WO2025159990A1

WO2025159990A1 - Engineered tissues and bioreactors and methods for producing them

Info

Publication number: WO2025159990A1
Application number: PCT/US2025/012108
Authority: WO
Inventors: Mark Skylar-Scott; Luca ROSALIA; Fredrik Samdal SOLBERG; Allison Sihan Jia; Jonathan Weiss; Mark Cutkosky; Samuel FRISHMAN; Katelyn MOSLE
Original assignee: Leland Stanford Junior University
Current assignee: Leland Stanford Junior University
Priority date: 2024-01-22
Filing date: 2025-01-17
Publication date: 2025-07-31
Anticipated expiration: 2026-07-22

Abstract

Provided herein are methods for producing a cultured tissue that mimics a natural tissue or organ. In some embodiments, the methods comprise culturing cells in a three-dimensional (3D) pattern on a scaffold, and applying an exogenous force to the cells while the cells are cultured. The applied exogenous force induces the cultured cells to form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ. Also provided herein are bioreactors for producing a cultured tissue that mimics a natural tissue or organ. In certain embodiments, the bioreactors comprise: a scaffold for culturing cells in 3D pattern, and an exogenous force applicator to exert an exogenous force to the cells cultured in the 3D pattern. Additional embodiments provide cultured tissues that mimic natural tissues or organs as produced according to the methods disclosed herein.

Description

ENGINEERED TISSUES AND BIOREACTORS AND METHODS FOR PRODUCING

THEM

GOVERNMENT SUPPORT CEAUSE

[0001] This invention was made with government support under contract AY1 AX000002-01 awarded by the Advanced Research Projects Agency - Health and under contract DGE-1656518 (FELLOWSHIP) awarded by the National Science Foundation. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims the priority benefit of United States Provisional Application No. 63/623,715, filed January 22, 2024, and which is incorporated herein by reference in its entirety.

BACKGROUND

[0003] Organ transplants are used to replace failing or diseased organs and are life-saving in many instances. For many reasons, availability is limited for organs suitable for transplants. For example, worldwide about 50,000 people per year need heart transplants; however, only about 5,000 heart transplants are performed.

[0004] With the advancement of tissue engineering and three-dimensional (3D) printing technologies, producing cultured tissues/organs is attempted to fill the gap between the demand and supply of organs suitable for transplant. However, a major challenge is to produce engineered tissues that mimic the properties, such as functional and structural properties, of naturally occurring tissues or organs. For example, cardiac tissue organization is complex and engineered cardiac tissue would require precise alignment of cardiomyocytes such that the engineered cardiac tissues mimic the cell contraction and function of the natural heart.

[0005] Current approaches include a spectrum of hemodynamic, electrical, and mechanical stimulation. For example, cardiomyocytes align in the direction of higher stress. (Lu et al. (2021), Theranostics, 11(13):6138-6153 and Lind et al. (2017), Nature Mater, 16, 303-308.) However, this is only demonstrated in a two-dimensional (2D) setting, which cannot produce twisting and contractile cardiac mechanics found in the 3D heart. SUMMARY

[0006] In certain aspects, the disclosure provides methods for producing cultured tissues that mimic a natural tissues or organs, particularly, the functional and structural properties of natural tissues or organs.

[0007] In some embodiments, the methods comprise culturing cells in a 3D pattern on a scaffold, and applying an exogenous force to the cells while the cells are cultured. The applied exogenous force induces the cultured cells to form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ.

[0008] In certain aspects, the disclosure relates to bioreactors for producing cultured tissues that mimic natural tissues or organs. In certain embodiments, the bioreactors comprise: a scaffold for culturing cells in a 3D pattern, and an exogenous force applicator to exert an exogenous force to the cells cultured in the 3D pattern.

[0009] In further aspects, the disclosure provides cultured tissues that mimic natural tissues or organs as produced according to the methods disclosed herein. In certain such cultured tissues, a scaffold comprising cells cultured in a 3D pattern is subjected to an exogenous force that induces the cultured cells to form a cultured tissue that mimics the 3D cellular pattern and biomechanics of a natural tissue or organ.

[0010] In certain embodiments, the disclosure provides methods and devices for producing a cultured tissue that mimics the 3D cellular pattern and biomechanics of a natural heart.

[0011] Further embodiments of the disclosure provide methods for transplanting a cultured tissue that mimics a natural tissue or organ produced according to the methods described herein.

[0012] Even further embodiments of the disclosure provide methods for evaluating test agents by contacting the test agents to a cultured tissue that mimics a natural tissue or organ produced according to the methods described herein. Certain such methods comprise contacting the cultured tissues with the test agent and analyzing the effect of exposure to the test agent on the cultured tissue.

INCORPORATION BY REFERENCE

[0013] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0015] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0016] FIGS. 1A-1E. Exemplary bioreactors. A and B show top view of exemplary bioreactors that could be used to produce a cultured tissue in a tubular shape. C shows the side view of an exemplary bioreactor that could be used to produce a cultured tissue in a tubular shape. D shows the effects of rotating/twisting the scaffold at 90° and 180° angles.

[0017] FIGS. 2A-2B. Exemplary scaffold for a cultured tissue in a tubular shape. A shows silicone-gelbrin adhesion and resulting scaffold. B shows silicon fibrin adhesion and resulting scaffold.

[0018] FIGS. 3A-3C. Exemplary bioreactors. A and C show side views of exemplary scaffolds that could be fitted into a bioreactor used to produce a cultured tissue in a ventricular shape. B shows top view of an exemplary scaffold that could be fitted into a bioreactor used to produce a cultured tissue in a ventricular shape.

[0019] FIGS. 4A-4C. Exemplary scaffold for a ventricular cultured tissue. A and C show side views of exemplary scaffolds that could be fitted into a bioreactor used to produce a cultured tissue in a ventricular shape. B shows top view of an exemplary scaffold that could be fitted into a bioreactor used to produce a cultured tissue in a ventricular shape.

[0020] FIG. 5. The computational finite element analysis of the human-size ventricle.

[0021] FIGS. 6A-6F. Silicone-tissue co-printing and 3D mechanostimulation workflow.

[0022] FIGS. 7A-7G. 3D bioprinting and 3D mechanostimulation.

[0023] FIGS. 8A-8C. Tissue alignment resulting from 3D mechanostimulation.

[0024] FIGS. 9A-9D. Exemplary core-double shell nozzle. A shows nozzle channels for the bioink (outer shell), core ink, and scaffold ink (inner shell) in front/back view. B shows nozzle drawing in side view. C shows nozzle inlets (top view). D shows nozzle outlet (bottom view).

[0025] FIGS. 10A-10E. Exemplary process of co-printing of scaffold and tissue of ventricular shape (endocardial configuration) using concentric tube robotics. DETAILED DESCRIPTION

[0026] Before the methods, devices, and compositions of the present disclosure are described in greater detail, it is to be understood that the methods, devices, and compositions are not limited to the embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing the embodiments only, and is not intended to be limiting, since the scope of the methods, devices, and compositions will be limited only by the appended claims.

[0027] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both the limits, ranges excluding either or both of those included limits are also included.

[0028] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and devices belong. Although any methods, devices, and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods, devices, and compositions, representative illustrative methods and devices now described.

[0030] It is noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only,” and the like in connection with the recitation of claim elements or use of a “negative” limitation. [0031] It is appreciated that certain features of the methods, devices, and compositions which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and devices, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each combination was individually and explicitly disclosed, to the extent that such combinations embrace operable methods, devices, and/or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods, devices, and compositions and are disclosed herein just as if each such sub-combination was individually and explicitly disclosed herein.

