A bioreactor for quantitative cardiac Anti-fibrotic drug assessment

Abstract

This disclosure related to a 3D biomimetic bioreactor platform. The 3D biomimetic bioreactor platform induces and quantifies cardiac fibrosis through synchronized multi-modal stimulation, maintaining native tissue preservation and inducing fibrosis from zero to any predefined ratio. The system combines electrical, mechanical, and biochemical cues to modulate tissue behavior, with real-time data acquisition on conduction velocity, tissue impedance, and strain-response curves. Advanced technologies, including vascularized microjet and 3D magnetic actuator, enable precise control over mechanical stretching and temporal modulation of strain. This innovative platform empowers researchers to study cardiac fibrosis mechanisms, assess treatment efficacy, and optimize therapeutic strategies for associated cardiovascular disorders, revolutionizing the field of cardiovascular research.

Description

A Bioreactor for Quantitative Cardiac Anti-Fibrotic Drug Assessment

[0001] The present disclosure pertains to a novel dynamic bioreactor system specifically designed for in vitro and ex-vivo investigation of anti-fibrotic therapeutic efficacy and the reprogramming of cardiac myofibroblast transdifferentiation into fibroblasts. More particularly, the system is tailored to study native intact cardiac fibrosis and its reversal mechanisms, a crucial aspect of artificial cardiovasclar research, with a focus on elucidating the underlying biology of heart failure and developing effective treatments for this debilitating condition.

[0002] Heart failure represents a significant global health concern, with interstitial fibrosis being a major contributing factor to its progression. To develop effective therapies, it is essential to understand the mechanisms underlying cardiomipathy signaling and evaluate potential anti-fibrotic treatments. In vitro or ex vivo models that accurately recapitulate the pathological conditions of cardiac fibrosis are crucial for advancing cardiomipathy research and drug development.

[0003] The present invention pertains to the field of cardiac disease quantitative modeling, where cardiac fibrosis is characterized by excessive accumulation of extracellular matrix components, by means of mechanibiology and mechanotransduction phenomena, leading to increased myocardial stiffness and compromised cardiac function. Conventional in vitro and ex-vivo models often suffer from limitations in replicating the complex microenvironmental and mechanical dynamics inherent to native three layers cardiac tissue, thereby restricting their utility in investigating fibrotic regression and assessing potential therapeutic agents. To overcome these deficiencies, the instant invention provides a novel bioreactor system that utilizes three layers native cardiac tissue slices, with a thickness exceeding 400 micrometers, thereby providing a more physio-pathologically relevant and reliable platform for modeling cardiac fibrosis and evaluating anti-fibrotic interventions.

[0004] US Patent No. 9,902,929 to Jinjin Wu et al., entitled "Bioreactor for three-dimensional tissue perfusion culture," discloses a bioreactor designed for perfusion culture of three-dimensional tissues and cells. The bioreactor comprises a reaction chamber, a reservoir, and a peristaltic pump connected via infusion tubes, with the reaction chamber including various components such as a cover body, box body, cut-off device, overflow plate, screen frame, static pressure tank, drainage chute, and drainage trench. This invention addresses issues related to cell death caused by non-uniform flow fields and uneven nutrient transfer in existing perfusion cultures, making it suitable for three-dimensional culture of tissues and cells and improving the quality of tissue engineering products.

[0005] US Patent No. 9,034,571 to Joel L. Berry et al., entitled "Three-dimensional, prevascularized, engineered tissue constructs, methods of making and methods of using the tissue constructs," discloses three-dimensional (3D) prevascularized engineered tissue constructs, 3D prevascularized engineered tissue models of cancer, and bioreactors and bioreactor arrays incorporating these tissue constructs. The patent also describes methods for creating these tissue constructs, using them for drug discovery, and other related applications.

[0006] China patent application No. 201811352918.7, entitled "Engineered artificial structure for generating vascular network as well as construction method and application of artificial structure," discloses an engineered artificial structure capable of generating a vascular network, along with its construction method and applications. The artificial structure comprises hydrogel microspheres, vascularized growth factor slow-release microspheres, vascularized cells, and a carrier material, which can be cultured in a bioreactor to obtain a vascular network or transplanted into the body to regenerate the vascular network. This technology has applications in tissue engineering, regenerative medicine, in-vitro physiological model construction, and drug research.

[0007] US Patent No. 10,683,476 to Kevin David Costa et al., entitled "Method and apparatus to prepare cardiac organoids in a bioreactor system," discloses a bioreactor system for preparing cardiac organoid chambers and subsequent testing. The bioreactor system includes a first vessel with a hollow interior and open top, a first cover with an opening, a cannula with a lumen extending from the open first end to the open second end, and a porous ring coupled to the cannula. A balloon catheter with an inflatable balloon is adapted to pass through the lumen of the cannula and can be axially adjusted to prepare the cardiac organoid chamber about the inflated balloon and porous ring.

[0008] While dynamic bioreactor systems have advanced, a need remains for improved models in in-vitro and ex-vivo anti-fibrotic drug assessments. Current over-simplified models inadequately replicate cardiac fibrosis, hindering accurate cardiomyopathy research and drug development. Thus, more sophisticated, physio-pathologically relevant models are required. This disclosure provides a novel invention addressing these limitations. Specifically, the invention provides a dynamic bioreactor system creating a more accurate cardiac fibrosis model. This model incorporates advanced features, precisely controlling mechanical stimulation, fluid flow, and extracellular matrix composition. This improved model facilitates enhanced drug efficacy and toxicity assessment, accelerating novel cardiomyopathy therapeutic strategies. It also provides a valuable tool for investigating complex cardiac fibrosis factors, advancing our understanding of this disease.

[0009] This summary is intended to provide an overview of the subject matter of this invention, and is not intended to identify essential elements or key elements of the subject matter, nor is intended to be used to determine the scope of the claimed implementations. The proper scope of this invention may be ascertained from the claims set forth below in view of the detailed description and the drawings.

[0010] In accordance with certain preferred embodiment of present invention, this disclosure proposes a bioreactor. The bioreactor is designed to control and quantify the viability, fibrosis induction, and fibrosis reversal of 3D multi-layer cardiac tissue through multi-modal functional stimulation. A first chamber within the bioreactor houses a first set of cardiac tissue and is configured to maintain its viability and functional integrity for predefined periods of time. A second chamber within the bioreactor houses a second set of cardiac tissue and is designed to induce varying degrees of fibrosis in this tissue over predefined periods, with fibrosis induction being precisely controlled using synchronized electromechanical and biochemical conditioning and predefined electromechanical stimulation protocols that allow for gradual fibrosis progression. The bioreactor includes a functional stimulus delivery system that provides mechanical, electrical, and biochemical stimuli to the cardiac tissue sets, including adjustable electrical signals to simulate normal and pathological conditions, mechanical forces and dynamic pressure to induce controlled strain and stress, and biochemical cues such as growth factors and pharmaceutical agents to facilitate fibrosis induction. The bioreactor also features a 3D magnetic actuator system that dynamically applies mechanical stress and strain to the cardiac tissue sets in a time-dependent manner, mimicking cyclic forces experienced by the myocardium in vivo and enabling controlled fibrosis induction with adjustable protocols for various fibrosis levels. A fluid microjet system within the bioreactor vascularizes the tissue, ensuring adequate oxygenation and nutrient supply while maintaining a stable environment during fibrosis induction and reversal. The bioreactor incorporates integrated sensors, data acquisition, and imaging systems for real-time monitoring of tissue mechanics, electrophysiology, and biochemical markers, including conduction velocity, excitability threshold, and optical mapping of voltage and ion concentration. A software component analyzes data from these systems, generating quantitative curves to assess the cardiac tissue functionally using advanced bidomain modeling and machine-learning algorithms to predict fibrosis progression, including real-time curve fitting for fibrosis induction and reversal. The bioreactor induces controlled fibrosis in the cardiac tissue through synchronized multi-modal stimulation, including electrical, mechanical, and biochemical cues, and accommodates biochemical and immunological agents to study the impact of anti-fibrotic drugs on fibrosis, using real-time monitoring and curve fitting to assess drug effectiveness in reversing fibrosis.

[0011] In one another aspect, the present disclosure is further directed to a real-time feedback method for bioreactors. The real-time feedback method for controlling and reversing fibrosis in a bioreactor involves continuously monitoring the degree of fibrosis in a tissue sample using parameters such as conduction velocity, excitation threshold, and tissue compliance to quantitatively assess fibrosis progression. This monitoring includes tracking changes in fibrosis through real-time curve fitting of electrophysiological and mechanical data to accurately determine the extent of fibrosis. The method then identifies the quantitative stage of fibrosis based on the monitored degree of fibrosis, categorizing it into one of several stages including early fibrosis, moderate fibrosis, advanced fibrosis, and scar tissue. In response to the identified stage of fibrosis, the method adjusts at least one of the following: the electrical functional stimulation intensity, which is modified based on the stage of fibrosis to stimulate cardiac tissue function, promote electrical conductivity, and prevent excessive fibrosis accumulation, with real-time feedback provided by tissue impedance and conduction velocity measurements. The mechanical loading intensity is also adjusted based on the stage of fibrosis to simulate physiological cardiac strain and promote normal tissue alignment, enabling graded induction of fibrosis. Additionally, the method adjusts drug delivery for antifibrotic treatment, administering the drug at a rate and concentration determined by the stage of fibrosis, with the capability to deliver TGF-β inhibitors, anti-inflammatory agents, and collagen-modulating drugs at optimized doses for effective fibrosis reversal. The method continuously checks for changes in functional markers, such as conduction velocity, electrical impedance, tissue stiffness, and strain-stress response curves, and adjusts the balance between electrical and mechanical functional stimulation accordingly. Finally, the method makes synchronous continuous adjustments to the electrical functional stimulation intensity, mechanical loading intensity, and drug delivery to maintain the tissue in an optimal state for fibrosis reversal, ensuring a graded reversal of fibrosis from nearly scar tissue back to native tissue phenotype.

[0012] In a yet another aspect, the disclosure is further directed to a novel multi-modal stimulation method. The multi-modal functional stimulation method for inducing and reversing fibrosis in in-vitro and ex-vivo native cardiac tissue involves providing at least one 3D functional electrical stimulation component that is designed to induce stimuli to multi-layer cardiac tissue by means of a plurality of electrodes penetrating into the tissue. This electrical stimulation component utilizes biphasic and monophasic pulses with a predefined stimulation protocol, enabling accurate control of electrical signals for fibrosis induction across a range of stages, from early fibrosis to advanced fibrosis. The method also involves providing a microjet apparatus that constructs micro-channels in the tissue for the delivery of nutrients, oxygenation, and biochemical agents, such as TGF-β and angiotensin II, to induce controlled fibrosis, ensuring long-term tissue viability during both induction and reversal of fibrosis. Additionally, the method provides at least one functional mechanical stimulation component, including 3D preloading with static and dynamic pressure, baseline strain, and dynamic increases in strain during active cycles, which simulates myocardial mechanical forces and has adjustable parameters to induce fibrosis and simulate pathological conditions from early fibrosis to advanced fibrosis. The method further involves providing at least one biochemical environment component selected from a group consisting of exogenous fibrotic agents, including TGF-β, angiotensin II, and macrophage, which are applied in varying concentrations to induce controlled levels of fibrosis at different stages. The multi-modal functional stimulation method is configured to induce a quantitative range of fibrosis levels, including preservation of native myocardial phenotype, induction of early fibrotic transition, generation of a balanced native and fibrotic tissue model, and induction of advanced fibrotic state. Furthermore, the system is configured to facilitate drug screening and evaluate anti-fibrotic therapies using real-time data-driven curve fitting techniques, allowing for precise monitoring and assessment of the effects of various treatments on cardiac tissue fibrosis.

