth device 310. In some embodiments, processor 334 includes an FPGA and can execute the scheduler via Verilog or other suitable language. In some embodiments, an operator can use user interface 340 to set the pulse interval of system 300. User interface 340 is configured to send pulse interval information to the scheduler, and the scheduler is configured to generate a time table for accurately determining when specific wells within tissue growth device 310 receive pulses (e.g., for stimulation). Thus, the scheduler can be used to provide an average accurate stimulation timing with short-term deviations for continuous well stimulation.

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

[0203] Some embodiments described in this specification relate to computer-readable media. The computer -readable medium (or processor-readable medium) is non-transitory in the sense that it does not include a propagating signal itself (transmission medium, e.g., a propagating electromagnetic wave that carries information through space or a cable). The medium and the computer code (which may also be called code) may be designed and constructed for a particular purpose. Examples of non-transitory computer -readable media include magnetic storage media such as hard disks, floppy disks, and magnetic tapes; optical storage media such as compact discs / digital versatile discs (CD / DVDs), compact disc read-only memory (CD-ROMs), and holographic devices; magneto-optical storage media such as optical discs; carrier signal processing modules; and hardware devices specially configured to store and execute program code, such as application-specific integrated circuits (ASICs), programmable logic devices (PLDs), read-only memories (ROMs), and random access memories (RAMs), but are not limited thereto. Other embodiments described in this specification may relate to computer programs that can include, for example, the instructions and / or computer code described in this specification.

[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 that are executed by a computer using an interpreter. For example, embodiments may be written in Python, R, Java, JavaScript, C++, or other programming languages. It can be executed using languages and development tools. Another example of computer code includes, but is not limited to, control signals, encryption codes, and compression codes.

[0205] Although the present teachings have been described in connection with various embodiments and examples, the present teachings are not intended to be limited to such embodiments or examples. Rather, the present teachings include various alternatives, modifications, and

[0206] equivalents, as will be understood by those skilled in the art. Although various embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or achieving the results and / or one or more of the advantages described herein. Accordingly, each of such variations and / or modifications is to be regarded as within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will appreciate that all of the parameters, dimensions, materials, and configurations described herein are for purposes of illustration and that the actual parameters, dimensions, materials, and / or configurations will The method is targeted. Further, if such functions, systems, articles, materials, kits, and / or methods are not mutually inconsistent, any combination of two or more such functions, systems, articles, materials, kits, and / or methods is included within the scope of the invention of the present disclosure.

[0207] All definitions defined and used herein are to be understood as controlling dictionary definitions, definitions of incorporated by reference documents, and / or ordinary meanings of defined terms.

[0208] The indefinite articles "a" and "an" used herein and in the claims are to be understood as meaning "at least one" unless explicitly indicated to the contrary. The ranges recited herein include the values at both ends.

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

[0210] As used herein, the term "POMaC" refers to poly(octamethylene maleate(anhydride) citrate)(POMaC), or a POMaC prepolymer containing a mixture of 1,8-octanediol, citric acid, and maleic anhydride. "Synthesis and characterization of a biodegradable elastomer featuring a dual crosslinking mechanism" by Tran et al., Soft Matter, January 1, 2010, 6 thesis and characterization of a biodegr adable elastomer featuring a dual crossl inking mechanism", Soft Matter, January 1, 2010, 6 ​​​​​​Reference may be made to Volume 11, Numbers 2449 - 2461, which is hereby incorporated by reference in its entirety. Incorporated.

[0211] As used in this specification and the claims, the phrase "and / or" is to be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements recited in "and / or" are to be construed likewise, i.e., as "one or more" of the elements so conjoined. Other elements not specifically identified by the "and / or" clause may optionally be present, whether or not related to those elements specifically identified. Thus, by way of non - limiting example, a reference to "A and / or B" when used in combination with an open - ended term such as "comprising" may refer, in one embodiment, to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), and so forth. "Either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Is to be understood to mean... Of the elements so conjoined, i.e., as "one or more" of the elements so conjoined. Are to be construed likewise... Regardless of whether or not related to those elements specifically identified. Thus, by way of non - limiting example, a reference to "A and / or B"... When used in combination with an open - ended term such as "comprising" In one embodiment, only A (optionally including elements other than B), in another embodiment Only B (optionally including elements other than A), in yet another embodiment Both A and B (optionally including other elements), and so forth.

[0212] As used in this specification and the claims, "or" is to 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 inclusive, i.e., it is to be interpreted as including at least one, and in some cases two or more, of the elements or items in the list, and optionally other items not listed. Conversely, when specifically... Have the same meaning as "and / or" as defined above. For example, when separating items in a list... "Or" or "and / or" is inclusive... Of the elements or items in the list, and optionally other items not listed. Including at least one, and in some cases two or more, of the elements or items in the list, and optionally other items not listed. Conversely, when specifically... Only the terms shown, such as "only one of" or "exactly one of", and when used in the claims, "consisting of" means including exactly one of the elements of several elements or lists. Generally, the terms used in this specification, "or", when used in the claims, is an exclusive term, such as "either", "one of", "only one of", "exactly one of", preceded by, is to be construed as indicating exclusive alternatives (i.e., "one or the other, but not both"). When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law. When used in this specification and the claims, the phrase "at least one" in relation to a list of one or more elements is to 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 each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements.