[0032] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods, devices, and compositions. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0033] Recent developments in biofabrication have enabled the development of engineered tissues for various organ systems, supporting applications in drug testing and regenerative medicine. However, current approaches do not allow for dynamic mechanical maturation of engineered tissues in 3D. Although uniaxial mechanostimulation techniques have shown promise in generating anisotropic tissues, they fail to recapitulate the biomechanics of complex tissues. As a result, existing biofabricated tissues lack the ability to replicate complex 3D alignment patterns essential for functional biomimicry.

[0034] Although ID mechanostimulation approaches have led to the development of anisotropic tissues, such as skeletal muscle, tendons and ligaments, and cartilage, these techniques are not sufficient to engineer biomimetic tissues with 3D biomechanical function, such as the heart. In fact, the heart offers a notable example of how replicating the complex 3D alignment at both the cellular and tissue levels is crucial to achieving its physiologic function. In the native cardiac ventricle, myocardial cells are aligned along complex 3D helical patterns to maximize cardiac output.

Uniaxial cell contraction along these complex patterns generates shortening in both the circumferential and longitudinal directions, as well as twisting of the left ventricle. [0035] Methods for inducing alignment in engineered cardiac constructs can be broadly divided into passive and active approaches. Passive techniques rely on anisotropic materials on which cardiomyocytes are seeded, while active stimulation involves the use of dynamic forces to promote tissue maturation. Passive conditioning is often achieved by modulating substrate stiffness and topography or applying static stresses to cardiac tissues. For example, substrates like polyacrylamide gel or poly dimethyl siloxane with elastic moduli of 10-20 kPa have been shown to promote cell alignment and increase cell spread area. Additionally, modifying substrate topography can enhance the anisotropic organization of cardiac tissue. In 3D constructs, fiber-infused anisotropic gel scaffolds drive cell alignment in a 3D-printed ventricle. Static stress facilitates sarcomere rearrangement and increases internal tension, advancing tissue maturation. For instance, one study demonstrated that maintaining hiPSC-CM-derived microtissues under static tension for two weeks using nylon tabs led to improved cell alignment, cardiomyocyte hypertrophy, enhanced contractility, increased passive stiffness, and better force-frequency relationships. Similarly, static stresses applied to 3D-printed cardiac tissue grafts from hiPSC-CMs improved cell alignment, contractile force, extracellular matrix organization, and upregulated cardiac-specific gene expression. Recently, bioprinted anisotropic building blocks of hiPSC-CMs have been developed by leveraging the shear forces experienced by the printed cells upon extrusion and programming anisotropy by varying the printed path. Based on this approach, applied shear stress, stretching or extension force, and post-print deformation can be manipulated to create aligned structures in 3D cardiac tissues.

[0036] Active mechanical stimulation can further influence cardiomyocyte maturation, alignment, and contractile function. Uniaxial tensile forces are applied using mechanical, dielectric or pneumatic actuators. Variations in these systems were developed to apply different regimes of uniaxial mechanical stimulation, including simple or cyclic uniaxial stretch or compression, for up to 8 days of stimulation. In hiPSC-CM monolayers, contractile stress was found to increase with higher strain magnitudes, but reached a plateau at 15% strain. These studies supported that cyclic stimulation results in increased cellular elongation and enhanced physiological phenotypes. Other studies have shown that active stimulation can support cardiomyocyte alignment in 3D constructs as well as 2D. However, these studies limit their mechanical stimulation to uniaxial loading, which is not representative of the complex 3D mechanics of the native heart.

[0037] Certain embodiments of the disclosure describe methods to drive programmable 3D alignment of bioprinted tissue using tunable soft robots. This method combines the advantages of active mechanical stimulation and tailorable by programmably tuning the dynamic triaxial tissue strain. In certain cases, to produce 3D patterns of biohybrid constructs comprising tissue and silicone, biological tissue was co-printed with a biocompatible, room-temperature vulcanizing silicone. This produced a tissue with an adhesive and integrated silicone soft robot. Pressurization of the silicone provided expanding forces to the cells, while twisting of the silicone-tissue construct was applied by a servo motor. Each of these 3D mechanostimulation modalities was found to result in a specific pattern of cellular orientation, as observed via confocal imaging. Although soft robots had demonstrated their ability to recapitulate the motion and function of organ tissues in a physiological manner, the methods described herein demonstrate their application in 3D biomanufacturing and bioprinting to address complex tissue engineering challenges.

[0038] Thus, the disclosure presents the development of a soft robotics-driven approach for programmable 3D alignment in bioprinted tissues. By integrating a silicone-based soft robot with biological tissue using a custom core-double shell nozzle, the application of dynamic, exogenous 3D forces can effectively promote cell alignment within the engineered tissues. Confocal imaging confirmed that the stimulated samples exhibited notable anisotropy compared to their unstimulated counterparts. In addition, the cellular orientation patterns seen with various mechanostimulation modalities indicate the versatility and promise of this approach in improving the functional mimicry of complex tissues. Particularly, pressurization of the constructs resulted in circumferential cellular alignment, whereas the combination of expansion and twisting led to angles analogous to those observed in native heart tissue. This research paves the way towards the development of biomimetic tissues with complex patterns of cellular alignment, bridging the gap between tissue engineering and pressing clinical challenges.

METHODS - PRODUCING A CULTURED TISSUE THAT MIMICS A NATURAL TISSUE OR ORGANS

[0039] The term “cultured tissue” as used herein refers to 3D cell aggregate. In addition to cells, a cultured tissue may comprise reinforcements/framework, such as columns, fibers, rings, and the like. Further, a cultured tissue may comprise extracellular matrix material, for example, extracellular matrix proteins, such as collagens, elastins, fibronectins and laminins, fibrillins, fibulins, matrilins, tenascins, and thrombospondins.

[0040] The term “mimics a natural tissue or organ” indicates that a cultured tissue produced according to the methods disclosed herein exhibits 3D cellular pattern and biomechanics of a natural tissue or organ. Thus, the cultured tissue exhibits properties of a natural tissue or organ, such as functional or structural properties of a natural tissue or organ. Thus, the term “a cultured tissue mimics a natural heart” indicates that the cultured tissue has cells that are present in a natural heart, for example, cardiomyocytes, and that are arranged in a 3D pattern such that the cardiomyocytes contract and relax to produce heart-beating effects similar to the effects observed in a natural heart. Similarly, the term “a cultured tissue mimics a natural muscle” indicates that the cultured tissue has cells present in a natural muscle, for example, myocytes, and that are arranged in a 3D pattern such that the myocytes contract and relax to produce muscle contraction and relaxation similar to that observed in a natural muscle. Further, the term “a cultured tissue mimics a natural artery” indicates that the cultured tissue has cells present in a natural artery, for example, various layers of cells in the basement membrane, tunica intima, tunica media, and tunica externa, and that are arranged in a 3D pattern such that the cells produce arterial structural and function similar to that observed in a natural artery.

[0041] The term “biomechanics of a tissue or organ” refers to structure, function, and motion of a tissue or organ. For example, the heart pumps blood throughout the body and specific cardiac mechanics is responsible for such function. Particularly, pumping of blood in a heart requires specific biological processes and mechanical stress generated by specifically located and functional cardiomyocytes in combination with other cardiac structures, such as extracellular matrix structures that control the heart function. Thus, “biomechanics of the heart” refers to the structure, function, and motion of the natural heart that is controlled by the specific location and function of cardiomyocytes and other cells within the heart.

[0042] The term “mechanostimulation” as used herein refers to application of 3D, exogenous, dynamic expansion, torsional, and/or other types of forces to a cultured tissue. Forces exerted on a cultured tissue for such mechanostimulation are designed to induces the cultured cells to form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ. [0043] In some aspects, the disclosure provides a method for producing a cultured tissue that mimics a natural tissue or organ, the method comprising: a) culturing cells in a 3D pattern on a scaffold, b) applying an exogenous force to the cells while the cells are cultured in step a). The applied exogenous force induces the cultured cells to form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ.