[0013] In another aspect, the disclosure is further directed to a method for quantitatively controlling reprogramming and reversing fibrosis in cardiac tissue. The method for quantitatively controlling reprogramming and reversing fibrosis in cardiac tissue involves inducing fibrosis in cardiac tissue within a bioreactor system using synchronized 3D mechano-electrical stimulation protocols, wherein fibrosis is initiated in the tissue across a gradual range from early fibrosis to advanced fibrosis. This method also includes quantifying fibrosis at each stage of fibrosis using real-time curve fitting of electrophysiological and mechanical data, including conductivity changes, tissue compliance, and strain-stress response curves, and correlating each stage of fibrosis to a quantitative fibrosis fraction function and mechanical properties of the tissue, including contractility and stiffness. Additionally, the method involves administering specific antifibrotic drugs to induce mechanical modulation of tissue stiffness and contractility, wherein the administration of the antifibrotic drugs is mediated by specific signaling pathways that influence myofibroblast-to-fibroblast transition, fibrotic ECM deposition, and mechanotransduction. The method further includes monitoring the reprogramming of tissue layers fibrosis within the tri-layer cardiac tissue, including layer-specific reprogramming and differentiation of cardiac cells under mechanical stimulation, and modulating each layer's stiffness based on drug treatments. A mesh network of fluid microjet penetrated channels is also provided within the tri-layer cardiac tissue to deliver TGF-β and other fibrotic agents directly into the tissue, wherein the mesh network mimics natural ECM structures and exerts localized strain and stress to enhance tissue mechanical integrity and fibrosis induction. The method continuously tracks real-time mechanical responses and electrophysiological markers, generating real-time quantitative curves, and adjusts treatment protocols for fibrosis reversal based on the stage of fibrosis, including changes in drug delivery, electrical stimulation, and mechanical loading. Furthermore, the method modulates and synchronizes multi-layered cardiac tissue by applying coordinated three-dimensional mechanical, electrical stimuli, and biochemical cues within a bioreactor, wherein the strain and stress profiles of each individual tissue layer are independently regulated to achieve antifibrotic remodeling across the cardiac tissue. The real-time curve fitting analysis is utilized to predict the effects of antifibrotic drug treatment, optimizing the dosage and timing for fibrosis resolution by targeting the TGF-β pathway, kinase signaling, conexine 43, and mechanotransduction pathways. One specified drug used is pirfenidone, which regulates TGF-β signaling to modulate the activation of myofibroblasts and the deposition of ECM, and inhibits fibrosis-associated kinases, such as conexine 43, MAPK, and mTOR, to reduce mechanical stiffness and contraction of the tissue. This specific antifibrotic drug treatment is used in conjunction with the previously described method for quantitatively controlling reprogramming and reversing fibrosis in cardiac tissue, allowing for a targeted approach to fibrosis resolution by modulating the TGF-β pathway and other key signaling pathways involved in fibrosis.

[0014] Furthemore, the present invention relates to a bioreactor for assessment of physiopathological conditions of a 3D thri-layer heart tissue. The bioreactor for assessing physiopathological conditions of a 3D tri-layer heart tissue and inducing quantitative fibrosis development, as well as measuring mechanical and electrical parameters of the heart tissue, comprises several key components. A sealed tank is used to hold a liquid solution, which is an essential part of the bioreactor's functionality. The tank has a first gas valve attached to it, allowing for the regulation of gas flow into the tank. At least one pH sensor is placed inside the liquid solution to monitor its acidity levels. Additionally, at least two pressurized compressed gas capsules are connected to the tank by means of second gas valves, providing oxygen and carbon dioxide as needed. A cylinder and piston set driven by a first linear actuator is also connected to the tank, containing the liquid solution and enabling precise control over its movement. The bioreactor includes a gas pressure sensor attached to the tank to measure the pressure of the gas above the liquid. Multiple chambers are connected to the tank, where the heart tissue is placed for assessment and experimentation. Each chamber has at least one liquid pressure sensor connected to it to monitor the pressure of the liquid within. Furthermore, each chamber has a liquid valve connected to its output, which in turn has a flow sensor attached to control and measure the flow of the liquid. A multi-input multi-output closed-loop controller is utilized to emulate mechanical conditions on the heart tissue by controlling various parameters such as shear stress, static pressure, and flow rate of the liquid solution. This controller uses feedback from the first linear actuator, gas pressure sensor, first and second gas valves, liquid pressure sensors, liquid valves, and flow sensors to achieve precise control over the bioreactor's environment. The bioreactor also features mechanical actuator apparatus embedded inside each chamber, comprising at least two force applicators that apply forces to the heart tissue using magnetic coils and neodymium magnets. A measurement and control system is integrated into the mechanical actuator apparatus to measure and control mechanobiology parameters related to the heart tissue. This system includes at least one high-speed camera for capturing images of visual indicia associated with the force applicators, allowing for real-time adjustments to be made based on image processing and feedback from the cameras. The position of the force applicators is determined through this process, enabling precise control over the forces applied to the heart tissue. A processor is used to statistically determine the stiffness of the heart tissue based on calculated changes in length and forces applied by the mechanical actuator apparatus, taking into account user input and feedback from the measurement system over a specific time period. The bioreactor also includes a high-pressure fluid microjet apparatus for injecting liquid solutions or medicinal substances directly into the heart tissue. This apparatus consists of a second cylinder and piston set driven by a second linear actuator, generating a high-pressure jet of fluid that can be directed at the heart tissue using a fluid delivery system. The apparatus creates an artificial channel for circulation of the fluid and additional substances through a three-way valve and tube, with a syringe pump connected to the three-way valve enabling precise control over the delivery of these substances. Finally, an electrical measuring and stimulation apparatus is included in the bioreactor for measuring electrical impedance and conduction velocity of the heart tissue, as well as stimulating the heart tissue as needed.

[0015] The inventive and non-obvious bioreactor system of the present invention provides a unique and comprehensive platform for studying heart tissue function and disease, enabling researchers to investigate the complex interactions between mechanical, electrical, and biochemical parameters that regulate heart tissue behavior. The system's ability to simulate physiological and pathological conditions, induce fibrosis levels, and measure mechanical and electrical parameters makes it an invaluable tool for understanding heart tissue mechanobiology and developing novel therapeutic strategies for cardiovascular diseases. The bioreactor system's capabilities include: simulating physiological and pathological conditions, such as hypertension, hypoxia, and inflammation; inducing fibrosis levels in the heart tissue; measuring mechanical parameters, such as stiffness, shear stress, and flow rate; measuring electrical parameters, such as impedance and conduction velocity; stimulating the heart tissue with electrical signals; delivering medicinal substances, such as drugs, cells, or growth factors, directly into the heart tissue using the high-pressure fluid microjet apparatus.

[0016] The present invention has numerous applications in cardiovascular research, including: studying heart tissue mechanobiology and disease mechanisms; developing novel therapeutic strategies for cardiovascular diseases; testing the efficacy of medicinal substances and devices on heart tissue; investigating the effects of mechanical and electrical stimulation on heart tissue function and disease.

[0017] The current state of the art in cardiac tissue modeling is plagued by significant shortcomings, particularly with regards to the accuracy and reliability of stem cell-derived tissue models. Despite advances in stem cell technology and tissue engineering, these models fail to recapitulate the complex biology of native cardiac tissue, leading to inadequate understanding of signaling pathways, cellular density, extracellular matrix (ECM) composition, connexin distribution, contractility, stiffness, and mitochondrial function. Limitations of Stem Cell-Derived Tissue Models are as follow:

[0018] Inadequate Signaling Pathways: Stem cell-derived tissue models often exhibit disrupted or insufficient activation of canonical mechanotransduction pathways, including RhoA / ROCK signaling, focal adhesion kinase (FAK), and Integrin signaling. Furthermore, non-canonical pathways such as Wnt / β-catenin and TGF-β signaling are also poorly represented in these models, leading to a lack of understanding of their role in cardiac tissue biology.

[0019] Insufficient Cellular Density: The cellular density of stem cell-derived tissue models is often lower than that of native cardiac tissue, resulting in inadequate representation of the complex interactions between cardiomyocytes, fibroblasts, and endothelial cells.

[0020] Inadequate ECM Composition: The ECM composition of stem cell-derived tissue models is often simplistic and fails to recapitulate the complex architecture of native cardiac tissue, leading to impaired cell-matrix signaling and non-physiological fibrosis-like responses.

[0021] Reduced Connexin Density: The connexin density in stem cell-derived tissue models is often reduced, resulting in asynchronous electrical activity and poor conduction properties, which are critical for understanding cardiac arrhythmias and electrophysiology.

[0022] Impaired Contractility: The contractile force generated by stem cell-derived tissue models is often substantially lower than that of native cardiac tissue, due to the lack of mature sarcomeres, improper alignment of cardiomyocytes, and reduced mechanical coupling between cells.

[0023] Inadequate Tissue Stiffness: The stiffness of stem cell-derived tissue models can be artificially high or low, failing to mimic the natural progression of fibrosis in native tissues, which is critical for understanding disease mechanisms and developing effective therapies.

[0024] Mitochondrial Dysfunction: The mitochondrial density and function in stem cell-derived tissue models are often impaired, leading to inadequate energy production, redox imbalance, and increased susceptibility to stress-induced fibrosis.

[0025] In light of these limitations, there is a pressing need for improved cardiac tissue models that can accurately recapitulate the complex biology of native cardiac tissue and maturity. Such models would enable researchers to better understand the underlying mechanisms of cardiac disease, develop more effective therapies, and improve patient outcomes.

[0026] This invention relates to a 3D three layers biomimetic bioreactor platform engineered to induce and quantify cardiac fibrosis through synchronized multi‐modal stimulation. The system is designed for Native Tissue Preservation and Fibrotic Induction. So, the bioreactor can maintain a single cardiac tissue under physiologically relevant preload conditions for up to several days and progressively induces and characterizes fibrosis (from 0% to 100% in 25% increments) using combined electrical, mechanical, and biochemical cues.

[0027] The disclosed system encompasses a comprehensive platform for quantifying fibrosis regression through precise manipulation of electrical, mechanical, and biochemical factors. Electrical stimulation employs carefully engineered waveforms, amplitude, frequency, and electrode configurations to modulate tissue behavior. Additionally, mechanical stimulation utilizes pneumatic and vacuum pressure to apply anisotropic strain, thereby establishing a dynamic 3D preloading environment. To modulate fibrosis progression, dose escalation strategies are implemented using agents such as TGF-β and angiotensin II alongside BNP and OXYTOCIN and antifibrotic drugs. The system concurrently acquires real-time data on conduction velocity, tissue impedance, and strain-response curves to establish a comprehensive understanding of tissue response. Computational validation is further achieved through bidomain modeling and ML-driven nonlinear regression models, enabling accurate prediction and quantification of fibrosis severity.

[0028] The disclosed bioreactor system embodies an innovative platform for quantifying fibrosis progression in cardiac tissue cultures. Precise manipulation of electrical, mechanical, and biochemical factors empowers researchers to induce controlled fibrosis and assess treatment efficacy. The bioreactor architecture comprises multiple chambers housing cardiac tissue strips. These chambers are equipped with stimulus delivery systems to provide tailored mechanical, electrical, and biochemical stimuli. Advanced vascularized microjet technology ensures adequate oxygenation and nutrient supply to thick-sectioned tissues, sustaining viability and promoting physiologically relevant responses.

[0029] To replicate the complex forces experienced by the myocardium in vivo, the bioreactor system employs a 3D magnetic actuator. This feature enables precise mechanical stretching, temporal modulation of strain, and quantitative fibrosis induction. Real-time monitoring of tissue mechanics, electrophysiology, and biochemical markers is facilitated through integrated sensors and imaging systems. The accompanying software analyzes these data, generating quantitative curves to assess treatment efficacy and identify potential biomarkers.

[0030] The system's capacity to accommodate immune cells fosters research on the role of inflammation in fibrosis. The inclusion of immune cells allows researchers to investigate the impact of anti-fibrotic drugs on immune cell populations and the resolution of fibrosis. The invention can implement novel biomimetic and bio-fibrotic stimulating protocols for inducing and characterizing cardiac tissue remodeling in a three-dimensional (3D) bioreactor. Specifically, the disclosed protocols are designed to precisely control the transition from native preserved tissue to fully fibrotic tissue by applying a combination of electrical, mechanical, and biochemical stimuli. The protocols further incorporate frequency-dependent and time-dependent stimulation protocols, which enable real-time, quantitative analysis of dynamic parameters through curve fitting techniques. A detailed description of these protocols is provided below, accompanied by illustrative tables, which provide a comprehensive understanding of the invention and its various aspects.