[0213] By this definition, elements other than the specifically identified elements may, in some cases, be present in the list of elements referred to by the phrase "at least one", regardless of whether they are related to the specifically identified elements. 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") in one embodiment, B does not exist (and in some cases includes elements other than B), and at least one A, in some cases does not necessarily include every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. By this definition, elements other than the specifically identified elements may, in some cases, be present in the list of elements referred to by the phrase "at least one", regardless of whether they are related to the specifically identified elements. 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") in one embodiment, B does not exist (and in some cases includes elements other than B), and at least one A, and in some cases does not include B (and in some cases includes elements other than B), and at least one A, and in some cases In another embodiment, it contains two or more A's. In some cases, there is no A (and in some cases it contains elements other than A), and it contains at least one B, and in some cases two or more B's. In yet another embodiment, it contains at least one A, and in some cases two or more A's, and at least one B, and in some cases two or more B's (and in some cases it contains other elements), etc. can be referred to.

[0214] Similar to the above specification, in the claims, all transitional phrases, such as "comprising", "including", "carrying", "having", "containing", "involving", "holding", "consisting of", etc. are open-ended, that is, they mean including but not limited to. It should be understood that, as described in the Manual of Patent Examining Procedure, Section 2111.03 of the United States Patent and Trademark Office, only the transitional phrases "consisting of" and "consisting essentially of" are exclusive or semi-exclusive

[0215] transitional phrases, respectively. The claims should not be read as prescribing the order or elements as defined therein unless otherwise specified in the spirit thereof. 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 falling within the spirit and scope of the following claims and their equivalents are claimed.

[0216] Example 1 Here, an innovative multi-material process for a scalable and functional platform A theory has been proposed in the form of a 96-well plate. Three classes of materials are integrated into the platform. An array of soft elastic micro-wires is used not only as an anchor for tissue formation but also as a sensor for recording tissue contraction. Conductive carbon electrodes are embedded in the plate to drive electrical stimulation for tissue maturation and to regulate tissue contraction during drug testing. Most of the device is made of rigid polystyrene plastic to eliminate drug-absorbing polydimethylsiloxane (PDMS). This platform has a higher throughput than current state-of-the-art devices, and significantly reduces manufacturing costs and tissue production costs. An array of soft elastic micro-wires is used not only as an anchor for tissue formation but also as a sensor for recording tissue contraction. Conductive carbon electrodes are embedded in the plate to drive electrical stimulation for tissue maturation and to regulate tissue contraction during drug testing. Most of the device is made of rigid polystyrene plastic to eliminate drug-absorbing polydimethylsiloxane (PDMS). This platform has a higher throughput than current state-of-the-art devices, and significantly reduces manufacturing costs and tissue production costs. Our tissue culture platform (herein called the Biowire II plate) is made of tissue culture plastic (polystyrene), resulting in a final product that does not contain PDMS and minimizes drug absorption. This platform is based on the footprint of a standard 96-well plate and is compatible with widely used plate readers. The device is 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 (Figs. 2A - 2C).

[0217] The plate cap and the 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: 5x1x0.2 mm) centered in each bottomless well (Fig. 2A). Human pluripotent stem cell-derived cardiomyocytes (hCM) and human cardiac fibroblasts (ratio 10:1) are seeded in a hydrogel matrix in the microwells of the bottomless 96-well plate. The device is 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 (Figs. 2A - Fig. 2C). The plate cap and the 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: 5x1x0.2 mm) centered in each bottomless well (Fig. 2A). Human pluripotent stem cell-derived cardiomyocytes (hCM) and human cardiac fibroblasts (ratio 10:1) are seeded in a hydrogel matrix in the microwells of the bottomless 96-well plate. Human pluripotent stem cell-derived cardiomyocytes (hCM) and human cardiac fibroblasts (ratio 10:1) are seeded in a hydrogel matrix in the microwells of the bottomless 96-well plate. Kus (3.0 mg / mL -1 collagen and 15% (v / v) Matrigel) into the micro wells. To provide fixed points for tissue remodeling and compression, two elastic micro wires (diameter: 0.1 mm) arranged in parallel were cast throughout the micro wells (Figure 2B). Over time, the hCM / fibroblast / hydrogel mixture seeded in the micro wells self-assembled into a 3D tissue that was spanned and fixed by the two parallel wires (Figure 2C). Within one week, the tissue began to contract and the elastic polymer wires bent . Using the calibration curve generated by a standard force sensor (MicroSquishier, CellScale), the displacement of the polymer wire could be correlated with the contraction force of the tissue . Although the entire device manufacturing process may 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 the base plates is simultaneous. For these components, according to the specifications shown in Figures 8B and 8C, standard soft lithography techniques were used to design and fabricate an SU-8 master mold. Next, PDMS was poured into the SU-8 master and the PDMS mold was fabricated by cross-linking the PDMS at room temperature for 48 hours. Then, the PDMS mold was used to manufacture the polymer wires and the polystyrene base plates were made hot embossable