[0044] The cells cultured in the methods disclosed herein can comprise one or more cells suitable for producing a cultured tissue that mimics a desired natural tissue or organ. For example, to prepare a cultured tissue that mimics a natural heart, cells found in the natural heart, such as cardiomyocytes, epithelial cells, and connective tissue cells can be cultured in a 3D pattern that mimics the arrangement of these cells in the natural heart.

[0045] Similarly, depending on the desired natural tissue or organ, any suitable cells could be cultured that mimic the arrangement of such cells in a natural tissue or organ. Such cells include but are not limited to muscle cells, epithelial cells, connective tissue cells, blood cells, germ cells, stem cells, endothelial cells, immune cells, glandular cells, and the like.

[0046] In specific embodiments, a cultured tissue mimics the natural heart. However, any suitable tissue or organ can be produced according to the methods disclosed herein. Certain such tissues or organs include spleen, kidney, urinary duct, heart, blood, gonad, muscle, skeleton, dermis, connective tissue, liver, pancreas, pharynx, esophagus, stomach, intestinal tract, lung, thyroid, parathyroid, or thymus.

[0047] In some cases, the cells used to produce a cultured tissue are obtained from a known subject. In some cases, a subject from which such cells are derived requires a transplant of the concerned tissue or organ. A cultured tissue or organ produced from such cells contain cells that are autologous to the cells of the subjects from which the cells are obtained.

[0048] In some cases, the cultured cells are co-printed with a suitable scaffold material. A suitable scaffold material can be biocompatible and provides suitable microenvironment for optimal cell growth and function of the cultured cells. In some cases, the scaffold material can be biodegradable such that it gets degraded and replaced with the growing cultured cells. In some cases, the growing cultured cells substantially replace the scaffold material such that the final cultured tissue comprises none to very little of the scaffold material.

[0049] In some cases, the scaffold-tissue constructs are co-printed using a core-double shell nozzle, that can extrude multi-material concentric tubes. Extrusion to the core may be provided to allow the patency of the constructs during and after the printing process.

[0050] In some cases, the scaffold-tissue constructs are printed using concentric tube robotics or other techniques.

[0051] In some cases, the scaffold and tissues can be manufactured separately and then combined for the active scaffold to provide complex and biomimetic mechanostimulation via exogenous forces to the tissue. Adhesives or other types of mechanical couplers may be used to integrate the tissue into the active scaffold.

[0052] A suitable scaffold material also provides mechanical support for the cultured cells and is also sufficiently flexible to allow movement of the cultured cells under an exogenous force applied to the cells. Certain non-limiting examples of such scaffold materials include silicone and silicone derivatives, alginate, ovalbumin, titanium, porous polyethylene glycol, polycaprolactone, silk gland fibroin, collagen, elastin, PolyHIPE polymer, chitosan, collagen-coated poly-lactide-co-glycolide- gelatin/chondroitin/hyaluronate, poly-lactic-co-glycolic acid, hydroxyapatite, bone morphogenetic protein, coralline hydroxyapatite, P-tricalcium phosphate, silk fibroin, gelatin, collagenglycosaminoglycan, fibrin, and gelbrin.

[0053] Certain scaffolding materials that could be used in the methods disclosed herein are described by Loh et al. (2013), Tissue Eng Part B Rev , 19(6):485— 502 and the references cited therein, the contents of which are herein incorporated by reference in their entireties. Additional materials that could be used as scaffold materials in the methods disclosed herein are well known in the art and use of such materials is within the purview of the disclosure.

[0054] In certain embodiments, the scaffold material is silicon in an appropriate shape designed based on the desired tissue or organ. For example, a silicon tubing can be used to prepare cultured tissue in a tubular shape, which can be used for preparing a cultured tissue that mimics a natural blood vessel or any other natural tissues or organs that have tubular structures, such as a vascular tissue, muscles, epithelial tissues, channels, ducts, and the like. Similarly, silicon in ventricular shape can be used to produce a ventricular shaped cultured tissue, which can be used to produce a cultured tissue that mimics a natural heart.

[0055] An exogenous force can be applied to the cultured cells via applying such forces to the scaffold material. In this description, the word ‘exogenous’ is used to encompass the forces applied to the tissue that are distinct from the ‘endogenous’ forces arising from the cardiomyocyte contraction. These exogenous forces may be applied using actuators that are surrounding the scaffold or are embedded within the scaffold. For example, exogenous forces applied to a silicone tube that lies adjacent to or contacting co-printed cells would be transmitted to the cultured cells. [0056] In some cases, the exogenous force is applied using one or more of: pneumatic actuators, electric actuators, hydraulic actuators, piezoelectric actuators, shape memory alloys, electromagnetic actuators, thermal actuators, and muscle wire, and nitinol actuators.

[0057] In some cases, the exogenous force is applied to the scaffold material via supporting structures, such as fibers, columns, rings, etc. that are embedded in the scaffold. For example, specifically arranged fibers in a ventricular shaped silicone sleeve can be used to apply an exogenous force to the silicone sleeve. (FIGS. 3A-3C and 4A-4C.) [0058] The exogenous force applied to a cultured tissue is designed to induce the cultured cells to form a cultured tissue that mimics a desired 3D cellular pattern for optimal mechanical and electrical function of an engineered heart tissue: for example optimized ejection fraction and electrical synchronicity. In some cases, this may mimic the biomechanics of natural tissue, such as the heart, but in other cases may be optimized for arbitrary organ shape, such as a sphere or cylinder. Thus, the exogenous force can be one or more of: expanding force, twisting force, and elongation force that would induce the cells in the cultured tissue to for the 3D cellular pattern and biomechanics of the desired tissue or organ.

[0059] The term “expanding force” refers to a force that induces expansion of the 3D pattern of the cultured cells. For example, if cells are cultured in a tubular shape, expansion force would cause an increase in the diameter of the tube thereby inducing expansion of the tubularly arranged cells. [0060] The term “twisting force” refers to a force that induces relative rotation or twisting of the 3D pattern of the cultured cells. For example, if the cells are cultured in a tubular shape, twisting force would cause twisting of the tube so that the cultured cells move off the longitudinal axis as compared to the cells’ position without the exogenous twisting force.

[0061] The term “elongation force” refers to a force that induces increase in the length of the 3D pattern of the cultured cells. For example, if cells are cultured in a tubular shape, elongation force would increase the length of the tube thereby inducing stretching of the cells along the longitudinal direction.

[0062] Cardiomyocytes align in the direction of higher stress. Therefore, if a cultured tissue comprising cardiomyocytes is subjected to exogenous forces in specific directions or patterns, they would induce the cardiomyocytes in the 3D pattern to align in the direction of the applied exogenous forces.

[0063] Certain aspects of the biomechanics of the natural heart are described by Nakatani (2011), J Cardiovasc Ultrasound, 19(1): 1-6. As described by Nakatani, in a natural heart, specific arrangement of cardiomyocytes in the sub-endocardium and sub-pericardium produces specific clockwise and anti-clockwise rotation of heart walls to produce the various contractions and relaxations that lead to the pumping of the blood in and out of the heart.

[0064] To reproduce such effects in a cultured tissue, in certain embodiments of the methods disclosed herein, cardiomyocytes, such as IPSC-derived cardiomyocytes, are cultured between a ventricular shaped silicone outer sleeve and a silicone inner sleeve as shown in FIGS. 4A-4C. The outer silicone sleeve can comprise fibers arranged in a left-handed helix that can be used to apply counterclockwise twisting (when observed from the base) thereby mimicking rotation of myocardial fibers in subpericardium of a natural heart. The inner silicone sleeve can comprise fibers arranged in a right-handed helix can be used to apply clockwise twisting (when observed from the base) thereby mimicking rotation of myocardial fibers in sub endocardium of a natural heart. Such fibers can be made from silicone and provide sufficient support and flexibility.