[0031] The bioreactor system's comprehensive capabilities transcend traditional approaches, empowering researchers to gain profound insights into cardiac fibrosis mechanisms and optimize therapeutic strategies for associated cardiovascular disorders.

[0032] OVERVIEW OF TISSUE COMPOSITION PROTOCOLS

[0033] Below Multi-Modal Mechano-Stimulation Protocols for Cardiac Tissue is a structured multi-modal stimulation protocols in a 3D biomimetic bioreactor for inducing and characterizing myocardial fibrosis in 25% increments—from 100% native tissue to 25% native tissue (i.e., 0%, 25%, 50%, and 75% fibrosis). These protocols combine synchronized electrical, mechanical, and biochemical stimuli with real-time data acquisition and AI-driven computational validation. This invention provides a reproducible, high-precision platform for inducing myocardial fibrosis in a 3D biomimetic bioreactor. By integrating multi-modal stimulation—electrical, mechanical, and biochemical—fibrosis is generated in defined increments (0%, 25%, 50%, 75% fibrotic states corresponding to 100%, 75%, 50%, and 25% native tissue, respectively). Real-time electrophysiological and biomechanical data (e.g., conduction velocity, impedance, strain-response curves) are continuously captured and processed via advanced bidomain modeling and machine-learning algorithms, ensuring predictable and quantifiable fibrosis progression.

[0034] In the present invention an improved method for cultivating Living Cardiac Tissue (LCT) cultures has been implemented, wherein mechanical cues are integrated during the cardiac tissue processing step. Specifically, the application of specialized mechanical strain stress enhances tissue preservation and induces quantitative fibrosis, thereby achieving a dual effect. This innovation enables the long-term cultivation of LCT cultures for periods of up to several days, wherein a first set of tissues remains preserved in their native state while a second set of tissues is progressively remodeled into fibrotic tissue. The resultant capability for parallel comparative studies under controlled experimental conditions provides valuable insights into the transition from healthy to fibrotic myocardium, thereby facilitating the understanding and analysis of cardiac tissue pathology.

[0035] The present invention further comprises a 3D magnetic actuator system for dynamically applying mechanical stress and strain to a biomimetic interface in a time-dependent manner, thereby enhancing the fidelity of said interface. The actuator system is configured to provide: (a) precise mechanical stretching, wherein the cyclic mechanical forces experienced by the myocardium in vivo are mimicked with high accuracy; (b) temporal modulation, wherein the strain, stress, and stretch protocols are dynamically adjusted over the culture period to simulate physiological or pathological conditions; and (c) quantitative fibrosis induction, wherein a graded induction of fibrotic tissue is enabled by applying predetermined mechanical loads, thereby simulating pathological remodeling processes. The actuator system allows for the precise control of mechanical stimuli, including but not limited to, frequency, amplitude, duration, and waveform, thereby enabling the recreation of complex in vivo-like mechanical environments for the study of cardiac tissue biology and disease modeling.

[0036] The present invention provides a 3D mechanical stimulation system that not only maintains the viability and functional integrity of native tissue for extended periods of time, but also enables the controlled induction of fibrosis in other tissue preparations. The system further comprises means for continuous monitoring and real-time electrophysiological assessments, thereby facilitating the collection of detailed physiological data and bridging the gap between traditional 3D cell cultures and the complex in vivo cardiac environment. This integrated approach allows for the simultaneous preservation of native tissue architecture and function, while also enabling the study of pathological processes such as fibrosis, under controlled and physiologically relevant conditions. The system thus provides a unique platform for investigating cardiac tissue biology and disease mechanisms, and enables the development of novel therapeutic strategies for the treatment of cardiovascular disorders.

[0037] MODALITIES

[0038] A. Stimulation for viability and Fibrotic Induction:The present invention utilizes a combined experimental-computational approach to validate and optimize the electrical and mechanical stimulation protocols. The methodology for simulating electrophysiological responses of native and fibrotic cardiac tissues is described herein. A simulation framework is provided, which enables prediction of tissue-level responses to applied electrical and mechanical stimuli, quantification of dynamics, and analysis of fibrotic progression through mathematical modeling and curve fitting techniques. This framework allows for the optimization of stimulation protocols to induce desired electrophysiological responses and fibrotic transformations in cardiac tissues.

[0039] B. Computational Framework:The present invention comprises a simulation model that includes two primary systems, one of which is a cardiac tissue culture array. The cardiac tissue culture array is modeled using a 2D Bidomain Model, specifically a Cardiac tissue Wedge Preparation, which simulates conduction and action potential (AP) propagation in cardiac tissue sections having dimensions of approximately a few centimeters. The model incorporates three distinct layers to accurately represent cardiac tissue transmural gradients, namely: an Endocardial (Endo) layer of approximately 0.1 cm; a Midmyocardial (M) layer of approximately 0.6 cm; and an Epicardial (Epi) layer of approximately 0.25 cm. To simulate pacing stimuli, a few millisecond pulses are transmembranously applied at pacing cycle lengths (PCLs) of few hundreds to few thousands millisecond, with the amplitude set to twice the diastolic threshold. Furthermore, a unipolar recording electrode is positioned above the epicardial surface to capture electrocardiogram (ECG) signals that resemble left precordial leads, thereby allowing for the simulation of clinically relevant cardiac electrophysiological responses.

[0040] C. Incorporation of Fibrotic Tissue Dynamics:The present invention incorporates a modeling framework that simulates the progressive effects of fibrosis on cardiac tissue. Specifically, the model comprises five distinct tissue conditions, which are designed to represent various stages of fibrotic progression. These conditions include: a fully preserved physiological tissue state, comprising 100% native tissue; a transitional state, comprising 75% native tissue and 25% early myofibroblast / fibrotic transition tissue; a mixed state, comprising an equal proportion of native tissue and fibrotic tissue (50%:50%); an advanced fibrotic state, comprising 25% native tissue and 75% fibrotic tissue; and a fully fibrotic pathological state, comprising 100% fibrotic tissue. By simulating these distinct tissue conditions, the model enables the investigation of the progressive effects of fibrosis on cardiac electrophysiology and the evaluation of potential therapeutic interventions.

[0041] D. Electrical Stimulation Protocols and Parameter Adjustments:The present invention utilizes microelectrode arrays (MEA) to apply electrical stimulation to the tissue preparation, wherein biphasic pulse waveforms are precisely controlled to induce specific electrophysiological responses. In particular, the electrical stimulation protocols are designed to replicate: tissue fibrillation (TF)-like arrhythmogenic stress, thereby simulating complex arrhythmic conditions; rate-dependent conduction abnormalities, allowing for the investigation of rate-induced electrophysiological changes; and fibrotic-dependent electrophysiological changes, enabling the study of fibrosis-related alterations in cardiac conduction. The electrical stimulation protocols are implemented using a carefully optimized electrode placement and application strategy, comprising: bipolar stimulating electrodes positioned on endocardial surfaces for simulations of sinus rhythm, thereby ensuring accurate representation of physiological conditions; time-dependent pacing cycles for progressive fibrosis modeling, allowing for the investigation of dynamic changes in cardiac electrophysiology; and current amplitude adjustments based on impedance measurements performed before and after fibrosis induction, thereby ensuring optimal stimulation parameters and minimizing potential artifacts. By utilizing this controlled electrical stimulation approach, the present invention enables the precise investigation of complex electrophysiological phenomena, including the effects of fibrosis on cardiac conduction and arrhythmogenesis.

[0042] E. Mechanical Stimulation Protocols and Parameter Adjustments:The present invention utilizes mechanical actuation systems to apply mechanical stimulation to the tissue preparation, wherein controlled mechanical forces are precisely regulated to induce specific mechanobiological responses. In particular, the mechanical stimulation protocols are designed to replicate: tissue fibrillation (TF)-like mechano-genic stress, thereby simulating complex mechanical conditions; strain-dependent deformation abnormalities, allowing for the investigation of strain-induced mechanobiological changes; and fibrotic-dependent mechanobiological changes, enabling the study of fibrosis-related alterations in cardiac mechanics. The mechanical stimulation protocols are implemented using a carefully optimized actuator placement and application strategy, comprising: uniaxial or biaxial stretching systems positioned on epicardial or endocardial surfaces for simulations of physiological loading conditions; time-dependent cyclic loading protocols for progressive fibrosis modeling, allowing for the investigation of dynamic changes in cardiac mechanobiology; and force amplitude adjustments based on stiffness measurements performed before and after fibrosis induction, thereby ensuring optimal stimulation parameters and minimizing potential artifacts. By utilizing this controlled mechanical stimulation approach, the present invention enables the precise investigation of complex mechanobiological phenomena, including the effects of fibrosis on cardiac mechanics and remodeling.

[0043] F. Rate-Dependent Variability and Dynamic Adaptation:The present invention utilizes a method for investigating time-dependent changes in fibrotic remodeling, wherein rate-dependent stimulation is applied at varying intervals to simulate physiological conditions. Specifically, the rate-dependent stimulation protocol comprises: a steady-state (SS) interval, wherein the stimulation frequency is maintained at a constant level to assess the steady-state response of the tissue; a diastolic interval (DI), wherein the stimulation frequency is varied to mimic the natural diastolic period and evaluate the effects on cardiac function; and a cardiac tissue-cardiac tissue interval (V1V2), wherein the stimulation frequency is adjusted to simulate the natural heartbeat and investigate the effects on cardiac tissue function. By analyzing the mathematical fitting characteristics of curves, the present invention enables the prediction and determination of various physiological parameters, including: conduction velocity (CV), which is a critical determinant of cardiac function and arrhythmia susceptibility; mechanical response time, which reflects the contractile properties of the cardiac tissue; Effective refractory period (ERP) and functional refractory period (FRP) modifications, which are essential for understanding arrhythmia mechanisms and conduction abnormalities; myofibroblast-to-cardiomyocyte ratios, which provide insight into the cellular composition and fibrotic remodeling of the cardiac tissue; and correlations between fibrosis severity, conduction slowing, and APD heterogeneity, which are vital for understanding the underlying mechanisms of arrhythmogenesis and cardiac dysfunction.

[0044] The present invention provides a comprehensive framework for investigating the complex relationships between rate-dependent stimulation, fibrotic remodeling, and cardiac function, thereby enabling the development of novel therapeutic strategies for the treatment of cardiovascular diseases.

[0045] G. 3D Anisotropy and Biomechanical Integration:The present invention utilizes a three-dimensional (3D) fiber orientation in the vertical cross-section of cardiac tissue, thereby simulating the mechanical anisotropy that occurs in real-life physiological conditions. Specifically, the 3D fiber orientation has been utilized to mimic the natural arrangement of cardiac fibers, allowing for a more accurate representation of the mechanical properties of the heart. The boundary conditions and tissue conductivity properties are established in accordance with the standard bidomain model, which provides a well-established framework for modeling the electrical activity of cardiac tissue. By utilizing this model, the present invention ensures a high degree of accuracy and consistency in simulating the electrical and mechanical behavior of cardiac tissue. Furthermore, excitation imaging techniques can be employed to visualize the propagation of membrane potential in both normal and fibrotic tissues. This allows for a detailed examination of the effects of fibrosis on electrical conduction and provides valuable insights into the underlying mechanisms of cardiac function and dysfunction.

[0046] 3D ELECTRO-MECHANICAL PROTOCOLS FOR FIBROSIS MODELING

[0047] The present invention implements an integrated 3D framework for modeling fibrosis and analyzing electrophysiological properties, comprising a combination of experimental electrical, mechanical, and biochemical stimulation protocols, as well as a computational simulation approach. This comprehensive system provides a platform for innovative fibrosis modeling and electrophysiological analysis.

[0048] A. Electrical Stimulation Protocol:The electrical stimulation protocol of the present invention utilizes microelectrode arrays (MEAs) integrated into a bioreactor, wherein the electrodes are positioned to facilitate uniform field stimulation across tissue wedge. The protocol employs biphasic pulse waveforms with variable parameters, including Amplitude, Current, Pulse Width and Frequency.The electrical stimulation is applied in a pre- and post-mechanical conditioning regimen, with frequency-dependent modulation. For example, an atrial fibrillation (AF)-like stimulation protocol may be implemented, comprising rapid pacing cycles to induce arrhythmogenic stress.