[0218] . To minimize the effect of thermal expansion, we chose to cross-link the PDMS mold at room temperature instead of by heating. Therefore, the resulting PDMS mold ​​​​​​​​Maintain dimensions designed to span a large surface area, such as the footprint of a 96-well plate. However, during the hot embossing of the base plate, thermal expansion still occurs. Initially, it was found difficult to maintain the pre-determined plate dimensions without shrinking across the entire large footprint. Even a shrinkage of 0.1 mm over a length of 100 mm can cause significant misalignment between the wires and the micro-wells. Therefore, the degree of shrinkage after hot embossing was determined empirically 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 a polystyrene-based base plate, the back of the PDMS mold of the base plate was bonded to a 6-inch silicon wafer by plasma bonding or corona etching. After bonding to the silicon wafer, the mechanical properties of the PDMS master were further enhanced by exposing it to 150 °C for 30 minutes. The polystyrene sheet was pressed against the PDMS master using a hot embosser to form and customize the polystyrene plate base. Fabrication of the PDMS master for hot embossing is much less expensive than conventional metal etching methods for creating masters. Since PDMS is softer than metal, there is a possibility of shape distortion during hot embossing, but this problem was found to be avoidable by using a low compression force at high temperature during embossing. One important advantage of PDMS over metal-based masters is that the embossed polysty is much less expensive than conventional metal etching methods for creating masters. Since PDMS is softer than metal, there is a possibility of shape distortion during hot embossing, but this problem was found to be avoidable by using a low compression force at high temperature during embossing. One important advantage of PDMS over metal-based masters is that the embossed polysty The ability to temporarily distort the lens so that it can be easily released from the master. This is a major problem in that when a hard material (polystyrene) is pressed against another hard material (metal master) for embossing the fine elements must have exactly vertical walls to prevent the two hard materials from engaging.

[0221] The plate has a pair of built-in carbon electrodes around all the microwells to generate a local electric field throughout each tissue. Pacing the heart tissue with an external electrode has been shown to ensure consistency in drug testing, both in maturing the tissue over time and in standardizing the beating frequency. For each column of wells, two long carbon electrodes were placed along the vertical boundaries of the wells Figure 8C shows the design of the base plate. The rectangular posts around the microwells were designed to hold the carbon electrodes in the PDMS master mold prior to hot embossing (Figure 3A). The carbon electrodes were embedded into the polystyrene sheet during hot embossing and exposed on the plate surface and side walls that form the microwells (Figures 3B, 3C). These exposed surfaces were placed so as to be as close as possible to the tissue and were sufficient to generate an electric field along the length of the tissue Figure 8C shows the design of the base plate. The rectangular posts around the microwells were designed to hold the carbon electrodes in the PDMS master mold prior to hot embossing (Figure 3A). The carbon electrodes were embedded into the polystyrene sheet during hot embossing and exposed on the plate surface and side walls that form the microwells (Figures 3B, 3C). These exposed surfaces were placed so as to be as close as possible to the tissue and were sufficient to generate an electric field along the length of the tissue (Figure 3B, Figure 3C). These exposed surfaces were considered to be placed as close as possible to the tissue and were sufficient to generate an electric field along the length of the tissue to generate an electric field along the length of the tissue. Embedding electrodes with a thickness of 400 μm into the polystyrene base allowed current to flow with minimal resistance. This is beneficial in contrast to other methods including our first tests using sputtering (e.g., sputtered gold electrodes) which resulted in a thin conductive surface with a nanometer-scale thickness (Figure 9A). Such is beneficial in contrast to other methods including our first tests using The conductive surface is difficult to uniformly coat the entire large surface area and is easily damaged by scratches resulting in high electrical resistance and lack of long-term stability due to delamination (Fig. 9B). Compared with the voltage applied from the power source, the proportion of the overall voltage drop of the embedded carbon electrode was negligible and consistent across the entire well plate (Fig. 10A) . .

[0222] Rapid wire casting on microwells

[0223] Scalably casting soft and elastic polymer micro wires across an array of microstructures can be difficult. To avoid this, a method of directly forming and crosslinking polymer wires on a polystyrene-based microwell structure was developed. To do this, a PDMS mold of the wire array was placed on top of the base plate such that the microchannels on the mold faced the plate (Fig. 4A) . A poly(oxytrimethylene maleic acid (anhydride) citric acid) (POMaC) prepolymer, which is 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 microchannels by capillary action . In particular, the prepolymer solution continuously filled the channels on the microwells even when the cross-section of the microchannels was not completely closed (Fig. 4B). Following complete perfusion, the P OMaC prepolymer was exposed to UV light to generate an array of crosslinked elastomer wires, after which the PDMS mold was removed. To reduce the influence of batch-to-batch variation of the bulk polymer solution on the mechanical rigidity of the UV-crosslinked polymer wires, the UV crosslinking energy was drawn into the microchannels by capillary action. In particular, the prepolymer solution continuously filled the channels on the microwells even when the cross-section of the microchannels was not completely closed (Fig. 4B). Following complete perfusion, the P OMaC prepolymer was exposed to UV light to generate an array of crosslinked elastomer wires, after which the PDMS mold was removed. To reduce the influence of batch-to-batch variation of the bulk polymer solution on the mechanical rigidity of the UV-crosslinked polymer wires, the UV crosslinking energy was drawn into the microchannels by capillary action. In particular, the prepolymer solution continuously filled the channels on the microwells even when the cross-section of the microchannels was not completely closed (Fig. 4B). Following complete perfusion, the P OMaC prepolymer was exposed to UV light to generate an array of crosslinked elastomer wires, after which the PDMS mold was removed. To reduce the influence of batch-to-batch variation of the bulk polymer solution on the mechanical rigidity of the UV-crosslinked polymer wires, the UV crosslinking energy was drawn into the microchannels by capillary action. In particular, the prepolymer solution continuously filled the channels on the microwells even when the cross-section of the microchannels was not completely closed (Fig. 4B). Following complete perfusion, the P OMaC prepolymer was exposed to UV light to generate an array of crosslinked elastomer wires, after which the PDMS mold was removed. To reduce the influence of batch-to-batch variation of the bulk polymer solution on the mechanical rigidity of the UV-crosslinked polymer wires, the UV crosslinking energy was The Young's modulus of the resulting bulk polymer (measured with a myograph as described) was adjusted for each batch of polymer to be 33 ± 3 k Pa. The UV-crosslinked POMaC wires consistently adhered to the polystyrene base and multiple micro wires were cast across the entire plate and spanned across 96 microwells (Figure 4C). By this method, 24 wires that spanned across 96 microwells in a single step could be attached. The plate base cast with micro wires was fully assembled in a thermal bonding step with a hot embosser, and a bottomless 96-well plate was fused onto the polystyrene plate base cast with polymer micro wires . Next, the assembled plate was subjected to several post-treatment steps including washing overnight with distilled water, air drying, packaging , and gamma sterilization. The elasticity of the micro wires could be further fine-tuned by another thermal crosslinking by baking the entire plate at 70 °C for several days . The plates were stored at 4 °C and protected from light before use to minimize exposure to unnecessary heat and light prior to use.