[0065] In the left and right-handed helices used in the outer and inner silicone sleeves, different parameters can be modified. For example, number of helices and helix pitch can be varied as desired. For example, the number of helices/fibers can be from about 5 to 30, such as 5, 10, 15, 20, 25, or 30 fibers. The pitch of the helices can vary from 5 cm to 100 cm, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 cm.

[0066] Also, material properties of the actuatable sleeve and helices can be varied. For example, the actuatable sleeve and helices can have the Young’s modulus from about 100 kPa to about 100 MPa, such as 100 MPa, 1 MPa, 10 MPa, 20 MPa, 30 MPa, 40, MPa, 50 MPa, 60 MPa, 70 MPa, 80 MPa, 90 MPa, or 100 MPa.

[0067] In some cases, the orientation of helices can be symmetric. Alternatively, the orientation of the helices can be asymmetric, i.e., it can be modified to mimic exact anatomical patterns, either in a generalized or patient-specific fashion.

[0068] A ventricular cultured tissue can be treated with pressurization of the silicone sleeve. Actuation pressures, for example, from about 0.5 to 15 psi, can be used to enable motion of the sleeve and tissue alignment.

[0069] FIG. 5 shows the effects of pressurization of the silicone sleeve on the cultured tissue. Particularly, this figure shows the computational finite element analysis of the human-size ventricle. The left panel shows the un-deformed sleeve and the right panel shows the deformed geometry. Color map illustrates the von mises stress of the sleeve, illustrating the ability of the sleeve to recapitulate the motion of the healthy heart in a physiologic manner.

[0070] The frequency and duration of exogenous force can be adjusted based on the desired tissue or organ. For example, to prepare a cultured tissue that mimics a natural heart, the outer and inner silicone sleeve can be subjected to exogenous force at a frequency from about 0.5 to 5 Hz, such as at a frequency from about 0.5 to 2 Hz, particularly, at a frequency of about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 Hz. Also, the exogenous force can be applied over a period of 1 week to 2 weeks, such as 1 week, 2 weeks, 3 weeks, or 4 weeks. [0071] Depending on the desired natural tissue or organ, the cells in a scaffold can be cultured from about 1 week to about 4 months, such as 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3 months, or 4 months.

[0072] Depending upon the desired natural organ or tissue, a cultured tissue can be subjected to the exogenous force on a continuous basis or an intermittent basis. For example, a cultured tissue can be subjected to periods where an exogenous force is applied separated from periods where an exogenous force is not applied. Alternatively, a cultured tissue can be continuously subjected to an exogenous force. Also, when an exogenous force is applied, its type and/or intensity can be varied. [0073] In certain embodiments, after a time in culture, it may be desirable to remove the cultured tissue from the force actuators, for example, by cutting, releasing, removing, or sliding the tissue off from the actuators. For example, after about 1 week to 3 months of culturing, the exogenous forces may have sufficiently driven cardiomyocyte alignment. Alternatively, instead of manually removing the actuators, the actuators may also be derived from biodegradable materials, such as collagenase sensitive poly(ethylene glycol) diacrylate that incorporates a collagenase-sensitive peptide in its backbone. After the actuators are removed or degraded, the cultured tissue would retain the 3D cellular pattern and biomechanical properties.

[0074] The term “an exogenous force is continuously applied” includes the application of exogenous force in cycles of short duration of time, for example, from 0.5 second to 10 seconds; however, the exogenous force is not interrupted for a longer duration, such as more than 5 minutes. A cycle of exogenous force can have shorter periods of time where an exogenous force is not applied. For example, in a 2 second cycle, an exogenous force can be applied in the first half a second, followed by a gap of one second, followed by an exogenous force applied for another half a second. Appropriate cycles can be designed based on the desired natural tissue or organ to be produced. [0075] In certain embodiments, a cultured tissue is subjected to an exogenous force to induce the cultured cells to mimic the 3D cellular pattern and biomechanics of the natural heart. To that end, appropriate cells, such as cardiomyocytes, can be printed or embedded in a scaffold comprising silicone and, optionally, additional materials, such as extracellular matrix proteins. Once the printed cultured cells in scaffold are produced, the exogenous force is applied to mimic the forces experienced by heart cells in a natural beating heart. Thus, the exogeneous forces are applied in cycles without interruption. In a natural beating heart, in a given cycle of a heartbeat, the heart cells contract for about 30% of the time and relax for the remainder of the time. Accordingly, to induce the cultured cells to mimic natural heart cells, exogenous forces are applied in uninterrupted cycles, where within each cycle, the exogenous force is applied from between 20% to 50% of the cycle and the cells are allowed to relax for remainder of the cycle. Each cycle can be from about half a second to about two seconds.

[0076] In addition to exogenous force, electrical stimulation can also be applied to the cultured cells, for example, through a conductive silicone material. Electrical stimulation from any conductive material to the tissue can be applied from about 1A to about 10A, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 A. Electrical stimulation from any conductive materials to the tissue can be applied from 1 V/cm to about 10 V/cm, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 V/cm.

[0077] In certain embodiments, applying the electrical stimulation comprises providing biphasic current to a conductive material mounted in the scaffold. This electrical stimulation may be performed in phase or out of phase with the mechanical stimulation.

[0078] In addition to the exogenous force, the cultured cells can also be subjected to hydrodynamic stimulation. The hydrodynamic stimulation can be a continuous or pulsatile flow. The flow can be varied from about 0.5 L/min to about 5 L/min, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 L/min. The hydrodynamic stimulation is designed so as to induce the cultured cells to experience the natural shear forces that some cells, for example, endothelial cells, experience in a natural tissue or organ. [0079] In the methods of producing a cultured tissue, the cultured cells or tissue can be cultured in an appropriate medium. Also, the medium can be changed at a suitable time depending on the type of cells cultured.

[0080] Non-limiting examples of the media that could be used to culture cells in the methods disclosed herein include: Dulbecco's Modified Eagle Media (DMEM), Minimal Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimal Essential Medium (G-MEM), Basal Medium Eagle (BME), DMEM/Ham's F12, Advanced DMEM/Ham's F12, Iscove’s Modified Dulbecco's Media and Minimal Essential Media (MEM), Ham's F-10, Ham's F-12, Medium 199, and RPMI 1640 Media.

[0081] The media can comprise appropriate nutrients and supplements, such as fetal bovine serum (FBS) or a combination of one or more growth factors, cytokines, and the like.

[0082] A person of ordinary skill in the art can determine an appropriate medium for culture specific cells and such embodiments are within the purview of the disclosure.

DEVICES - A BIOREACTOR FOR PRODUCING A CULTURED TISSUE THAT MIMICS A NATURAL TISSUE OR

ORGAN [0083] The devices of producing cultured tissues disclosed herein can be used to apply to the cultured tissues exogenous forces in 3D. Conventional methods of culturing tissues can only apply forces in 2D, for example, having the effect of stretching the cultured tissues in one plane or in ID along one axis. However, natural tissue motion, for example, natural cardiac motion occurs in 3D. Therefore, it is important that cultured tissue, for example, cultured tissue that mimics a natural heart comprises cells that are aligned along complex 3D trajectories within the natural organ or tissue, such as natural heart.

[0084] In the natural heart, these 3D trajectories resemble helices, i.e., the cells are aligned along helices. This arrangement causes the heart to twist and contract both radially and longitudinally to pump the blood in and out of heart chambers. Therefore, to reproduce cardiac function in a cultured tissue, such cultured tissue must follow the alignment of the complex 3D forces observed in the natural heart.

[0085] The devices and methods disclosed herein can be used to apply external forces to cultured cells to re-create the complex force trajectories of a natural tissue or organ, for example, the natural heart. In some cases, this can be achieved by a combination of expansion, for example, via pressurization, and/or twisting, e.g., via servo motor rotation or via complex 3D motion of a mechanically-programmed scaffold, such as mechanically programmed soft robotic actuators.