[0049] B. Mechanical Stimulation Protocol:The mechanical stimulation protocol of the present invention employs a programmable uniaxial and biaxial stretch mechanism, providing mechanical cues with variable parameters, including Stretch Percentage, Stress / Force, Cycle Duration and Application Timing.These mechanical stresses are designed to mimic in vivo hemodynamic forces and are varied in a time-dependent manner to simulate diastolic intervals and cardiac tissue stress.

[0050] C. Biochemical Stimulation Protocol:The biochemical stimulation protocol of the present invention involves the administration of biochemical inducers, such as TGF-β or angiotensin II, in controlled concentrations to promote the transition from a native phenotype to myofibroblast activation and fibrosis. The dosing regimen is coordinated with the mechanical and electrical protocols to achieve a real-time dynamic response.

[0051] FREQUENCY-DEPENDENT AND TIME-DEPENDENT PROTOCOLS

[0052] The present invention embodies a novel approach to tissue stimulation, wherein frequency-dependent and time-dependent protocols are employed. This approach comprises applying stimulation protocols at multiple time intervals, including:

[0053] Pre-conditioning phase:An initial stage characterized by baseline measurement acquisition and the application of an inaugural stimulation.

[0054] Stimulation phase:A subsequent stage marked by repeated cycles of combined mechanical, electrical, and biochemical stimuli.

[0055] Post-conditioning phase:A final stage wherein reassessment of the tissue response is conducted, accompanied by dynamic curve fitting.

[0056] Throughout the protocol, measurements are recorded in a continuous manner. Subsequently, mathematical curve fitting techniques are utilized to extract distinctive signatures corresponding to each stimulation rate and time interval. This enables the prediction of fibrosis severity and the estimation of the myofibroblast-to-cardiomyocyte ratio, thereby facilitating a more accurate assessment of tissue composition and pathology.

[0057] DETAILED PROTOCOL DESCRIPTIONS

[0058] The present invention utilizes a novel multi-modal stimulation protocol for the in vitro study of cardiac tissues study, encompassing a range of native tissue and fibrosis compositions. These protocols are designed to mimic physiological conditions and induce controlled levels of fibrosis, thereby providing a versatile tool for cardiovascular research, drug discovery, and tissue engineering applications. The multi-modal stimulation protocols comprise electrical, mechanical, and biochemical components, which are tailored to specific objectives, including:

[0059] Preservation of Native Myocardial Phenotype:Maintenance of fully preserved myocardial characteristics over extended culture periods (up to several days) while ensuring optimal electrophysiological and mechanical properties.

[0060] Induction of Early Fibrotic Transition:Introduction of an early fibrotic state characterized by a predominantly native myocardial phenotype with minimal fibrosis.

[0061] Generation of Balanced Native and Fibrotic Tissue Model:Creation of a model with equal proportions of native and fibrotic tissue for direct comparative analysis.

[0062] Induction of Advanced Fibrotic State:Establishment of an advanced fibrotic state characterized by high tissue stiffness and conduction heterogeneity.

[0063] The multi-modal stimulation protocols are described in detail below:

[0064] Protocol A: Preservation of Native Myocardial Phenotype (0% Fibrosis)

[0065] Electrical Stimulation: Biphasic pulses with an amplitude of 1.0-1.5 V, a current of 0.2-0.6 mA, and a frequency of 1-2 Hz.

[0066] Mechanical Stimulation: 3D preloading with pneumatic pressure (5-7 kPa) and vacuum pressure (3-5 kPa), baseline strain at 5%, and dynamic increases to 7% during active cycles.

[0067] Biochemical Environment: No exogenous fibrotic agents are applied, maintaining conditions that support native tissue integrity.

[0068] Protocol B: Induction of Early Fibrotic Transition (25% Fibrosis)

[0069] Electrical Stimulation: Biphasic pulses with an amplitude of 1.5-2.0 V, a current of 0.3-0.8 mA, and a frequency of 1.5-2.5 Hz.

[0070] Mechanical Stimulation: 3D preloading with pneumatic pressure (7-9 kPa) and vacuum pressure (5-6 kPa), baseline strain at ~6%, and dynamic increases to 8% during active cycles.

[0071] Biochemical Environment: Low-dose TGF-β (0.5-1.0 ng / mL) and minimal angiotensin II are applied to initiate early myofibroblast transition.

[0072] Protocol C: Generation of Balanced Native and Fibrotic Tissue Model (50% Fibrosis)

[0073] Electrical Stimulation: Biphasic pulses with an amplitude of 2.0-2.5 V, a current of 0.6-1.2 mA, and a frequency of 2-3 Hz.

[0074] Mechanical Stimulation: 3D preloading with pneumatic pressure (10-12 kPa) and vacuum pressure (7-9 kPa), baseline strain at ~10%, and dynamic increases to 12% during active cycles.

[0075] Biochemical Environment: Moderate-dose TGF-β (1.5-2.5 ng / mL) and angiotensin II are applied to achieve consistent 50% fibrosis.

[0076] Protocol D: Induction of Advanced Fibrotic State (75% Fibrosis)

[0077] Electrical Stimulation: Biphasic pulses with an amplitude of 2.5-3.0 V, a current of 0.8-1.5 mA, and a frequency of 2.5-3.5 Hz.

[0078] Mechanical Stimulation: 3D preloading with pneumatic pressure (12-15 kPa) and vacuum pressure (9-11 kPa), baseline strain at ~12%, and dynamic increases to 15% during active cycles.

[0079] Biochemical Environment: High-dose TGF-β (2.5-3.5 ng / mL) and angiotensin II are applied to robustly drive fibrotic remodeling.

[0080] As described above, the present invention utilizes the multi-modal stimulation protocols on myocardial tissues, which provide several advantages. In particular, the protocols of the present invention enable controlled fibrosis, wherein specific levels of fibrosis can be induced, ranging from minimal to advanced. Additionally, the protocols exhibit physiological relevance, wherein the cardiac tissues mimic native myocardial tissue characteristics. A further advantage of the present invention is its versatility, wherein the multi-modal stimulation protocols can be applied to a variety of applications, including cardiovascular research, drug discovery, and regenerative medicine. The multi-modal stimulation protocols of the present invention comprise a combination of electrical, mechanical, and biochemical environment stimulations. Specifically, Table 1 sets forth exemplary Electrical Stimulation Protocols, Table 2 sets forth exemplary Mechanical Stimulation Protocols, and Table 3 sets forth exemplary Biochemical Environment Protocols. A comprehensive overview of the Multi-Modal Stimulation Protocols is provided in Table 4. The protocols of the present invention have significant potential for advancing the understanding of cardiovascular disease and developing novel therapeutic strategies. Therefore, the multi-modal stimulation protocols of the present invention represent a significant improvement over existing methods.

[0081] [Table 1] Electrical Stimulation ProtocolsParameterProtocol A (100% Native)Protocol B (75% Native)Protocol C (50% Native)Protocol D (25% Native)WaveformBiphasic pulsesBiphasic pulsesBiphasic pulsesBiphasic pulses with enhanced amplitudeAmplitude (*10)1.0–1.5 V1.5–2.0 V2.0–2.5 V2.5–3.0 VCurrent0.2–0.6 mA0.3–0.8 mA0.5–1.0 mA0.6–1.5 mAFrequency1–2 Hz1.5–2.5 Hz2–3 Hz2.5–3.5 HzElectrode ConfigurationUniform grid-arrayEnhanced edge-arrayMixed central & peripheralOptimized MEA spatially varying

[0082] [Table 2] Mechanical Stimulation ProtocolsParameterProtocol A (100% Native)Protocol B (75% Native)Protocol C (50% Native)Protocol D (25% Native)3D Preloading - Pneumatic Pressure5–7 kPa7–9 kPa9–11 kPa12–15 kPa3D Preloading - Vacuum Pressure3–5 kPa5–6 kPa6–8 kPa8–10 kPaStrain - Baseline5%~6%~8%~12%Strain - Dynamic7%8%10%15%Cycle Details - Frequency~1 Hz~1.5 Hz~2 Hz~1–1.5 HzCycle Details - Duration (Active)60 seconds55 seconds50 seconds45 secondsCycle Details - Duration (Recovery)30 seconds25 seconds20 seconds15 secondsLoadingBiaxial stretchBiaxial stretch optimizedAnisotropic biaxial stretchEnhanced biaxial stretch adaptive

[0083] [Table 3] Biochemical Environment ProtocolsParameterProtocol A (100% Native)Protocol B (75% Native)Protocol C (50% Native)Protocol D (25% Native)Inhibitors / InducersNo exogenous fibrotic agentsLow-dose TGF-β, minimal Angiotensin IIModerate doses of TGF-β, Angiotensin IIHigh-dose TGF-β, Angiotensin IIDoseMaintained to support native integrityGradual escalation synchronized with stimulation cyclesSynchronized application with electrical / mechanical cyclesAggressive, synchronized application with stimulation cycles

[0084] [Table 4] Multi-Modal Stimulation ProtocolsFibrosis LevelObjectiveElectrical StimulationMechanical StimulationBiochemical Environment100% Native Tissue (0% Fibrosis)Preserve native tissue properties for 14 days.- Waveform: Biphasic pulses- Amplitude: 1.0–1.5 V- Current: 0.2–0.6 mA- Frequency: 1–2 Hz- Electrode Configuration: Uniform grid-array microelectrodes- Pneumatic Pressure: 5–7 kPa (systolic)- Vacuum Pressure: 3–5 kPa (diastolic)- Strain: 5% baseline, dynamically increased to 7%- Cycle Frequency: ~1 Hz- Cycle Duration: 60s active, 30s recoveryNo exogenous fibrotic agents; conditions maintained to support native tissue integrity.75% Native Tissue (25% Fibrosis)Induce early fibrosis while preserving native characteristics.- Waveform: Biphasic pulses- Amplitude: 1.5–2.0 V- Current: 0.3–0.8 mA- Frequency: 1.5–2.5 Hz, with periodic bursts- Electrode Configuration: Enhanced edge-array electrodes- Pneumatic Pressure: 7–9 kPa- Vacuum Pressure: 5–6 kPa- Strain: ~6% baseline, dynamically increased to 8%- Cycle Frequency: ~1.5 Hz- Cycle Duration: 55s active, 25s recoveryLow-dose TGF-β (0.5–1.0 ng / mL) and minimal angiotensin II to initiate early myofibroblast transition.50% Native Tissue (50% Fibrosis)Generate a balanced model with equal native and fibrotic tissue.- Waveform: Biphasic pulses- Amplitude: 2.0–2.5 V- Current: 0.5–1.0 mA- Frequency: 2–3 Hz, with intermittent high-frequency bursts (up to 7 Hz)- Electrode Configuration: Mixed central and peripheral microelectrodes- Pneumatic Pressure: 9–11 kPa- Vacuum Pressure: 6–8 kPa- Strain: ~8% baseline, dynamically increased to 10%- Cycle Frequency: ~2 Hz- Cycle Duration: 50s active, 20s recoveryModerate doses of TGF-β (1–2 ng / mL) and angiotensin II to drive fibrotic phenotype.25% Native Tissue (75% Fibrosis)Induce advanced fibrosis with high tissue stiffness and conduction heterogeneity.- Waveform: Biphasic pulses with enhanced amplitude- Amplitude: 2.5–3.0 V- Current: 0.6–1.5 mA- Frequency: 2.5–3.5 Hz, with bursts up to 9–10 Hz- Electrode Configuration: Optimized MEA with spatially varying electrode density- Pneumatic Pressure: 12–15 kPa- Vacuum Pressure: 8–10 kPa- Strain: ~12% baseline, dynamically increased to 15%- Cycle Frequency: ~1–1.5 Hz with intermittent high-frequency bursts- Cycle Duration: 45s active, 15s recoveryHigh-dose TGF-β (2–3 ng / mL) and angiotensin II to robustly drive fibrotic remodeling.