[0224] Characterization and usage method of the plate

[0225] The cast polymer wires in the plate were used as both the fixation points of the tissue and the force sensors for monitoring the changes in shrinkage. To do so, the correlation between the displacement of the wires and the generation of force was characterized. The inventors used a commercially available microscale mechanical test system, MicroSquisher (CellScale), to obtain the force-displacement calibration curve The probe tip was attached to the polymer wire in a similar manner to the tissue adhesion (Figures 5A-5D). The probe tip is attached to the polymer wire at the center point of the customized polymer wire with a curvature of 1 / 2 mm. The force and displacement were recorded with a MicroSquisher as the wire was moved relative to the target. A force-displacement curve was generated (Figure 5A). The mechanical properties of the polymer wire were determined by thermal crosslinking. Further fine-tuning can be achieved through post-processing. Force-displacement curves after 2, 4, and 6 days of thermal cross-linking. The fitted polynomials are shown in Figures 5B-5D. Consistency was demonstrated across the entire 96-well plate. When comparing wells in the same row, there was no significant difference (Figure 11C). , and the force required to displace 150 μm were also reproducible among all wells (Figure 1 1B). Furthermore, the tensile and relaxation curves of typical polymer wires show minimal hysteresis. The microtissues cultured in each 96-well plate were able to grasp the wire (Figure 11C). The cardiac tissue also expressed sarcomeric α-actinin (green) and F- Actin (red) staining revealed well-aligned sarcomere structures (Figure 6B). 6-well plates are a widely adopted tissue culture format, so Biowire plates The company also uses commercially available equipment, such as the SpectraMax imaging site for automated imaging and analysis. We showed that this corresponds to the meter (Figure 6C).

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

[0227] Next, the usefulness of the platform for in situ measurements (e.g., force-displacement) is characterized. Tarryzation was performed and a proof-of-principle test was carried out to show that the platform can support long-term culture of heart tissue in a manner similar to Biow ire I. To show that the platform can support long-term culture of heart tissue in a manner similar to Biowire I, the electrical function responses of heart tissue cultured on the platform were tracked under electrical stimulation and treatment with clinically relevant drugs. To generate heart tissue, human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes co-cultured with human cardiac fibroblasts were seeded onto a hydrogel in a 96-well plate device . Tissue compaction was observed in the first 7 days (Figures 7A and 7B), dramatic changes were seen in the first 2 days, and stabilization continued after 3 days. Electrical field stimulation of the heart tissue was initiated on day 7 and continued for an additional 12 days. During this period, the electrical function of the tissue was improved as observed by a significantly increased maximum capture rate (Figures 7C and 7D). The plate facilitated non-destructive evaluation of tissue contraction movements, and the evaluation showed that the resting tension, active force, contraction, and relaxation times remained stable after 12 days of electrical stimulation (Figures 7E - 7G). Application of thapsigargin (50×10 −6 m), an inhibitor of sarcoplasmic reticulum function in cardiomyocytes, in the presence of electrical pacing (1.5 Hz) prolonged the contraction and relaxation times of the tissue (Figure 7H). Furthermore, despite a constant stimulation frequency, the frequency of tissue beating was halved upon drug application (Figure 7H) . -6

[0228] Discussion

[0229] In this study, a new method was developed for rapidly casting an array of elastic microstructures (e.g., wires) bridged on an array of microfabricated forms (e.g., microwells). By thermal bonding with a hot embosser, multiple parts of the plate were fused together, and carbon ​​​​​​Both the N electrode and the polymer wire were embedded in the platform to create a controlled microenvironment around the tissue. The function and response of the tissue could be manipulated by electrical stimulation and tracked by the embedded force sensors. In this multi-well plate format, each tissue was cultured and analyzed individually. Although the entire manufacturing process, from fabrication to storage, takes six days, the 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 384-well plates. Furthermore, multiple plates can be fabricated in parallel to achieve a higher production throughput compatible with industrial production processes. Up to 96 tissues can be cultured on one plate, and the experimental throughput is significantly higher than that of many traditional biofunctional chip platforms. Furthermore, only 100,000 cells are required for heart tissue formation. Currently, cell seeding remains the rate-limiting step in high-throughput tissue production. However, the open-well design allows the use of automated cell dispensers widely used in the pharmaceutical industry to scale up cell seeding and manipulation on this multi-well plate platform. By post-processing the materials after plate assembly, the deflection-force profile of this system could also be adjusted. Characterizing the mechanical properties of the elastic micro-wires allows for inferring the function readout of the tissue's contraction motion. The mechanical properties of the POMaC wires are reproducible across all wells of a 96-well plate, which means that ...