[0086] Accordingly, certain aspects of the disclosure provide a bioreactor for producing a cultured tissue that mimics a natural tissue or organ, the bioreactor comprising: a scaffold for culturing cells in a 3D pattern, an exogenous force applicator to apply the exogenous force to the scaffold so as to exert the exogenous force to the cells cultured in the 3-dimensional pattern.

[0087] In the bioreactors disclosed herein, under the applied exogenous force, the cultured cells form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ.

[0088] The cultured cells can be in a scaffold in a 3D pattern in a tubular shape. The scaffold can be operably connected to a motor that can twist the tubular shaped scaffold, as shown in FIGS. 1 A to 1C. For example, the motor can rotate/twist the tubular scaffold by 90° or 180° angles as shown in FIG. ID.

[0089] The motor can be configured to apply the exogenous force at regular intervals or cycles. For example, each cycle of applying an exogenous force can be from about 0.5 seconds to 10 seconds, such as 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds. In some cases, the forces can be applied from about 20% to about 70% of the duration of the cycle. In some cases, the amplitude of the mechanical strain will be changed or ramped gradually over time, for example starting at 1%, 2, 5, or 10% strain and ending at 20%, 25%, 30%, 35%, 40%, 45%, or 50% strain. In addition to amplitude ramping, the frequency of strain may be changed gradually over time, increasing from 0.25, 0.5, 1.0 Hz to 1, 2, 3, 4, 5, 6 Hz over the course of from 1 day and 30 days.

[0090] In some cases, a bioreactor comprises a scaffold comprising cultured cells in the 3D pattern in a ventricular shape. The cultured cells in such ventricular shaped scaffold can be used to produce cultured tissue that mimics a natural heart. In certain such cases, the applied exogenous force comprises one or more of: expanding force, twisting force, and elongation force.

[0091] A suitable exogenous force applicator that could be used in the bioreactors disclosed herein can be one or more of: pneumatic actuators, electric actuators, hydraulic actuators, piezoelectric actuators, shape memory alloys, electromagnetic actuators, thermal actuators, and muscle wire, and ni tinol actuators.

[0092] In some cases, in addition to the exogenous force applicator, a bioreactor can further comprise an electrical stimulator for applying an electrical stimulation to the cultured cells. Such electrical stimulator can be configured to apply electrical stimulation from about 1 A to about 10A, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 A and from about 1 V/cm to about 10 V/cm, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 V/cm.

[0093] In certain embodiments, the electrical stimulator is configured to apply biphasic current to a conductive material mounted in the scaffold. In other embodiments, a point stimulator such as a platinum electrode, or patterned iPSC-derived nodal cells may be used to define the origin of electrical pacing. In some cases, the amplitude of the electrical pacing will be changed or ramped gradually over time to ensure continued propagation of cardiac conduction, for example starting at 5 V/cm, and ending at 10 V/cm. In addition to amplitude ramping, the frequency of pacing may be changed gradually over time, increasing from 0.25, 0.5, 1 Hz to 1, 2, 3, 4, 5, 6 Hz over the course of from 1 day to 30 days.

[0094] In certain embodiments, in addition to the exogenous force applicator, a bioreactor can further comprise a hydrodynamic stimulator for applying a hydrodynamic stimulation to the cultured cells. A hydrodynamic stimulation can be a continuous or pulsatile flow.

[0095] The bioreactor disclosed herein can comprise connections to the culture chamber for supplying or removing a culture medium to the cultured cells. The supply and/or removal of a culture medium could be performed manually or automatically using pumps, for example, automatically controlled pumps. COMPOSITIONS - A CULTURED TISSUE THAT MIMICS A NATURAL TISSUE OR ORGAN

[0096] The methods and devices described herein can be used to produce cultured tissues that mimic a natural tissue or organ.

[0097] Accordingly, certain aspects of the disclosure provide a cultured tissue that mimics a natural tissue or organ as produced from the methods described herein. In certain such methods, a cultured tissue is produced using the devices described herein.

[0098] In some cases, A cultured tissue that mimics a natural tissue or organ, the cultured tissue comprising a scaffold comprising cells cultured in a three-dimensional (3D) pattern, wherein an exogenous force is applied to the cultured cells, and wherein the applied exogenous force induces the cultured cells to form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ. In some cases, the cultured tissue mimics a natural heart.

[0099] In specific embodiments, a cultured tissue mimics the natural heart.

[0100] However, a cultured tissue can be produced according to the methods disclosed herein such that the cultured tissue mimics any suitable tissue or organ. Certain such tissues or organs include spleen, kidney, urinary duct, heart, blood, gonad, muscle, skeleton, dermis, connective tissue, liver, pancreas, pharynx, esophagus, stomach, intestinal tract, lung, thyroid, parathyroid, or thymus. In some cases, the scaffold used to culture the cells comprises reinforcement fibers. Depending on the natural tissue or organ that the cultured tissue mimics, the cells cultured in the scaffold can comprise one or more of: cardiomyocytes, muscle cells, epithelial cells, connective tissue cells, blood cells, germ cells, stem cells, endothelial cells, immune cells, and glandular cells. For example, cells cultured in a scaffold to produce a natural heart can comprise cardiomyocytes, epithelial cells, and connective tissue cells cultured in a 3D pattern that mimics the arrangement of these cells in a natural heart.

[0101] A suitable scaffold material used to culture the cells can be made from silicone and silicone derivatives, alginate, ovalbumin, titanium, porous polyethylene glycol, polycaprolactone, silk gland fibroin, collagen, elastin, PolyHIPE polymer, chitosan, collagen methacrylate, fibrinogenmethacrylate, collagen-coated poly-lactide-co-glycolide-gelatin/chondroitin/hyaluronate, poly- lactic-co-glycolic acid, hydroxyapatite, bone morphogenetic protein, coralline hydroxyapatite, |3- tricalcium phosphate, silk fibroin, gelatin, collagen-glycosaminoglycan, fibrin, and gelatin- fibrinogen blend. [0102] In some cases, the cultured cells are co-printed with a suitable scaffold material. In some cases, the scaffold material can be biodegradable such that it gets degraded and replaced with the growing cultured cells. In some cases, the growing cultured cells substantially replace the scaffold material such that the final cultured tissue comprises none to very little of the scaffold material. [0103] In some cases, the cultured tissue is in a tubular shape. Such tissue can mimic a natural blood vessel or any other natural tissues or organs that have tubular structures, such as a vascular tissue, muscles, epithelial tissues, channels, ducts, and the like.

[0104] In some cases, the cultured tissue is in a ventricular shape. Such tissue can mimic a natural heart.

[0105] The cultured tissue disclosed herein changes its shape when under an exogenous force and reverts to its original shape when the exogenous force is removed. This property is designed to confer to the cultured tissue 3D cellular pattern and biomechanics of the natural tissue or organ.

METHODS - TRANSPLANTING A CULTURED TISSUE THAT MIMICS A NATURAL TISSUE OR ORGAN

[0106] As noted above, cultured tissues produced by the methods described herein mimic a natural tissue or organ such that the cultured tissues can be used as tissue or organ for transplant.

[0107] Accordingly, certain embodiments of the disclosure provide a method of transplanting in a subject that requires an organ or tissue transplant a cultured tissue produced by a method disclosed herein. The subject can be a human or a non-human animal.

[0108] In specific embodiments, a transplanted cultured tissue mimics a natural heart.

[0109] However, any suitable tissue or organ can be transplanted with a cultured tissue produced according to the methods disclosed. Certain such tissues or organs include spleen, kidney, urinary duct, heart, blood, gonad, muscle, skeleton, dermis, connective tissue, liver, pancreas, pharynx, esophagus, stomach, intestinal tract, lung, thyroid, parathyroid, or thymus.

[0110] In specific embodiments, cells are obtained from a subject and cultured according to the methods disclosed herein to produce a cultured tissue. Such cultured tissue can then be transplanted into the subject. Because the cells are from the subject into which the cultured tissue is transplanted, the cells are autologous to the subject and, hence, the risk of graft rejection is minimized.