[0085] DETAILED EXPLANATION OF THE FEEDBACK SYSTEM FOR DIFFERENT STAGES OF FIBROSIS

[0086] The real-time feedback system in the bioreactor continuously monitors and adjusts the mechanical and electrical stimulation protocols to target different stages of fibrosis progression and reversal. As fibrosis develops and the tissue response changes, the system makes real-time adjustments to ensure optimal treatment and promote effective fibrosis reversal. This dynamic system is divided into several stages based on the degree of fibrosis (e.g., 25%-100%) and adapts its protocols accordingly.

[0087] The real-time feedback system integrates into the bioreactor, designed to continuously monitor and adjust mechanical and electrical stimulation protocols to target different stages of fibrosis progression and reversal. This dynamic system adapts its treatment strategies based on the degree of fibrosis, ensuring optimal conditions for tissue remodelling and fibrosis reversal. Here’s a breakdown of how the feedback system works through the different stages of fibrosis:

[0088] Stage 1: Early Fibrosis (25%-50%):At this stage, fibrosis begins to form in the cardiac tissue, causing slight increases in tissue stiffness and minor changes in electrical conduction. The feedback system detects these initial changes in both mechanical and electrical properties and adjusts the protocols to limit further fibrosis progression and encourage tissue remodeling as follow:

[0089] Electrical Stimulation Adjustments:Low-frequency electrical pacing (e.g., 1-2 Hz) is applied to stimulate cardiomyocyte function and prevent excessive fibrosis accumulation. The feedback system monitors the V1V2 / S1S2 intervals to identify any initial delays in conduction, which are indicative of early fibrosis. Modulation of pacing intensity ensures that the stimulation is gentle enough to promote healthy tissue recovery without overstressing the tissue, which could accelerate fibrosis.

[0090] Mechanical Stimulation Adjustments:Low mechanical stretch (e.g., 2-3% strain) is applied to simulate physiological cardiac strain and promote normal tissue alignment. The feedback system carefully tracks tissue compliance, ensuring that it remains flexible and that the initial signs of fibrosis are reversed before they advance further. Passive mechanostimulation (e.g., stretching the tissue in the direction of normal heart movement) encourages fibroblast reprogramming into myocytes while preventing further fibroblast proliferation.

[0091] Feedback System Actions, Early fibrosis detection:The system compares stress-strain curves and electrical conduction data (e.g., V1V2 / S1S2) against baseline values to identify deviations, signaling the onset of fibrosis. Adjust stimulation protocols based on the feedback: If slight delays in conduction are detected, the system will apply gentler electrical and mechanical pulses to ensure that fibrosis does not worsen. The system ensures that no excessive mechanical forces are applied, reducing the risk of inducing scar tissue formation while promoting tissue regeneration.

[0092] Stage 2: Moderate Fibrosis (50%-75%):In this stage, fibrosis has become more pronounced with greater tissue stiffness and slower conduction velocities. The feedback system now focuses on controlling fibrosis progression and introducing treatment protocols that can reverse or halt the progression of fibrosis.

[0093] Electrical Stimulation Adjustments:Medium-frequency electrical pacing (e.g., 2-5 Hz) is applied to enhance myocardial contraction while breaking down fibrotic tissue. The feedback system evaluates changes in V1V2 / S1S2 intervals to detect slower conduction as fibrosis worsens. The system monitors these changes closely to adjust pacing intensity and frequency. Higher intensity electrical pulses are applied in specific regions of the tissue that have shown signs of fibrosis, stimulating fibroblast reprogramming and preventing excess collagen accumulation.

[0094] Mechanical Stimulation Adjustments:The system increases mechanical stretch (e.g., 3-5% strain) to simulate more pathological conditions and encourage tissue remodeling. Active mechanostimulation (e.g., providing forces directly on the tissue to mimic contraction) helps the tissue return to a more functional state by breaking down the collagen matrix. Passive mechanical loading continues to align the tissue, while increasing compression forces help to remodel the tissue and prevent the fibrosis from becoming irreversible.

[0095] Feedback System Actions:Monitoring fibrosis progression: The system continually checks the compliance of the tissue to ensure moderate fibrosis does not progress into severe fibrosis.

[0096] Dynamic adjustment of parameters:If the conduction velocity slows further or the tissue becomes too stiff, the system increases electrical stimulation intensity or modifies mechanical stretch to encourage tissue regeneration and reverse fibrosis.

[0097] Drug delivery optimization:Antifibrotic drugs or gene therapies may be added, with dose adjustments based on feedback from the system's conduction and compliance data.

[0098] Stage 3: Advanced Fibrosis (75%-100%):At this stage, fibrosis is fully established, leading to severe tissue stiffness, impaired conduction, and possibly arrhythmic behaviour. The feedback system now works to restore tissue function as much as possible, focusing on maximizing fibrosis reversal and restoring normal conduction.

[0099] Electrical Stimulation Adjustments:High-frequency electrical pacing (e.g., 5-10 Hz) is applied to break down scar tissue and stimulate myocardial regeneration. The feedback system evaluates the degree of conduction delay across the three ventricular layers and adjusts the stimulation frequency and intensity to reverse fibrosis by targeting regions of slow conduction. Electrical stimulation maps help the system identify areas of extreme fibrosis and apply targeted, high-frequency pulses that break down the fibrotic scar tissue.

[0100] Mechanical Stimulation Adjustments:High mechanical strain (e.g., >5% strain) is applied to simulate heart failure conditions and stimulate tissue recovery by restoring elasticity and regenerating functional myocardium. Compression and shear forces are used to disrupt collagen deposition, stimulating the fibroblasts to revert to myocytes and decrease collagen production. Active mechanostimulation is paired with electrical pacing to induce coordinated contraction, which encourages the restructuring of fibrotic tissue and the formation of functional myocardium.

[0101] Feedback System Actions:Aggressive fibrosis reversal: The system constantly monitors electrical conduction and measures tissue compliance. When conduction delays are detected, the system boosts stimulation intensity (both electrical and mechanical) to reverse fibrosis more aggressively.

[0102] Drug optimization and adjustments:Antifibrotic drugs and gene therapies are applied at optimal concentrations, based on real-time data from the feedback system.

[0103] The system makes necessary adjustments to drug delivery rates, mechanical loading protocols, and electrical pacing to achieve the best possible outcomes for fibrosis reversal and tissue remodeling.

[0104] The overall feedback system workflow for controlling fibrosis stages in Living Cardiac Tissue comprises a series of interconnected steps, including:

[0105] Fibrosis Detection: continuously monitoring the conduction velocity (V1V2 / S1S2) and tissue compliance to identify the fibrosis stage, wherein the fibrosis stage is determined based on predetermined thresholds for conduction velocity and tissue compliance;

[0106] Protocol Adjustment: adjusting, in response to the identified fibrosis stage, at least one of: electrical stimulation intensity, wherein the intensity is selected from a range of low, medium, and high frequencies; mechanical loading intensity, wherein the intensity is selected from a range of passive stretch, compression, and active stimulation; and drug delivery for antifibrotic treatment, wherein the drug is delivered at a rate and concentration determined based on the fibrosis stage; and Real-Time Monitoring, continuously checking for changes in histological markers, such as collagen deposition, conduction changes, and tissue stiffness, and making continuous adjustments to the electrical stimulation intensity, mechanical loading intensity, and drug delivery to maintain the tissue in an optimal state for fibrosis reversal.

[0107] [Table 5] Feedback Adjustments for Different Fibrosis StagesFibrosis StageElectrical Stimulation AdjustmentMechanical Stimulation AdjustmentDrug Delivery AdjustmentStage 1 (0% - Healthy Tissue)Low-Frequency Stimulation (1 Hz) to maintain normal conduction.Physiological Strain (2%) to support normal contractility.No drug intervention needed.Stage 2 (25% - Early Fibrosis)Low-Medium Frequency Stimulation (1-2 Hz) to prevent further fibrosis.Low-Moderate Strain (3%) to preserve compliance.Early antifibrotic agents (e.g., TGF-β inhibitors).Stage 3 (50% - Moderate Fibrosis)Medium-Frequency Stimulation (3-5 Hz) to break fibrosis networks.Moderate Strain (4-5%) to enhance myocyte realignment.Pirfenidone or collagen inhibitors to slow fibrosis.Stage 4 (75% - Severe Fibrosis)High-Frequency Stimulation (5-10 Hz) to restore conduction pathways.High Strain (>6%) to stimulate ECM remodeling.Increased Pirfenidone dose (600-1000 mg / day) to reverse fibrosis.Stage 5 (99%-100% - Scar Tissue)No Response to Electrical Stimulation.Tissue Stiffness Too High for Mechanical Adaptation.Scar tissue requires ECM degradation enzymes for partial recovery.

[0108] The controller of the present invention provides a closed-loop feedback mechanism for controlling fibrosis stages in LCT, wherein the system continuously monitors and adjusts the treatment protocol to optimize the reversal of fibrosis. This workflow enables the creation of controlled levels of fibrosis, thereby providing a valuable tool for cardiovascular research, drug discovery, and regenerative medicine. Table 5 sets forth exemplary Mechanical Feedback Adjustments for Different Fibrosis Stages.

[0109] QUANTITATIVE CURVE FITTING AND DATA EXTRACTION

[0110] The present invention utilizes a quantitative curve fitting analysis to model the progression of fibrosis through various stages. Fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) components, leading to tissue stiffening and impaired organ function. The quantitative curve fitting analysis provides a robust framework to describe the continuum from healthy tissue to severe fibrosis, with each stage represented by distinct mathematical models. This section focuses on the application of multiexponential, second-order, first-order, and zero-order models to describe the mechanical behavior of tissue at different stages of fibrosis, with emphasis on the critical role of mechanobiology.

[0111] Stage 1 (Native (100%) Live Tissue - Multiexponential Model):In healthy tissue, the ECM exhibits a complex interplay of various components, resulting in intricate mechanical behavior. A multiexponential model is employed to capture this complexity, considering multiple relaxation processes. This model reflects the diverse mechanical responses of healthy tissue, encompassing various cellular and matrix interactions. The use of a multiexponential model at this stage provides a comprehensive understanding of the mechanical behavior of native tissue.

[0112] Stage 2 (25% Fibrosis - Third Exponential Component Emergence):As fibrosis initiates, there is a subtle increase in ECM deposition, introducing a third exponential component to the model. The introduction of higher order components accounts for the early fibrotic changes, reflecting the initial alterations in tissue mechanics due to ECM accumulation. This stage marks the beginning of significant changes in tissue behavior, which can be accurately modeled using the third exponential component.

[0113] Stage 3 (50% Fibrosis - Second-Order Model):With the progression to moderate fibrosis, the tissue's mechanical behavior transitions to a second-order system, indicative of increased stiffness and damping. This model encapsulates the dynamic response of tissue undergoing significant fibrotic remodeling, where both inertia and damping play pivotal roles. The second-order model provides a detailed understanding of the tissue's viscoelastic properties at this stage.

[0114] Stage 4 (75% Fibrosis - First-Order Model):In advanced fibrosis, the tissue exhibits predominantly elastic behavior with minimal viscous effects. Here, the stiffness coefficient has markedly increased, reflecting the dense ECM network. The tissue's response is now primarily governed by elastic deformation, with rapid stress relaxation. A first-order model is employed to describe this stage, capturing the essential characteristics of the tissue's mechanical behavior.

[0115] Stage 5 (99% Fibrosis - Zero-Order (Scar / Death) Model):At the terminal stage, the tissue becomes extensively scarred, exhibiting near-infinite stiffness and negligible compliance. This state is modeled as a zero-order system. In this scenario, any applied force results in imperceptible displacement, signifying the loss of functional elasticity and the culmination of fibrotic progression. The zero-order model provides a clear understanding of the tissue's mechanical behavior at this stage.

[0116] In summary, the quantitative curve fitting analysis integrates mechanical and electrical stimulation with biophysical properties of tissue to provide a comprehensive framework for modeling the mechanical progression of fibrosis through distinct stages. By employing multiexponential, second-order, first-order, and zero-order models, we can delineate the transition from healthy tissue to severe fibrosis. The model applies multi-exponential decay for native (100%) live tissue; Third-exponential decay for 25% fibrosis; Second-order kinetics for 50% fibrosis; First-order kinetics for 75% fibrosis; and Zero-order kinetics for 99% fibrosis (scar formation and near cell death). At the final step (100%) fibrosis, the most critical factor is mass stiffness and mechanobiological behavior of matrix proteins, where viscoelasticity and impedance tend toward infinity. This comprehensive framework enables a detailed understanding of the mechanical progression of fibrosis and provides valuable insights into the underlying mechanisms of tissue stiffening and impaired organ function.