[0230] ... ... ... ... It shows the reliability of the casting method. The polymer wires are always placed at the same height. Therefore, in contrast to post-based platforms where the vertical position of the tissue is non-uniform, the height of the tissue is always constant and can be easily monitored. Non-destructive functional readout is important for long-term chronic drug research. In our application, we demonstrated the rapid casting of soft elastic micro-wires for heart tissue culture. However, more complex intertwined microstructures can be cast to enable the formation of more complex tissue structures in the same way. This platform also incorporates built-in electrodes that can continuously stimulate the tissue for at least 12 days to support tissue maturation. The highly conductive carbon electrodes minimize the voltage drop from the power source to the electrodes, thereby facilitating accurate control of the culture and test environment. The presence of the electrodes plays an important role in the rigorous investigation of drug responses. For example, when testing drugs in acute or chronic forms, the chronotropic effect of the drug can be separated from the inotropic effect by stimulating the tissue at physiologically relevant frequencies. Treatment with thapsigargin slowed down the contraction and relaxation of the tissue, and the pacing frequency decreased even though it was fixed. This means that the relaxation of the tissue was too slow to initiate the next depolarization until the relaxation of the previous contraction cycle ended, and therefore the frequency was reduced by half. Therefore, our platform can reproduce the known in vivo effects of this compound and suggests its application as a relevant in vivo screening platform. Here, scalable 3D heart tissue culture and contractility in a 96-well plate format are presented.

[0231] ​​​​​​​​​​​​​​​​A simple method for manufacturing an array of elastic micro-wires for reading was described. This arrayed tissue culture plate promotes micro-scale tissue culture with a minimum number of cells, promotes the maturation of cardiomyocytes with built-in carbon electrodes, and enables non-invasive functional evaluation for cardiotoxicity prediction. Based on ease of fabrication, high throughput, and the availability of highly faithful heart tissue, the featured platform provides a useful tool for evaluating the tissue response of pharmacological compounds.

[0232] Experimental Procedures

[0233] Preparation of POMaC prepolymer solution Built-in polymer sensors were prepared using POMaC. POMaC was synthesized by first preparing a prepolymer gel by polycondensation reaction. Citric acid (Caledon, A00019), maleic anhydride (Sigma, 63200), and 1,8-octanediol (Sigma, 03303) were combined in a 250 mL three-necked round-bottom flask with an equimolar amount of carboxylic acid (citric acid + maleic anhydride) relative to the alcohol (1,8-octanediol) functional group, and a ratio of citric acid to maleic anhydride of 1:4. The reaction solution was heated to form a melt polymerization solution, which was carried out at 150 °C with stirring at 200 rpm for 5 hours under a nitrogen stream. The resulting gel was dissolved in 1,4-dioxane (Sigma, D201863), followed by dropwise precipitation in deionized water to remove unreacted monomers or short-chain oligomers. Next, the aqueous phase was poured off, and the undissolved prepolymer was collected and concentrated. The purified prepolymer was in a ratio of 6:4 (w:w) with poly(ethylene glycol) dimethyl ether (PEGDM Mw~500) (Sig ma, 445886), and 5 (w / w)% photoinitiator, 2-hydroxy-1-[4 (hydroxyethoxy)phenyl]-2-methyl-1-propane (Sigma, 4108 96) and mixed. The resulting solution was stored at 4 °C until used for sensor casting .

[0234] Fabrication of a base plate with an embedded carbon electrode

[0235] A repeating pattern consisting of rectangular microwells (5 x 1 mm, L x W) and rectangular notches (1 x 0.5 m m, L x W) was designed in AutoCAD. The spacing (in all directions) between adjacent microwells was 9 mm to achieve compatibility with a conventional 96-well tissue culture plate . Using soft lithography, an SU-8 photoresist master mold was generated using a photomask obtained from a computer model, and a negative PDMS (Sylgard 184 silicone elastomer kit, Dow Coming, 01064291) master mold with a body height of 200 μm was fabricated . Briefly, the silicone elastomer base and hardener were vigorously mixed in a 5:1 (w:w) ratio in a cup, and the mixture was poured into the SU-8 master mold and cured at room temperature. Then, the PDMS master mold was fixed to the silicon wafer by plasma bonding or corona etching. To embed the electrodes in a polystyrene base plate, the carbon electrodes were first placed on the PDMS mold and fixed between the rectangular notches. The carbon electrodes were Ohio Carbon Blan k et or corona etching. To embed the electrodes in a polystyrene base plate, the carbon electrodes were first placed on the PDMS mold and fixed between the rectangular notches. The carbon electrodes were Ohio Carbon Blan k et or corona etching. To embed the electrodes in a polystyrene base plate, the carbon electrodes were first placed on the PDMS mold and fixed between the rectangular notches. The carbon electrodes were Ohio Carbon Blan k It was a custom computer numerical control (CNC) machined with k. The electrode was hydrostatic pressure pressed carbon / graphite (catalog number AR-14, Ohio Carb on Blank). Then, a blank polystyrene sheet and a silicon wa fer were placed on top and inserted into an EVG 520 Hot Embosser. The temperature and pressure were gradually increased to 190 °C and 3000 N, and the internal pressure was set to 5x10 -3 mbar . After the temperature and pressure returned to ambient conditions, the obtained carbon electrode-embedded patterned polystyrene base was removed from the PDMS mold. The edges were trimmed and it was ready for use.