METHODS - EVALUATING TEST AGENTS USING A CULTURED TISSUE THAT MIMICS A NATURAL TISSUE

OR ORGAN [0111] Certain embodiments of the disclosure provide methods of testing test agents for potential therapeutic use. Certain such methods utilize cultured tissues produced according to the methods described herein.

[0112] Accordingly, certain embodiments of the disclosure provide a method for evaluating a test agent, the method comprising contacting the test agent with a cultured tissue produced according to the methods disclosed herein and analyzing the effect of exposure to the test agent on the cultured tissue.

[0113] Drugs can be evaluated for metabolism (e g., evaluating test agent’s metabolism profiles), efficacy (e.g., screening for test agents that are effective as pharmaceuticals), toxicity, or interactions with other test agents.

[0114] Methods of evaluating metabolism of a test agent can comprise contacting the test agent to a cultured tissue produced according to the methods disclosed herein and analyzing the metabolism of the test agent via the cultured tissue.

[0115] Methods of evaluating test agent’s therapeutic efficacy can comprise contacting the test agent to a cultured tissue produced according to the methods disclosed herein and analyzing the therapeutic effects on the cultured tissue. In some cases, a plurality of test agents are evaluated to identify potentially efficacious test agents.

[0116] Such efficacy can also be determined on an individualized basis, for example, by producing cultured tissues from cells obtained from different subjects and evaluating drug efficacy on such cultured tissues from different subjects.

[0117] Methods of evaluating toxicity of a test agent can comprise contacting the test agent to cultured tissue produced according to the methods disclosed herein and analyzing the toxic effects of the test agent on the cultured tissue.

[0118] In some cases, a plurality of test agents are evaluated to identify potentially toxic test agents. Such toxicity can also be determined on an individualized basis, for example, by producing cultured tissues from cells obtained from different subjects and evaluating drug toxicity on such cultured tissues from different subjects.

[0119] Methods of evaluating interactions of a test agent with other test agent or agents comprise contacting the test agent and other test agent or agents with a cultured tissue produced according to the methods described herein and analyzing the effects of the combination of test agents on the cultured tissue. [0120] Such interactions between test agents can also be determined on an individualized basis, for example, by producing cultured tissues or organs from cells obtained from different subjects and evaluating drug interactions in such tissues or organs from different subjects.

[0121] Notwithstanding the appended claims, the present disclosure is also defined by the following embodiments:

[0122] Embodiment 1. A method for producing a cultured tissue that mimics a natural tissue or organ, the method comprising:

[0123] a) culturing cells in a three-dimensional (3D) pattern on a scaffold,

[0124] b) applying an exogenous force to the cells while the cells are cultured in step a),

[0125] wherein the applied exogenous force induces the cultured cells to form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ.

[0126] Embodiment 2. The method of Embodiment 1, wherein the cultured tissue mimics a natural heart.

[0127] Embodiment 3. The method of Embodiment 1 or 2, wherein the exogenous force comprises one or more of: expanding force, twisting force, and elongation force.

[0128] Embodiment 4. The method of any one of Embodiments 1 to 3, wherein the cultured cells comprise one or more of: cardiomyocytes, muscle cells, epithelial cells, connective tissue cells, blood cells, germ cells, stem cells, endothelial cells, immune cells, and glandular cells.

[0129] Embodiment 5. The method of any one of Embodiments 1 to 4, wherein the exogenous force is applied using one or more of: pneumatic actuators, electric actuators, hydraulic actuators, piezoelectric actuators, shape memory alloys, electromagnetic actuators, thermal actuators, and muscle wire, and nitinol actuators.

[0130] Embodiment 6. The method of any one of Embodiments 1 to 5, wherein the cultured cells in the 3D pattern are printed with a scaffold material to produce the cultured cells on the scaffold.

[0131] Embodiment 7. The method of Embodiment 6, wherein the cultured cells are coprinted with the scaffold material.

[0132] Embodiment 8. The method of Embodiment 7, wherein the co-printed is performed using a core-double shell nozzle. [0133] Embodiment 9. The method of Embodiment 6, wherein the cultured cells and the scaffold are separately produced and coupled together to form the 3D pattern comprising the cultured cells on the scaffold.

[0134] Embodiment 10. The method of any one of Embodiments 1 to 9, wherein the scaffold material is silicone, silicone derivative, alginate, ovalbumin, titanium, porous polyethylene glycol, polycaprolactone, silk gland fibroin, collagen, elastin, PolyHlPE polymer, chitosan, collagen-coated poly-lactide-co-glycolide-gelatin/chondroitin/hyaluronate, poly-lactic-co-glycolic acid, hydroxyapatite, bone morphogenetic protein, coralline hydroxyapatite, P-tricalcium phosphate, silk fibroin, gelatin, collagen-glycosaminoglycan, fibrin, gelatin-fibrin blend, xantham gum, or a cellular material.

[0135] Embodiment 11. The method of any one of Embodiments 1 to 10, wherein the cultured tissue achieves a desired 3D alignment to optimize the alignment for an arbitrary tissue geometry such as a tubular or spherical organ.

[0136] Embodiment 12. The method of any one of Embodiments 1 to 11, further comprising applying an electrical stimulation to the cultured cells.

[0137] Embodiment 13. The method of Embodiment 12, wherein applying the electrical stimulation comprises providing biphasic current to a conductive material mounted in the scaffold. [0138] Embodiment 14. The method of any one of Embodiments 1 to 11, further comprising applying hydrodynamic stimulation to the cultured cells.

[0139] Embodiment 15. The method of Embodiment 14, wherein the hydrodynamic stimulation comprises a continuous or pulsatile flow.

[0140] Embodiment 16. The method of any one of Embodiments 1 to 15, wherein the 3D pattern is a tubular shape.

[0141] Embodiment 17. The method of any one of Embodiments 1 to 15, wherein the 3D pattern is a ventricular shape.

[0142] Embodiment 18. The method of any one of Embodiments 1 to 15, wherein the 3D pattern is selected from a heart chamber shape, a four-chambered heart, or a patient-specific heart. [0143] Embodiment 19. A bioreactor for producing a cultured tissue that mimics a natural tissue or organ, the bioreactor comprising:

[0144] a scaffold for culturing cells in a 3-dimensional (3D) pattern,

[0145] an exogenous force applicator to exert an exogenous force to the cells cultured in the 3- dimensional pattern. [0146] Embodiment 20. The bioreactor of Embodiment 19, wherein, under the applied exogenous force, the cultured cells form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ.

[0147] Embodiment 21. The bioreactor of Embodiment 19 or 20, wherein the scaffold comprising cultured cells in the 3D pattern is in a tubular shape.

[0148] Embodiment 22. The bioreactor of Embodiment 19 or 20, wherein the scaffold comprising cultured cells in the 3D pattern is in a ventricular shape.

[0149] Embodiment 23. The bioreactor of Embodiment 19, 20, or 22, wherein the cultured tissue mimics a natural heart.

[0150] Embodiment 24. The bioreactor of any one of Embodiments 19 to 23, wherein the applied exogenous force comprises one or more of expanding force, twisting force, and elongation force.

[0151] Embodiment 25. The bioreactor of any one of Embodiments 19 to 24, wherein the exogenous force applicator comprises one or more of: pneumatic actuators, electric actuators, hydraulic actuators, piezoelectric actuators, shape memory alloys, electromagnetic actuators, thermal actuators, and muscle wire, and nitinol actuators.

[0152] Embodiment 26. The bioreactor of any one of Embodiments 19 to 25, wherein the cultured cells in the 3D pattern are printed with a scaffold material to produce the scaffold comprising cells cultured in the 3D pattern.

[0153] Embodiment 27. The bioreactor of any one of Embodiments 19 to 26, further comprising an electrical stimulator for applying an electrical stimulation to the cultured cells.