[0117] The present invention relates to bioreactors for culturing cells, particularly cardiac cells. One of the major challenges in cardiac research is creating an in vitro environment that accurately mimics the complexity and functionality of native heart tissue. The invention provides several significant advantages. Firstly, the utilization of native cardiac tissue piece maintains the intricate architectural complexity and cellular composition of the heart, thereby providing a more accurate and reliable model of human cardiac fibrosis.

[0118] Additionally, the system's capability to incorporate patient-specific tissues enables personalized disease modeling and therapeutic testing, whereby the relevance of findings is enhanced for individual patients, thus allowing for tailored treatment strategies.

[0119] Furthermore, the bioreactor's versatile design accommodates a range of tissue sizes and permits simulation of varying degrees of fibrosis through adjustable stimulation protocols, rendering it suitable for a broad spectrum of research and drug development applications.

[0120] Moreover, the integration of mechanical, electrical, and biochemical stimulation with advanced data analysis tools facilitates a comprehensive and holistic assessment of cardiac fibrosis and the efficacy of potential treatments, thereby enabling a more thorough understanding of the underlying mechanisms and optimizing therapeutic outcomes.

[0121] The bioreactor of the present invention provides several advantages over existing technologies, including:

[0122] Enhanced Electrophysiological Fidelity:The bioreactor maintains native cell-cell communication and structural heterogeneity and maturity and maturity is necessary for quantititative induction of fibrosis, ensuring that electrical conduction patterns closely mimic those in human myocardium. This is achieved through the use of LCT cultures, which preserve the natural interactions between cells and their environment.

[0123] Extended Viability and Functionality:The application of controlled mechanical cues preserves tissue integrity and maturity, and these tow advantages allowing long-term cultivation (up to 14 days) and repeated functional assays. This enables researchers to conduct prolonged studies on cardiac tissue, providing valuable insights into its behavior and response to various stimuli.

[0124] Quantitative Fibrosis Modeling:The bioreactor supports the graded induction of fibrosis, enabling the study of transitional states (e.g., 75% native / 25% fibrotic to 100% fibrotic tissue) and their electrophysiological consequences. This feature allows researchers to investigate the effects of fibrosis on cardiac function and identify potential therapeutic targets.

[0125] Integrated Multi-Modal Stimulation:The bioreactor combines electrical, mechanical, and biochemical stimuli to provide a robust framework for simulating in vivo conditions. This overcomes the limitations of stem cell-derived cardiomyocytes and static 3D constructs, enabling researchers to study cardiac tissue in a more realistic and dynamic environment.

[0126] Non-invasive Mechanobiology Profiling:Providing continuous real-time monitoring of mechanical and electrophysiological parameters without interrupting the culture, thereby allowing for dynamic profiling and predictive modeling of tissue responses to mechanical and biochemical stimuli.

[0127] Enhanced Electrophysiological Monitoring:Enabling enhanced electrophysiological monitoring through the capture of field potentials and MAPs in conjunction with mechanical data, which facilitates a deeper understanding of how fibrosis alters the electrical properties of cardiac tissue.

[0128] Customizable Load Application and Adaptation to Maturation:Allowing for customizable load application and adaptation to maturation through the use of a feedback-controlled stimulation unit that dynamically adjusts strain levels based on the tissue's mechanical response, thereby enabling the simulation of various physiological and pathological conditions.

[0129] Use of Thick Native Tissue:The bioreactor utilizes thick native tissue to better preserve tissue structure and functionality for long-term studies. This approach is crucial for accurately modeling fibrosis progression and the effects of therapeutic interventions.

[0130] Synchronized Electromechanical and Biochemical Conditioning:The system employs synchronized electromechanical stimulation protocols, combining electrical, mechanical, and biochemical cues in a coordinated manner to induce fibrosis across multiple stages, from 25% to 100%.

[0131] Fluid Microjet Penetrated Channel Network and Tubes:The bioreactor includes a network of Fluid Microjet Penetrated Channel within the tissue, facilitating the precise delivery of TGF-β and other fibrotic agents. These channels, in combination with microtube fabrication, apply controlled strain and stress to mimic natural tissue behavior and induce fibrosis at various levels.

[0132] Real-Time Curve Fitting for Fibrosis Assessment:Using real-time data analysis, the system generates quantitative fibrosis induction curves that track the progression from 25% fibrosis to 100% fibrosis, with machine-learning algorithms optimizing therapeutic adjustments.

[0133] The accompanying drawings depict various embodiments of the present invention, wherein identical reference numerals denote corresponding elements. It is to be understood that the scale, proportions, and relative positioning of these elements are not necessarily drawn to scale, but rather have been schematically represented for ease of recognition and understanding. The drawing figures illustrate one or more exemplary implementations of the present disclosure, solely by way of example and not by way of limitation.Fig.1

[0134] illustrates the general architecture of the invention.Fig.2

[0135] illustrates the details of main elements the invention and the fluid control system.Fig.3

[0136] illustrates the force actuator and measurement subsystem.Fig.4

[0137] illustrates high-pressure Tyrode jet subsystem.Fig.5

[0138] illustrates parallel apparatus system.

[0139] The following detailed description sets forth specific details and embodiments of the invention in order to provide a comprehensive understanding of the various aspects and features thereof. However, it will be apparent to a person skilled in the relevant art that certain embodiments may be practiced with fewer than all of these specific details, or with other methods, circuits, components, materials, or the like. Furthermore, well-known structures, electronic systems, and circuit design details have been omitted or simplified to avoid obscuring the description of the embodiments and to focus attention on the core inventive concepts, including novelty, inventive step, and applicability.

[0140] Throughout this specification and the appended claims, the terms "comprising" and "including" are used interchangeably, unless the context dictates otherwise, and are intended to be inclusive or open-ended, i.e., not excluding additional, unrecited elements or method acts. The phrases "in one embodiment" or "in an embodiment" do not necessarily refer to the same embodiment, but rather denote a particular feature, structure, or characteristic that may be combined in any suitable manner in one or more embodiments of the invention.

[0141] Additionally, the singular forms "a", "an", and "the" include plural referents, unless the context clearly indicates otherwise. The term "or" is used in its inclusive sense, i.e., "and / or", unless the context dictates otherwise, and is intended to convey a logical "OR" definition, wherein any one of the operands being true renders the result true.

[0142] In, the general block diagram of an exemplary embodiment of the invention has been shown, and inand, the details of each part have been depicted. According to, the heart or wedge of heart tissue100are placed inside the chamber180. A set of actuators and measuring tools102in connection with the chamber are able to apply electrical or mechanical signals104to the tissue and measure the parameters of the chamber environment such as pH and electrical conductivity and mechanical parameters such as static and dynamic pressure103inside the chamber. The material of the chamber180is made of clear plexiglass and therefore the cameras140and146are able to see different parts of the tissue using the optical lenses142and148. Controlling the actuators, capturing images and processing them, and measuring the parameters are done inside the computer170, and the cameras140and146transmit images with high frame rate to the computer170using high-speed interfaces144and150. The tissue electrical measuring device160is connected to the computer through the interface166and is connected to the impedance and conduction velocity probe164using connecting wires162and the probe is placed on the heart tissue and has a physical connection168with the tissue inside the chamber.

[0143] shows the schematic block diagram of exemplary embodiment of the bioreactor, actuators, sensors and relationship and connection between them. The bioreactor is designed so that it can independently enforce axial and shear stresses to the tissue and measure its mechanical characteristics, especially stiffness. The control of the actuators, pressure, flow and stress adjustment is done by the controller267and the controller sends and receives the settings and commands to the computer200through the connections201and202. The tank266has been filled with Tyrode’s solution liquid before starting the experiment. Tank266is connected to auxiliary tank210by tube268. The tank210is designed as a cylinder, and the piston209can be moved by the axis208and the linear actuator207(a High-Speed Linear Actuators is recommended for instance FA-RA-22-12-12 from Firgelli Automations),and therefore the volume of Tyrode’s solution in the main tank266or the level of the Tyrode’s solution269in the tank266has been controlled by the signal206sent from the controller267. The temperature of the Tyrode’s solution is sent by the probe229(for instance PT100 External Probe from Thermo-Scientific) and the transmitter230(for instance Thermocouple / RTD(PT100) Data Logger Model DL-GT-DT4947SD from IOThrifty) and and by means of signal231to the controller267.So, based on the measured temperature and the desired temperature of 37 degrees, controller 267, can set the Tyrode’s solution temperature by providing the power signal221tothe heater222. On the other hand, the temperature of the Tyrode’s solution has been stabilized by temperature feedback and the heater.

[0144] As mentioned before, the bioreactor has been designed to be able to independently control the shear stress and compressive (normal) stress on the tissue, therefore, no peristaltic pump has been utilized in its design. This function is obtained by controlling the pressure on the top surface of the Tyrode’s solution in the tank and by flow control in the chamber. For this purpose, output223and interface224are connected to pressure sensor226and pressure signal225is sent to controller267to measure the gas pressure entering the Tyrode’s solution (CO2 and O2 for pH control of Tyrode’s solution). The outlet213and the connecting tube214transmit the Tyrode’s solution pressure in the chamber257to the pressure sensor215and the static pressure signal inside the chamber212is sent to the controller267. The controller adjusts the gas solenoid valve227(for instance PFV-K13 proportional solenoid valve kit from Enfield Technologies, which is NSF certified) through signal228(0-5 V command), also, when needed through the rapid movement of the piston209in the cylinder210through the linear actuator207resulting the change in the level of the Tyrode’s solution liquid269, the controller267adjusts the static pressure of the Tyrode’s solution liquid in the desired value based on the output signal212(feedback from chamber). For this purpose, the controller must be a two-input and one-output controller which is able to set the desired static pressure in the chamber257by adjusting the solenoid valve227and changing the volume and level of the Tyrode’s solution through the fast response linear actuator207, and feedback from the pressure sensor226.

[0145] Since the pH value of the Tyrode’s solution must be set at the desired value, the pH meter / transmitter233(for instance Orion Star A221 Portable pH Meter from Thermo-Scientific) measures the pH value through the probe232(for instance Orion 927005MD Epoxy ATC Probe from Thermo-Scientific) and sends the value to the controller267through the signal234(RS-232), then the controller adjusts the desired pH value by adjusting the amount of oxygen and carbon dioxide gas injected into Tyrode’s solution.

[0146] The controller uses the signal235which is connected to the solenoid valve238which is connected between the oxygen tank236and the Tyrode’s solution tank through the tube241and also the signal244which is connected to the solenoid valve239which is connected between the carbon dioxide tank237and the Tyrode’s solution tank through the tube240. The controller adjusts the gas injection to the Tyrode’s solution in order to keep the pH of the Tyrode’s solution at a desired value constantly and for this purpose it uses the feedback from the pH probe232.

[0147] The Tyrode’s solution liquid transfer from the outlet260and the tube259to the inlet258of the chamber257. The tee249through the tube248has been utilized for injecting the medicinal substance247to the Tyrode’s solution by the syringe pump246and according to the signal245from the controller267, therefore, it is possible to inject the specified drugs through the flow of the Tyrode’s solution. The fibrotic heart tissue may need direct injection due to lack of functions of muscle and blood vessels. For this purpose, the user can inject medicine through the hole263on the surface of the chamber257and using the needle264through the high-pressure pump265into the heart tissue256. The hole263has been covered by an elastic cover, so when the needle removed the hole closes. Moreover, by using hole255, it is possible to enter the physical tissue sampling probe in this chamber. The high-pressure pump265provides a jet of Tyrode to the surface of the tissue which penetrate through the heart tissue and make an artificial vascular in the culture of the tissue. So, the Tyrode or drug can reach the internal parts of the tissue despite its width. The Tube268provides Tyrode to the high-pressure pump265. Mechanism and elements of the high-pressure pump has been depicted in the.

[0148] The heart tissue is placed on the holding device253and the forces254are applied to the tissue by the actuator255and the signal204sent from the controller267. The details of force and tension actuators and sensors will be explained in the next paragraphs and has been shown in.