[0236] Fabrication of PDMS mold and attachment of wires in one batch

[0237] To fabricate POMaC wires, a PDMS mold with 24 microchannels (cross-section: 100x100 μm) arranged in parallel was fabricated from the SU-8 master mold as described above. A silicone elastomer base and a curing agent were vigorously mixed in a cup at a ratio of 15: 1 (w:w), 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 microparticles. The PDMS mold was gently pressed onto the embossed polystyrene sheet after adjusting the positions for aligning the channel positions and directions and the positions of the polymer wires with the microwells . The POMaC prepolymer solution was injected into the PDMS mold with a syringe at the t of the PDMS mold Dispense at the upper edge and allow the prepolymer to perfuse by capillary action in the dark at room temperature for 48 hours. After perfusion, crosslink the polymer under UV light (1500 mJ / cm -2 ), and an elastomeric material network is generated. Remove the excess non-perfused POMaC polymer, and slowly peel the PDMS mold from the plate base, leaving the polymer wire attached to the polystyrene sheet.

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

[0239] Place the bottomless 96-well plate with the bottom facing up on a flat surface, and arrange the base plate with wires and electrodes facing it. Place pairs of polymer wires in the center of individual wells. Drop a single drop of acetone at the corners of the plate between the base and the plate to temporarily bond the plastic layer, and then completely bond the assembly by hot embossing. The bottom surface was gradually heated to 184 °C with a pressing force of 7,000 N and an internal pressure of 5 x 10 -3 mbar, and the top heating surface was maintained at 96 °C. After assembly processing

[0240] After embossing, fill the wells of the bonding plate with distilled water and leave it at a constant temperature (37 °C, 5% CO2

[0241] ) overnight. Then remove the water and air-dry the plate under aseptic conditions. After that, bake the plate at 70 °C for 6 days and perform gamma-ray sterilization. After sterilization, store the plate in the dark at 4 °C .

[0242] Characterization of the plate

[0243] To measure the Young's modulus of the bulk polymer, it was composed of a mechanical stretcher and a force transducer and the myograph device (Kent Scientific) was used to measure the strip of POMaC polymer cross-linked with UV light (1500 mJ cm -2 -2) (0.3 x 1 x 10 mm) in PBS solution. The Young's modulus was calculated using the slope of the obtained stress-strain curve. The polymer wire force / displacement curves in the wells were characterized using a commercial micro-mechanical tester, the MicroSquisher (CellScale). The test probe (diameter = 0.1524 mm) was modified with a customized chip (semi-elliptical, 0.5, 0.7, and 0.8 mm major axis to diameter ratio 4:1) made from an SU-8 master by soft lithography and attached to a tungsten probe provided using an adhesive (T-GSG-01 Titan Ge ll). The polymer wire was immersed in the medium for 7 days before testing. The test chip was placed in the center of the microwell, and then the polymer wire was displaced at the center using a probe tip that moved perpendicular to the long axis of the wire, and the corresponding force output (n ≥ 12) was recorded. The experimental data for each custom chip was fitted to a third-order polynomial to generate the standard force curve for each custom probe. To maintain consistency throughout the whole experiment, the force measured at a displacement of 100 mm was compared between the correct wires in each well. Since it was found that non-uniformity occurred in the outermost two columns due to the edge pressure, the outermost two columns were intentionally excluded from the measurement.

[0244] Voltage drop along the electrode

[0245] After assembling the plates, the carbon electrodes along the columns were connected to a voltage source (9V battery). A portable multimeter (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 difference in voltage between the carbon electrode and the voltage source by the voltage from the voltage source. Calculated by dividing the difference in voltage between the carbon electrode and the voltage source by the voltage from the voltage source.

[0246] Preparation of Hydrogel

[0247] Collagen hydrogel (500 μL) was prepared by combining high-concentration rat tail collagen (9.82 m g mL -1 153 μL, Corning), 15% (v / v) Matrigel (75 μ L, BD Biosciences), NaHCO₃ (50 μL at 2.3 mM, Sigm a), NaOH (5 μL at 10 mM, Sigma), deionized sterilized H₂O (167 μL) , and 1x M199 (50 μL, Sigma), resulting in a final collagen concentration of 3.0 mg mL -1 .

[0248] Preparation of Cardiomyocytes and Generation of Artificial Heart Tissue

[0249] Mainly ventricular cardiomyocytes (CM) were derived from the hiP SC line BJ1D using the monolayer differentiation protocol described above. On day 21 of differentiation, the cardiomyocytes were separated into single cells by a previously established method . Briefly, collagenase type II (Worthington, LS004205) at 1 mg mL -1 was added to the monolayer heart muscle cell culture and incubated overnight at room temperature. The isolated cells were mixed with cardiac fibroblasts (Lonza a, NHCF-V) at a cell number ratio of 10:1. The mixed cells were pelleted and 5.5 x10 7 cells / mL -1 were remixed with the collagen hydrogel. The cell-gel mixture was seeded at 2 mL per microwell. To maintain moisture until gelation was complete, droplets of medium were placed on the walls of the wells. The plate was incubated at 37 °C and 5% CO2 for 10 minutes and then medium was added. After seeding (day 0), the tissues were cultured for 7 days to be remodeled around the POMaC wire. To monitor the morphology of the tissues, bright-field images of the tissues were taken daily until day 7. By day 7, the tissues had contracted synchronously, and each time they contracted, the POMaC wire was bent. Batch imaging of tissue morphology