[0154] Embodiment 28. The bioreactor of Embodiment 27, wherein the electrical stimulation comprises providing biphasic current to a conductive material mounted in the scaffold.

[0155] Embodiment 29. The bioreactor of any one of Embodiments 19 to 28, further comprising a hydrodynamic stimulator for applying a hydrodynamic stimulation to the cultured cells.

[0156] Embodiment 30. The bioreactor of Embodiment 29, wherein the hydrodynamic stimulation comprises a continuous or pulsatile flow.

[0157] Embodiment 31. The bioreactor of any one of Embodiments 19 to 30, wherein the cultured cells in the 3D pattern are printed with a scaffold material to produce the cultured cells on the scaffold. [0158] Embodiment 32. The bioreactor of any one of Embodiment 31, wherein the scaffold material is silicone, silicone derivative, alginate, ovalbumin, titanium, porous polyethylene glycol, polycaprolactone, silk gland fibroin, collagen, elastin, PolyHIPE polymer, chitosan, collagen-coated poly-lactide-co-glycolide-gelatin/chondroitin/hyaluronate, poly-lactic-co-glycolic acid, hydroxyapatite, bone morphogenetic protein, coralline hydroxyapatite, P-tricalcium phosphate, silk fibroin, gelatin, collagen-glycosaminoglycan, fibrin, gelbrin, gelbrin-fibrin blend, xantham gum, or a cellular material.

[0159] Embodiment 33. A cultured tissue that mimics a natural tissue or organ as produced from a method of any one of Embodiments 1 to 18.

[0160] Embodiment 34. A cultured tissue that mimics a natural tissue or organ, the cultured tissue comprising:

[0161] a scaffold comprising cells cultured in a three-dimensional (3D) pattern, wherein an exogenous force is applied to the cultured cells, and wherein the applied exogenous force induces the cultured cells to form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ.

[0162] Embodiment 35. The cultured tissue of Embodiment 31, wherein the cultured tissue mimics a natural heart.

[0163] Embodiment 36. The cultured tissue of Embodiment 34 or 35, wherein the scaffold comprises reinforcement fibers.

[0164] Embodiment 37. The cultured tissue of any one of Embodiments 34 to 36, wherein the cultured cells comprise one or more of: cardiomyocytes, muscle cells, epithelial cells, connective tissue cells, blood cells, germ cells, stem cells, endothelial cells, immune cells, and glandular cells. [0165] Embodiment 38. The cultured tissue of any one of Embodiments 34 to 37, wherein the cultured cells in the 3D pattern are printed with a scaffold material to produce the cultured cells on the scaffold.

[0166] Embodiment 39. The cultured tissue of Embodiment 38, wherein the scaffold material is silicone, silicone derivative, alginate, ovalbumin, titanium, porous polyethylene glycol, polycaprolactone, silk gland fibroin, collagen, elastin, PolyHIPE polymer, chitosan, collagen-coated poly-lactide-co-glycolide-gelatin/chondroitin/hyaluronate, poly-lactic-co-glycolic acid, hydroxyapatite, bone morphogenetic protein, coralline hydroxyapatite, P-tricalcium phosphate, silk fibroin, gelatin, collagen-glycosaminoglycan, fibrin, and gelbrin. [0167] Embodiment 40. The cultured tissue of any one of Embodiments 34 to 39, wherein the 3D pattern is a tubular shape.

[0168] Embodiment 41. The cultured tissue of any one of Embodiments 34 to 39, wherein the 3D pattern is a ventricular shape.

[0169] Embodiment 42. The cultured tissue of any one of Embodiments 34 to 41, wherein the cultured tissue changes its shape when under an exogenous force and reverts to its original shape when the exogenous force is removed.

[0170] Embodiment 43. The cultured tissue of Embodiment 42, wherein the exogenous force is a mechanical force or an electrical force.

[0171] Embodiment 44. The cultured tissue of Embodiment 42, wherein the cultured tissue mimics a natural heart.

[0172] Embodiment 45. The cultured tissue of Embodiment 44, wherein the cultured tissue twists and shrinks when under an exogenous force and reverts to its original shape when the exogenous force is removed.

[0173] Embodiment 46. A method of transplanting into a subject a cultured tissue that mimics a natural tissue or organ as produced from a method of any one of Embodiments 1 to 18.

[0174] Embodiment 47. The method of Embodiment 46, wherein the subject is a human.

[0175] Embodiment 48. The method of Embodiment 46, wherein the subject is a non-human animal.

[0176] Embodiment 49. The method of any one of Embodiments 46 to 48, wherein the cells cultured to produce the cultured tissue are obtained from the subject into which the cultured tissue is transplanted.

[0177] Embodiment 50. A method of evaluating a test agent, the method comprising contacting the test agent to a cultured tissue that mimics a natural tissue or organ as produced from a method of any one of Embodiments 1 to 18, and analyzing the effect of exposure to the test agent on the cultured tissue.

[0178] Embodiment 51. The method of Embodiment 50, comprising evaluating the test agent for the test agent’s metabolism profile, therapeutic efficacy, toxicity, or interactions with other test agents.

[0179] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used.

EXAMPLES

Example 1 - Devices and methods for producing a cultured tissue in a tubular shape

[0180] FIGS. 1A-1C describe certain exemplary bioreactors that could be used to produce a cultured tissue in a tubular shape.

[0181] FIG. 1 A shows a top view of an exemplary bioreactor from an angled perspective. In this embodiment, the bioreactor comprises a compartment containing a silicone scaffold that is connected to a rotary or twisting mechanism. The rotary or twisting mechanism can be a servo motor and is operably connected to the silicone scaffold thereby allowing rotation or twisting of the silicone scaffold. On two sides of the silicone scaffold, the compartment contains graphite rods that could be used to apply electrical current/stimulation to a cultured tissue.

[0182] FIG. IB also shows a top view of an exemplary bioreactor from a slightly different perspective. This perspective more clearly shows the two graphite rods for conduction. In addition, inlets/outlets are shown that could be used for supplying and removing a media or reagents into the compartment.

[0183] FIG. 1C shows a side view of an exemplary bioreactor. This perspective shows the port of pressurization of the motor that produces rotary or twisting force on the cultured tissue. FIG. ID shows the cultured tissue under no rotation pressure (left panel), under rotation of 90° angle (middle panel), and the rotation of 180° angle (right panel), the twisting or rotating

[0184] FIG. IE shows the circumferential strain achieved by the cultured tissue at various levels of actuation pressures and for actuators with two different thicknesses (20G and 22G). The graph shows that strains up to 40% were measured in this Example.

[0185] 3D tubular tissue can be produced via 3D printing the desired cells within an appropriate scaffold material, such as silicone, or silicone combined with fibrin or gelbrin. In some embodiments, silicone can be printed simultaneously with the cultured cells. The silicone and cultured cells can be in two separate syringes that deposit the materials simultaneously during the 3D printing process.

[0186] In some cases, during the preparing the mixture of silicone and fibrin, about 10 to 50 mg/ml fibrinogen can be added to the bioink for printing in a bath with about 1 to 20 units/ml of thrombin to produce fibrin matrix. About 0.1 to 0.3% w/v xanthan gum can be added to the bioink to increase viscosity for printability. Alternatively, about 10-50 mg/ml fibrinogen and 2-10% gelatin can be mixed to produce gelbrin.

[0187] In some cases, the matrix comprises gelbrin mixed with silicone as shown in FIG. 2A. FIG. 2A demonstrates where a fast-switching nozzle was used to allow printing a filament composed of silicone-gelbrin-silicone, switching on the fly. The resultant fibers demonstrate the mechanical coupling between the silicone and the tissue, which will enable force transmission from the silicone to the tissue and the cultured cells.