[0149] Due to the importance of electrical impedance and conduction velocity and their relationship with fibrosis, the probes250can measure the impedance and conduction velocity of the heart tissue256at the desired frequencies and send it directly to the computer200through the connection251and the measuring apparatus252(for instance impedance analyzer model 262K from SinePhase which has provided API for further processing and ADInstrument DAQ and LabChart Software).

[0150] The camera set262is connected to the computer200by means of the high-speed interface261, and the details of its operation based on illustration depicted inare described in the next paragraphs.

[0151] As mentioned, this bioreactor is able to independently control the shear stress caused by Tyrode’s solution movement on heart tissue. For this purpose, the outlet219of the Tyrode’s solution from the chamber257is connected to the liquid solenoid valve218(a pinch valve type is recommended, for instance EQV proportional pinch valve from Enfield Technologies) by means of the tube220. Then, it is connected to the flow sensor216(for instance SLS-1500 liquid flow meter from Sensirion) and the flow signal205is sent to the controller and the controller will be able to keep the flow at the desired value at any time by adjusting the opening and closing of the solenoid valve218through the signal211. So, it is able to control the shear stress on the tissue caused by the movement of the Tyrode’s solution. The output217,after the flow sensor216,can be collected and be transferred to the Tyrode’s solution tank266or can be sent out of the system.

[0152] All control loops utilized in this invention can be independent or dependent. For example, the static pressure controller inside the chamber can be independent from the flow controller because it uses completely independent actuators and sensors to apply forces and receive feedback signals. Although the differential equations governing the mathematical model may be related between them, the design of the pressure and flow control mechanism is such that it allows following the set values of the user independently.

[0153] The controller267is generally a computer equipped with some USB Data Acquisition Interface Modules (for instance D8000 series from Omega) and so it is capable to perform various kind of controlling algorithms in a convenient approach.

[0154] shows the block diagram of the force actuators system and the mechanical parameter measurement system. Parts of this sub-system is placed inside the bioreactor chamber320and by means of applying the forces determined by the user and measurement of displacement, with the help of a camera(s), the system is able to measure mechanobiology parameters including stiffness and mechanical impedance and stress or force and strain or length related to the function of heart tissue in the state of fibrosis.

[0155] The tissue or more precisely tissue cuts from the heart350is placed on the holder352and is restrained by the holder arms330and332on both sides, the holder arms are connected to the sliders346and348. The sliders can slide inside the rails342and344, and at the end of each of the sliders, as shown in, a neodymium magnet has been installed (360and362), and there is also a landmark hacked on each of the sliders. Visual indicia334and336on the sliders are such that they can be seen by the camera of each chamber or set of cameras. Two magnetic coils338and340are placed outside the chambers and at the end of the location of the sliders. Therefore, they are able to apply, based on the current passing through each coil and the magnetic field resulting from them, the force of tension or pressure to the sliders and through them to the tissue or wedge of tissue. The current passing through each coil is adjusted by the controller358based on the signal318sent from the computer. In this invention, high-speed cameras are used for position feedback. The way the cameras are placed is such that they can see the visual signs on the sliders from the transparent surface of the chamber. Then, the images taken from one camera316or two cameras304and316are sent to the computer using the high-speed interface314or314and302, and the position of each of the sliders is determined by image processing. Therefore, by extracting the position and calculating the change in length, as well as the specific forces introduced by the coils, the stiffness of the tissue has been determined statistically. The processor program is able to determine the stiffness of the tissue by calculating the average length change and the average forces applied in a specific time period (for example, every one minute).

[0156] shows the block diagram of the Tyrode high-pressure pump which provide a jet of Tyrode to the surface of the tissue. The jet of Tyrode penetrates to the tissue and make an artificial vein which help circulation of Tyrode and drug in all internal parts of the tissue. Due to width of the tissue, normal and ordinary methods is not applicable for a fibrosis tissue. The high-pressure pump consists of a cylinder400and a piston420, the piston420push the liquid by means of a linear actuator402and404and a control command406from the controller267provides accurate pressure and flow of the output liquid jet. Output of the high-pressure is a three way416and guide the liquid jet418to the tissue. A pinch valve410regulate the Tyrode come form the tank266bye the tube412. Control signal408for the pinch valve410has been provided by the controller267. At the beginning stage of the test, the pinch valve410is open and the piston420moves toward the beginning position (left side in the), So the Cylinder400fills with the Tyrode, then the pinch valve closes and the setup is ready. It’s important to note that the high-pressure Tyrode jet can carry drugs, collagen cells, TGF-Beta, endothelial cells, etc. For this purpose, the three way416is connected to a syringe pump426by means of tube424and is controlled by the signal428from the main controller267.

[0157] shows the parallel array of an exemplary embodiment of the invention. In this implementation at least two apparatuses512and514(or in between510) are running parallel with each other. The parallel apparatuses512and514(or in between510) are assembled from the main parts necessary to execute experiment on the tissues. These parts generally consist of the parts shown inbut the Tyrode for all paralleled apparatus is supplied from one tank500and by means of tubes502,506and508. Control signals, measurement signals and image signals are gathered by means of bus516,518and520and is processed by means of522.

[0158] In this section an exemplary method for quantitatively controlling and reversing fibrosis in cardiac tissue using real-time curve fitting, mechanical modulation, and anti-fibrotic drug signalling by means of disclosed invention has been provided as follow:

[0159] Step 1: Fibrosis Induction and Quantification:Inducing fibrosis in cardiac tissue within a bioreactor system, wherein fibrosis is initiated in the tissue across a gradual range from 25% to 100% using synchronized 3D mechano-electrical stimulation protocols.

[0160] The quantification of fibrosis at each stage (25%, 50%, 75%, and 100%) is performed using real-time curve fitting of electrophysiological and mechanical data, including: Conductivity changes (e.g., conduction velocity, impedance), Tissue compliance, which correlates to stiffness, Strain-stress response curves, which track mechanical deformation during cyclic loading.

[0161] Each stage of fibrosis is represented by a quantitative fibrosis fraction (F(t)) and correlated to mechanical properties of the tissue, including contractility and stiffness. This is modelled using a multi-exponential system where the tissue’s response to electrical and mechanical stimuli is modelled based on a time-dependent function.

[0162] Step 2: Drug-Specific Signalling Pathways for Fibrosis Reversal:The administration of specific antifibrotic drugs, such as pirfenidone, induces mechanical modulation of tissue stiffness and contractility. This process is mediated by specific signalling pathways which influence fibroblast-to-myofibroblast transition, fibrotic ECM deposition, and mechanotransduction.

[0163] Pirfenidone, a known anti-fibrotic drug, regulates TGF-β signalling, which modulates the activation of myofibroblasts and the deposition of ECM. It also inhibits fibrosis-associated kinases, such as MAPK and mTOR, reducing the mechanical stiffness and contraction of the tissue.

[0164] As fibrosis progresses from early (25%) to advanced (100%), the drug-induced signalling pathways adapt in response to the stages of fibrosis, influencing cell behaviour, ECM remodelling, and tissue mechanics. For instance, pirfenidone’s effects on TGF-β and ACTA2 (α-SMA) expression are tracked in real time to correlate drug effects with fibrosis reversal through the application of real-time feedback control.

[0165] Step 3: Maturation and Immune System Modulation:As fibrosis progresses and is reversed in the bioreactor system, the maturation of tissue layers within the tri-layer cardiac tissue is monitored. The three tissue layers (epicardial, myocardial, and endocardial) are integrated with distinct biomechanical properties, and the system induces layer-specific maturation and differentiation of cardiac cells under mechanical stimulation.

[0166] Each layer’s stiffness is modulated differently based on drug treatments and immune responses. For example, in the epicardial layer, macrophage signalling and TGF-β activity influence tissue remodelling, while the myocardial layer responds primarily to contractile stimulation. These processes are further influenced by the immune environment in the bioreactor, which is modulated using cytokines and growth factors to induce immune modulation through immune cells such as macrophages and fibroblasts.

[0167] The immune modulation supports the resolution of fibrosis by transitioning inflammatory macrophages to a reparative phenotype, promoting collagen degradation and ECM remodeling. This is quantified using immune markers in the system.

[0168] Step 4: 3D Mesh Network for Tissue Integrity and Drug Delivery:A mesh network of fluid microjet penetrated channels is inserted within the tri-layer cardiac tissue, which delivers TGF-β and other fibrotic agents directly into the tissue. This mesh network mimics natural ECM structures and exerts localized strain and stress, enhancing tissue mechanical integrity and fibrosis induction.

[0169] The network of fluid microjet penetrated tubes embedded in the tissue provides targeted mechanical strain, stimulating the tissue at the microscopic level to maintain the stress-strain relationship while enabling the precise delivery of drugs like pirfenidone and other antifibrotic agents.

[0170] Step 5: Quantitative Feedback Control for Fibrosis Reversal:The system continuously tracks real-time mechanical responses (stiffness, compliance) and electrophysiological markers (conduction velocity, impedance), generating real-time quantitative curves. This data is used to adjust treatment protocols for fibrosis reversal, including changes in drug delivery, electrical stimulation, and mechanical loading based on the stage of fibrosis (25% to 100%).

[0171] The real-time curve fitting analysis is used to predict the effects of antifibrotic drug treatment, optimizing the dosage and timing for fibrosis resolution by targeting the TGF-β pathway, kinase signalling, and mechanotransduction pathways. The drug’s efficacy is validated by observing dynamic tissue responses and changes in electromechanical properties during the fibrosis reversal process.

[0172] Step 6: Synchronized Multi-Layer Biomechanics:The tri-layer cardiac tissue undergoes synchronized 3D mechanical-electrical stimulation within the bioreactor, wherein each layer’s strain and stress responses are modulated independently. The epicardial layer is subjected to higher mechanical stress due to the thicker ECM, while the endocardial layer experiences different mechanical forces to simulate the natural physiological conditions of cardiac tissue.

[0173] The synchronized stimulation ensures that each layer of the cardiac tissue develops appropriate mechanical properties and electrophysiological activity, facilitating fibrosis reversal from the epicardial to the endocardial layer, ensuring uniform resolution of fibrosis across the tissue.

[0174] This method integrates a quantitative, multi-step approach to fibrosis induction and reversal, utilizing real-time curve fitting and dynamic feedback control to modulate the biomechanical, biochemical, and electromechanical environment of the tissue. By incorporating specific drug signalling pathways, such as pirfenidone targeting TGF-β signalling, connexin 43 activation, and kinase modulation, this system provides a novel platform for the quantitative assessment of fibrosis resolution and anti-fibrotic drug screening, leading to a comprehensive, predictive anti-fibrotic therapeutic strategy.

[0175] The detailed description has set forth various implementations of the method and apparatus by means of block diagrams, schematics, and examples. A person skilled in the art will appreciate that each operation and function proposed in these block diagrams, schematics, and examples can be implemented individually or collectively using a wide range of hardware, software, firmware, components, or virtually any combination thereof.

[0176] Designing the circuitry and writing the code for software and firmware implementations would be well within the skill of an ordinary skilled artisan, given the details disclosed herein. Many of the methods and components described herein may involve additional acts, omit certain acts, or execute acts in a different order than specified, all of which are readily recognizable by a person skilled in the art. For example, the invention may not require a microcontroller and may be hardwired to provide some or all of the functionality disclosed herein.

[0177] The exemplary embodiments disclosed herein are presented solely for clarity and understanding, and are not intended to limit the scope of the subject matter of this invention. A person skilled in the art will recognize that various adaptations, modifications, and changes are possible, and that the scope of the invention is defined solely by the appended claims.

[0178] Therefore, the present disclosure is intended to be exemplary, rather than limiting, and all variations and modifications that fall within the spirit and scope of the appended claims are intended to be included therein.

[0179] The present invention has significant industrial applicability in various fields, including:

[0180] Drug Screening :The use of the present invention enables high-throughput testing and evaluation of anti-fibrotic drugs, allowing for the assessment of their effects on cardiac tissue mechanics, electrophysiology, and molecular profiles, thereby facilitating the identification of effective therapeutic agents.