[0250]

[0251] SpectraMax MiniMax 300 Imaging Cytometer was used to image the tissues in the well plate and monitor the daily morphological changes of the tissues. Nine photos were taken in each well of the 96-well plate and automatically stitched together to show the overall image of the well. For contraction analysis by imaging in the 4’,6-diamidino-2-phenylindole (DAPI) channel and bright-field microscopy (4x objective lens), spontaneous contraction and pacing (1 Hz) contractions were imaged. The blue channel (10x objective lens, λ = 350 nm, λ = 470 nm, 100 frames s ex = 470 nm, 100 frames s em = 470 nm, 100 frames s -1 -1, 5 ms exposure) was used to record and analyze the contraction behavior of the tissues, and custom MATLAB code was used to output the data to track the displacement of the wire. The average tissue width and the width of the tissue (Tw), and the attachment sites to the polymer sensor were measured using bright-field images in the relaxed state. The polymer sensor ​The passive displacement was evaluated in the blue channel. The total and passive deflections (mm) of the polymer sensor were generated as described in the plate characterization section and converted to force (mN) using the calibration curve. The final readings of total and passive force were interpolated according to Tw and custom chip size. The active force was the difference between the total and passive forces. Custom MATLAB code was used to calculate passive force, active

[0252] Immunostaining and confocal microscopy

[0253] Tissues were fixed with 4% paraformaldehyde, permeabilized with 0.2% Tween20, and blocked with 10% fetal bovine serum. Immunostaining was performed using the following antibodies, mouse anti-α-actinin (Abcam; 1:200) and donkey anti- mouse-Alexa Fluor 488 (Abcam; 1:400). F-actin fibers were stained using Phalloidin-Alexa Fluor 660 (Invitrogen; 1:200). Confocal microscopy images were acquired using an Olympus FluoView 1000 laser scanning confocal microscope (Olympus Corporation).

[0254] Tissue maintenance and drug testing by electrical stimulation

[0255] Each well of the 96-well plate contained 300 μL of medium, which was exchanged every 24 h. With the built-in force sensor and electrodes, the tissue could be evaluated in situ for its contractile movement and drug The measured values necessary for the calculation were obtained. During the inspection, the pacing voltage was increased up to 110% of the excitation threshold. One polymer wire (blue channel, magnification 10x) was consistently imaged for comparison. After taking the baseline video, 0.3 μL of tupesigargin (50 x 10 m in DMSO) was added to the well using a gel-loading pipette tip. -3 After 15 minutes, after tupesigargin became effective, the video was taken again. The video was analyzed using custom MATLAB software.

[0256] Statistical analysis

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

[0258] Prior art documents

[0259] P. Liang, F. Lan, A. S. Lee, T. Gong, V. Sanchez-Freire, Y. Wang, S. Diecke, K. Sallam, J. W. Knowles, P. J. Wang, P. K. Nguyen, D. M. Bers, R. C. Robbins, J. C. Wu, Circulation 2013, 727, 1677.

[0260] Y. Zhao, A. Korolj, N. Feric, M. Radisic, Exper t Opin. Drug Saf. 2016, 75, 1455.

[0261] S. Morgan, P. Grootendorst, J. Lexchin, C. Cun ningham, D. Greyson, Health Policy 2011, 100 , 4.

[0262] G. Gintant, P. T. Sager, N. Stockbridge, Nat. R ev. Drug Discovery 2016, 75, 457.

[0263] M. Tiburcy, J. E. Hudson, P. Balfanz, S. Schlic k, T. Meyer, M. L. Chang Liao, E. Levent, F. Raad , S. Zeidler, E. Wingender, J. Riegler, M. Wang, J. D. Gold, I. Kehat, E. Wettwer, U. Ravens, P. Di erickx, L. W. van Laake, M. J. Goumans, S. Khadj eh, K. Toischer, G. Hasenfuss, L. A. Couture, A. Unger, W. A. Linke, T. Araki, B. Neel, G. Keller, L. Gepstein, J. C. Wu, W. H. Zimmermann, Circula tion 2017, 135, 1832.

[0264] S. S. Nunes, J. W. Miklas, J. Liu, R. Aschar - Sob bi, Y. Xiao, B. Zhang, J. Jiang, S. Masse, M. Gagl iardi, A., Hsieh, N., Thavandiran, M., A., Laflamme , Nanthakumar, K., Gross, G., J., Backx, P., H., Kel ler, M., Radisic, Nat. Methods 2013, 10, 781.

[0265] Shim, J., Grosberg, A., Nawroth, J., C., Parke, K., K r, Bertoldi, K., J. Biomech. 2012, 45, 832.

[0266] Lind, J., U., Busbee, T., A., Valentine, A., D., F., S., P asqualini, H., Yuan, M., Yadid, S., J., Park, A., Koti kian, A., P., Nesmith, A., P., Campbell, H., C., Vlassak, J., J k, Lewis, J., A., Parker, K., K., Nat. Mater. 2017, 16 , 303.

[0267] Schaaf, S., Shibamiya, A., Mewe, M., Eder, A., St ohr, M., N., Hirt, T., Rau, W., H., Zimmermann, L., Conr adi, T., Eschenhagen, A., Hansen, PLoS One 2011 , 6, e26397.

[0268] Boudou, T., Legant, W., R., Mu, A., Borochin, M., A., N . Thavandiran, M., Radisic, Zandstra, P., W., Epstein, J., A. Margulies, K., B., Chen, C., S., Tissue Eng., Part A 2012, 18, 910. g., Part A 2012, 18, 910.

[0269] M.W. Toepke, D.J. Beebe, Lab Chip 2006, 6, 14 84.

[0270] E. Berthier, E.W. Young, D. Beebe, Lab Chip 2 012, 12, 1224.

[0271] R.T. Tran, P. Thevenot, D. Gyawali, J.C. Chiao , L. Tang, J. Yang, Soft Matter 2010, 6, 2449.

[0272] B. Zhang, M. Montgomery, M.D. Chamberlain, S. Ogawa, A. Korolj, Pahnke, L.A. Wells, S. Masse, J. Kim, L. Reis, A. Momen, S.S. Nunes, A.R. Wheel er, K. Nanthakumar, G. Keller, M.V. Sefton, M.R adisic, Nat. Mater. 2016, 15, 669.

[0273] B. Zhang, B.F.L. Lai, R. Xie, L. Davenport Huy er, M. Montgomery, M. Radisic, Nat. Protoc. 201 8, 13, 1793.

[0274] S. Suomalainen, V. Kytoniemi, M. Mehto, Repro ducible Dispensing of Live Cells with th e Thermo Scientific Multidrop Combi Reag ent Dis-penser, Thermal Fisher Scientific , Vantaa, Finland 2009.

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[0276] L. Yang, M. H. Soonpaa, E. D. Adler, T. K. Roepke , S. J. Kattman, M. Kennedy, E. Henckaerts, K. Bo nham, G. W. Abbott, R. M. Linden, L. J. Field, G. M . Keller, Nature 2008, 453, 524.

Claims

1. A substrate, At least one pair of electrodes at least partially embedded in the substrate, the pair of electrodes having a first electrode and a second electrode separated by a gap, at least one pair of electrodes; 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 being configured to grow tissue from cells seeded therein, the pair of electrodes being configured to apply an electrical stimulus to the tissue, at least one well; At least two elastic sensing elements disposed across the well such that there is a gap between the sensing elements and the bottom of the well, the sensing elements being configured to (a) enable attachment of the tissue formed therebetween, thereby bridging the tissue above the bottom of the well, (b) deform in response to the contractile force exerted by the tissue on the sensing elements, thereby simulating the physiological environment specific to the tissue and / or enabling measurement of the contractile force, at least two elastic sensing elements; A device comprising.

2. The device according to claim 1, comprising two or more wells.

3. The device according to claim 2, comprising 6 wells, 12 wells, 24 wells, 48 wells, or 96 wells.

4. The device according to any one of claims 1 to 3, wherein the pair of electrodes is completely embedded in the substrate.

5. The electrodes include conductive carbon, gold, platinum, palladium, stainless steel, tin, tungsten, titanium, or a combination thereof, the device according to any one of claims 1 to 4.

6. The device according to claim 5, wherein the conductive carbon is non-porous.

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

9. The device according to any one of claims 1 to 8, comprising two or more pairs of electrodes, with at least one well disposed between each pair of electrodes.

10. The device according to claim 9, wherein each of the electrodes is parallel or substantially parallel to each other. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 。

11. The electrode pair is connected to a stimulation device, and the stimulation device is configured to apply an electrical stimulation between the electrode pair. The device according to any one of claims 1 to 10.

12. Comprising two or more electrode pairs, with two or more wells arranged between each electrode pair, and the stimulation device is configured to independently control the electrical stimulation between each electrode pair. The device according to claim 11.

13. The device according to any one of claims 1 to 12, comprising 2 to 25 sensing elements per well.

14. The device according to any one of claims 1 to 13, wherein the sensing element contains a polymer.

15. The polymer is polylactic acid, poly(lactic-co-glycolic) acid, poly(caprolactone), polyglycolide, polylactide, polyhydroxybutyrate, polyhydroxyalkanoate, 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) citrate) (POMaC), POMaC without citric acid, poly(ε-caprolactone), polyurethane, silk, nanomachined materials, copolymers, blend polymers, or at least one of combinations thereof. The device according to claim 14.

16. The device according to claim 15, wherein the polymer is POMaC.

17. The polymer has adjustable mechanical properties during the polymerization reaction. The device according to claim 14.

18. The sensing element is porous, thereby enabling the delivery of nutrients and growth factors to heart tissue. The device according to any one of claims 1 to 17.

19. The device according to any one of claims 1 to 18, wherein the sensing element has an elasticity of about 20 kPa to 0.5 MPa.

20. The device according to any one of claims 1 to 19, wherein the sensing element is in wire shape.

21. The well is configured to have a longitudinal axis. The device according to any one of claims 1 to 20.

22. The sensing element is in a direction perpendicular, parallel, or oblique to the longitudinal axis of the well. ​ ​ ​ ​ ​ ​ The device according to claim 21, wherein... **Claim 23** The device according to any one of claims 1 to 22, wherein the substrate contains a polymer. **Claim 24** The device according to claim 23, wherein the polymer is rigid. **Claim 25** The device according to claim 23, wherein the polymer is polystyrene or polycarbonate. . **Claim 26** The device according to any one of claims 1 to 25, wherein the cells are seeded in a hydrogel. 。 **Claim 27** The cells are selected from cardiomyocytes, fibroblasts, skeletal muscle cells, hepatocytes, renal cells, chondrocytes, skin cells, contractile cells, blood cells, immune system cells, germ cells, nerve 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, according to any one of claims 1 to 26. The device according to any one of claims 1 to 26, wherein...

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