[0181] Disease Modeling :The present invention provides a controlled environment for studying the progression of cardiac fibrosis, permitting the investigation of the roles of mechanical stress, electrical activity, and immune cell involvement, and enabling a deeper understanding of the underlying mechanisms of the disease.

[0182] Therapeutic Development :The present invention allows for the assessment of the efficacy of interventions aimed at reversing fibrosis, including agents that promote myofibroblast to fibroblast transition, thereby facilitating the development of novel therapeutic strategies.

[0183] Personalized Medicine :The present invention enables the modeling of patient-specific cardiac conditions, permitting the tailoring of therapeutic strategies based on individual tissue responses, and allowing for the development of personalized treatment plans.

Claims

A bioreactor for controlling and quantifying 3D multi-layer cardiac tissue viability, fibrosis induction, and fibrosis reversal through multi-modal functional stimulation, comprising:a first chamber housing a first set of cardiac tissue, wherein the first chamber is configured to maintain the viability and functional integrity of the cardiac tissue for predefined periods of time;a second chamber housing a second set of cardiac tissue, wherein the second chamber is configured to induce varying degrees of fibrosis to the second set of cardiac tissue for predefined periods of time, wherein fibrosis induction is precisely controlled using synchronized electromechanical and biochemical conditioning, with predefined electromechanical stimulation protocols for inducing gradual fibrosis progression;a functional stimulus delivery system configured to provide mechanical, electrical, and biochemical functional stimuli to the sets of cardiac tissue, comprising:electrical functional stimulation means for applying electrical signals to the sets of cardiac tissue, wherein the electrical signals are adjustable in terms of frequency, amplitude, duration, and waveform to simulate normal and pathological electrical conditions in cardiac tissue, including the induction of fibrosis at specific stages;mechanical functional stimulation means for applying mechanical forces and dynamic pressure to the sets of cardiac tissue, wherein the mechanical forces and dynamic pressure are adjustable in terms of frequency, amplitude, duration, and waveform to induce controlled strain and stress that correlates with fibrotic tissue progression, including mechanical preloads for fibrosis induction;biochemical functional stimulation means for applying biochemical cues to the sets of cardiac tissue, including but not limited to growth factors such as TGF-β, hormones, and pharmaceutical agents to facilitate and quantify fibrosis induction across stages;a 3D magnetic actuator system for dynamically applying mechanical stress and strain to the sets of cardiac tissue in a time-dependent manner, wherein the actuator system is configured to mimic cyclic mechanical forces experienced by the myocardium in vivo, enabling gradual and controlled induction of fibrosis, with adjustable stress and strain protocols for inducing various fibrosis levels;a fluid microjet system for vascularizing the tissue and ensuring adequate oxygenation and nutrient supply to the cardiac tissue, wherein the fluid microjet system is configured to maintain a stable and physiologically relevant environment for the cardiac tissue during the fibrosis induction and reversal process;integrated sensors, data acquisition, and imaging systems for real-time monitoring of tissue mechanics, electrophysiology, and biochemical markers, comprising conduction velocity, excitability threshold, extracellular field potential, impedance, strain-stress response curve, and optical mapping of voltage and ion concentration;a software component for analysing data from the integrated sensors, data acquisition, and imaging systems, generating quantitative curves to functionally assess the sets of cardiac tissue, wherein the software component is configured to process data using advanced bidomain modelling and machine-learning algorithms to provide predictive insights on fibrosis progression, including real-time curve fitting for fibrosis induction and reversal;wherein the bioreactor is configured to induce controlled fibrosis in the sets of cardiac tissue through synchronized multi-modal functional stimulation, including electrical, mechanical, and biochemical cues, and wherein the bioreactor further comprises means for accommodating biochemical and immunological agents to investigate the impact of anti-fibrotic drugs on the induction, progression, and resolution of fibrosis, wherein real-time monitoring and curve fitting are employed to determine the effectiveness of antifibrotic drugs in reversing fibrosis.A real-time feedback method for controlling and reversing fibrosis in a bioreactor, comprising:continuously monitoring the degree of fibrosis in a tissue sample using at least one of conduction velocity, excitation threshold, and tissue compliance to assess fibrosis progression quantitatively, including monitoring changes in fibrosis through real-time curve fitting of electrophysiological and mechanical data;identifying the quantitative stage of fibrosis based on the monitored degree of fibrosis, wherein the stage is selected from a plurality of stages including early fibrosis, moderate fibrosis, advanced fibrosis, and scar tissue;adjusting, in response to the identified stage of fibrosis, at least one of:electrical functional stimulation intensity, wherein the intensity is adjusted based on the stage of fibrosis to stimulate cardiac tissue function, promote electrical conductivity, and prevent excessive fibrosis accumulation, with real-time feedback based on tissue impedance and conduction velocity;mechanical loading intensity, wherein the intensity is adjusted based on the stage of fibrosis to simulate physiological cardiac strain and promote normal tissue alignment, enabling graded induction of fibrosis;drug delivery for antifibrotic treatment, wherein the drug is delivered at a rate and concentration determined based on the stage of fibrosis, with the ability to administer TGF-β inhibitors, anti-inflammatory agents, and collagen-modulating drugs at optimized doses for fibrosis reversal;continuously checking for changes in functional markers and adjusting the balance between electrical to mechanical functional stimulation, including changes in conduction velocity, electrical impedance, tissue stiffness, and strain-stress response curves; andmaking synchronous continuous adjustments to the electrical functional stimulation intensity, mechanical loading intensity, and drug delivery to maintain the tissue in an optimal state for fibrosis reversal, ensuring graded reversal of fibrosis from almost scar tissue back to native tissue phenotype.A multi-modal functional stimulation method for inducing and reversing fibrosis in in-vitro and ex-vivo native cardiac tissue, comprising:providing at least one 3D functional electrical stimulation component designed to induce stimuli to multi-layer cardiac tissue by means of a plurality of electrodes penetrating into the tissue, including biphasic and monophasic pulses with a predefined stimulation protocol, enabling accurate control of electrical signals for fibrosis induction across a range of stages;providing a microjet apparatus to construct micro-channels in the tissue for the delivery of nutrients, oxygenation, and biochemical agents such as TGF-β and angiotensin II to induce controlled fibrosis, ensuring long-term tissue viability during induction and reversal;providing at least one functional mechanical stimulation component, including 3D preloading with static and dynamic pressure, baseline strain, and dynamic increases in strain during active cycles to simulate myocardial mechanical forces, with adjustable parameters to induce fibrosis and simulate pathological conditions from early fibrosis to advanced fibrosis;providing at least one biochemical environment component selected from a group consisting of exogenous fibrotic agents, including TGF-β, angiotensin II, and macrophage, applied in varying concentrations to induce controlled levels of fibrosis at different stages;wherein the multi-modal functional stimulation method is configured to induce a quantitative range of fibrosis levels, including preservation of native myocardial phenotype, induction of early fibrotic transition, generation of a balanced native and fibrotic tissue model, and induction of advanced fibrotic state, and wherein the system is configured to facilitate drug screening and evaluate anti-fibrotic therapies using real-time data-driven curve fitting techniques.A method for quantitatively controlling reprogramming and reversing fibrosis in cardiac tissue, comprising:inducing fibrosis in cardiac tissue within a bioreactor system using synchronized 3D mechano-electrical stimulation protocols, wherein fibrosis is initiated in the tissue across a gradual range from early fibrosis to advanced fibrosis;quantifying fibrosis at each stage of fibrosis using real-time curve fitting of electrophysiological and mechanical data, including conductivity changes, tissue compliance, and strain-stress response curves, and correlating each stage of fibrosis to a quantitative fibrosis fraction function and mechanical properties of the tissue, including contractility and stiffness;administering specific antifibrotic drugs, to induce mechanical modulation of tissue stiffness and contractility, wherein the administration of the antifibrotic drugs is mediated by specific signalling pathways that influence myofibroblast -to-fibroblast transition, fibrotic ECM deposition, and mechanotransduction;monitoring the reprogramming of tissue layers fibrosis within the tri-layer cardiac tissue, including layer-specific reprogramming and differentiation of cardiac cells under mechanical stimulation, and modulating each layer's stiffness based on drug treatments;providing a mesh network of fluid microjet penetrated channels within the tri-layer cardiac tissue to deliver TGF-β and other fibrotic agents directly into the tissue, wherein the mesh network mimics natural ECM structures and exerts localized strain and stress to enhance tissue mechanical integrity and fibrosis induction;continuously tracking real-time mechanical responses and electrophysiological markers, generating real-time quantitative curves, and adjusting treatment protocols for fibrosis reversal based on the stage of fibrosis, including changes in drug delivery, electrical stimulation, and mechanical loading; andmodulating and synchronizing multi-layered cardiac tissue by applying coordinated three-dimensional mechanical, electrical stimuli, and biochemical cues within a bioreactor, wherein the strain and stress profiles of each individual tissue layer are independently regulated to achieve antifibrotic remodeling across the cardiac tissue,wherein the real-time curve fitting analysis is utilized to predict the effects of antifibrotic drug treatment, optimizing the dosage and timing for fibrosis resolution by targeting the TGF-β pathway, kinase signalling, conexine 43 and mechanotransduction pathways.The method of claim 4, wherein the antifibrotic drug is pirfenidone, which regulates TGF-β signalling to modulate the activation of myofibroblasts and the deposition of ECM, and inhibits fibrosis-associated kinases, such as Conexine 43, MAPK and mTOR, to reduce mechanical stiffness and contraction of the tissue.A bioreactor for assessment of physiopathological conditions of a 3D thri-layer heart tissue and to induce quantitative fibrosis development and measure mechanical and electrical parameters of the heart tissue, comprising:a sealed tank for holding a liquid solution;a first gas valve attached to the tank;at least a pH sensor; the pH sensor in placed inside the liquid solution;at least two pressurized compressed gas capsules, the capsules contain oxygen and carbon dioxide and are connected to the tank by means of a set of second gas valves;at least a cylinder and piston set driven by a first linear actuator, the cylinder is connected to the tank and contains the liquid solution;a gas pressure sensor, the gas pressure sensor is attached to the tank;at least a plurality of chambers, the chambers are connected to the tank and the heart tissue is placed in the chambers;at least a liquid pressure sensor connected to each chamber;at least a liquid valve connected to output of each chamber;at least a flow sensor connected to output of each liquid valve;a multi-input multi-output closed loop controller, the close loop controller utilizes the first linear actuator to displace the liquid solution, the gas pressure sensor to measure pressure of gas above the liquid, the first and second gas valve to regulate gas injection to the liquid in the tank, the liquid pressure sensor to measure the pressure of liquid, the liquid valve and the flow sensor to control flow of the liquid, where in the multi-input multi-output closed loop controller emulate mechanical condition on the heart tissue and the controller is capable of independently controls shear stress, static pressure, and flow rate of the liquid solution on the heart tissue;at least a plurality of mechanical actuator apparatus embedded inside each chamber, comprises at least two force applicators configured to apply forces to the heart tissue, wherein the force applicators comprise magnetic coils and neodymium magnets, a measurement and control system for measuring and controlling mechanobiology parameters related to the heart tissue, wherein the measurement and control system comprises at least one high speed camera for capturing images of visual indicia associated with the force applicators, and wherein the measurement and control system uses image processing to determine the position of the force applicators and adjusts the forces applied to the heart tissue based on feedback from the cameras and the calculated changes in length, and a processor for statistically determining the stiffness of the heart tissue based on the calculated changes in length and forces applied by means of the mechanical actuator apparatus and based on user input and feedback from the measurement system over a specific time period;a high-pressure fluid microjet apparatus for injecting the liquid solution or medicinal substances directly into the heart tissue, the apparatus comprises a second cylinder and piston set driven by a second linear actuator configured to generate a high-pressure jet of fluid, a fluid delivery system for directing the high-pressure fluid jet to the heart tissue and creating an artificial channel for circulation of the fluid and additional substances, a three-way valve and a tube for guiding the fluid jet, and a syringe pump connected to the three-way valve, wherein the high-pressure fluid microjet apparatus is capable of controlling the delivery of additional substances, such as drugs, cells, or growth factors, into the fluid jet using the syringe pump connected to the three-way valve; andan electrical measuring and stimulation apparatus for measuring electrical impedance and conduction velocity of the heart tissue and stimulating the heart tissue.

Images