[0188] In some cases, the biological tissue, for example, gelbrin and IPSC-derived cardiomyocytes are co-printed with the silicone tubing, such that forces applied to the silicone tube will be transmitted to the biological tissue.

[0189] In some cases, the matrix comprises of a wholly-cellular ink.

[0190] In some cases, the matrix comprises fibrin mixed with silicone as shown in FIG. 2B. FIG. 2B demonstrates that geometries other than a simple filament can be generated, such as, a tubular structure. In the case of the human-size sleeve, a ventricle shape can be produced.

[0191] In some cases, the biological tissue, for example, fibrin and IPSC-derived cardiomyocytes are co-printed with the silicone tubing, such that forces applied to the silicone tube will be transmitted to the biological tissue.

[0192] The rotary motor is connected to a controller (e.g. Arduino board) and may be connected to a fan for cooling.

[0193] Once the tubular 3D cultured tissue is produced (via 3D printing or other methods), it can be mounted on the bioreactors. The tubular 3D cultured tissue can be mounted on the bioreactor manually; i.e., by securing the ends to the barbed connectors of the bioreactor. This can be achieved by biocompatible glue (e.g., 3M Vetbond Tissue adhesive) and/or elastic rings or bands and/or hose clamps. The tissue adhesive cures in seconds, therefore the mounting process does not take longer than 1 minute. [0194] Once the cultured tissue is mounted, forces can be applied. Expanding forces via pressurization of the silicone tube and/or twisting forces via rotation of the silicone using a servo motor can be applied.

[0195] Pressurization can be in the order of 0.5-10 psi, for example, about 5 psi. Twisting angles of 20-90° can be achieved. In some cases, pressurization and twisting can be synchronized. The frequency of actuation can be from 0.5 to 2 Hz, and active forces can be applied for 30-50% (duty cycle) of each given cycle. Biphasic electrical stimulation by means of the graphite rods can be applied (current I = from 5 to 10 A). Electrical stimulation can also be synchronized with the mechanical stimulation. The cultured tissue can be processed as such as from 1 to 4 weeks. During the culturing process, the duration and/or the strength of the mechanical and electrical stimulation can be varied. Also, the culture media can be changed regularly, for example, every 1 to 4 days, such as 2 days or 3 days.

[0196] For example, changing the media every 3 days can be achieved by means of a continuously active peristaltic pump or by a peristaltic pump that replaces about third of the media daily. Media can also be changed manually every 1 to 4 days.

[0197] The bioreactor comprising a tubular cultured tissue was used to demonstrate its ability to apply expansion forces only, twisting forces only, and expansion and twisting forces simultaneously. Such culture was maintained for 14 days using acellular tissue. However, computational finite element studies demonstrate that the bioreactors disclosed herein can achieve the desired complex 3D trajectories.

[0198] However, such testing culturing can be performed using a scaffold comprising cultured cells. For example, fibroblasts and WTC-11 iPSCs can be cultured and subjected to the exogenous forces, for example, the forces of expansion, twisting, or a combination thereof.

Cultured tissue in a tubular form can be used as a replacement of a natural tissue or organ, such as a natural blood vessel or any other natural tissues or organs that have tubular structures, such as a vascular tissue, muscles, epithelial tissues, channels, ducts, and the like.

Example 2 - Devices and methods for producing a cultured tissue in a ventricular shape

[0199] For both small or the human-size ventricular constructs, the mechanical stimulation can be provided by the embedded silicone. Therefore, the cultured tissue need not to be mounted in a bioreactor designed for 3D tissue construct, for example, the bioreactor described in Example 1 and FIGS. 1A-1E. [0200] A ventricular cultured tissue may comprise an external and internal silicone sleeve as shown in FIGS. 3A-3C and FIGS. 4A-4C. The cultured tissue can be cultured between the outer and inner silicone sleeves, for example, as shown in FIGS. 3A-3C and FIGS. 4A-4C. In certain embodiments, the outer silicone sleeve can comprise outer fibers as shown in FIGS. 4A-4C. Similarly, the inner silicone sleeve can comprise inner fibers as shown in FIGS. 4A-4C. The inner and outer fibers could be used to apply exogenous force, such as expanding, twisting, or elongation forces. These forces may replicate the complex 3D patterns of tissue alignment and mechanics of cardiac muscle cells or other cells, such as muscle cells, epithelial cells, connective tissue cells, blood cells, germ cells, stem cells, endothelial cells, immune cells, and glandular cells.

[0201] The human-scale silicone sleeve can be made of inflatable silicone co-printed with the biological tissue, for example, fibrin and IPSC-derived cardiomyocytes. Reinforcement fibers, made of silicone or other stiffer material, run along the surface of the silicone sleeve in a helical pattern. Under pneumatic pressure, the silicone sleeve will expand and twist. The twisting angle will depend on the number of reinforcement fibers, their material, and their thickness. Multiple configurations of the sleeve may be made.

[0202] In one configuration, the sleeve will be internal to the tissue (endocardial sleeve), recreating the alignment of cardiomyocytes in the innermost layer of the cardiac chamber. An example of such embodiments is shown in FIGS. 3A-3C.

[0203] Exemplary process of co-printing of scaffold and tissue of ventricular shape (endocardial configuration) using concentric tube robotics is also shown in FIGS. 10A-10E. FIG. 10A shows exemplary printing of the tissue layer. FIG. 10B shows exemplary printing of the outer surface of the scaffold. FIG. 10C shows exemplary printing of reinforcement fibers to direct the motion of the scaffold and the directionality of exogenous forces to the tissue. FIG. 10D shows exemplary printing of the inner surface of the scaffold. FIG. 10E shows exemplary printing of the top layer of the scaffold.

[0204] In an alternative configuration, the tissue will be sandwiched between two sleeves (one endocardial and one epicardial), recreating the alignment of cardiomyocytes in both the innermost and outermost layer of the cardiac chamber. An example of such embodiments is shown in FIGS. 4A-4C.

[0205] In certain such cases, the outer silicone sleeve can comprise fibers arranged in a left-handed helix that can be used to apply counterclockwise twisting (when observed from the base) thereby mimicking rotation of myocardial fibers in subpericardium of a natural heart. The inner silicone sleeve can comprise fibers arranged in a right-handed helix can be used to apply clockwise twisting (when observed from the base) thereby mimicking rotation of myocardial fibers in sub endocardium of a natural heart.

[0206] In the left and right-handed helices used in the outer and inner silicone sleeves, different parameters can be modified. For example, number of helices and helix pitch can be varied as desired. Also, material properties of the actuatable sleeve and helices can be varied.

[0207] In some cases, the orientation of helices can be symmetric. Alternatively, the orientation of the helices can be asymmetric, i.e., it can be modified to mimic exact anatomical patterns, either in a generalized or patient-specific fashion.

[0208] A ventricular cultured tissue can be treated with pressurization of the silicone sleeve. Actuation pressures, for example, from about 0.5 to 15 psi, can be used to enable motion of the sleeve and tissue alignment. FIG. 5 shows the effects of pressurization of the silicone sleeve on the cultured tissue.

[0209] In addition, electrical stimulation can also be provided through a conductive silicone material. Electrical stimulation can be applied from about 1A to about 10A or from about 1 V/cm to 10 V/cm. The human-size ventricle can be placed in a bath to enable media exchange at a similar rate as described in Example 1.

[0210] Moreover, in certain embodiments, the shape of the ventricular scaffold can be modified to mimic other cardiac chambers, such as right ventricle or left ventricle. In addition, a scaffold can also be prepared in the shape of the right atrium or left atrium. In addition, a scaffold can be prepared in the shape of a four-chamber heart or a patient-specific heart.

[0211] For the preparation of cardiomyocytes for preparing the cultured tissue that mimics a natural heart, hiPSC aggregates were cultured using the BioStat B-DCU twofold Flexible 120V (Sartorius) and UniVessel Glass CC 2 L SW automated bioreactor system (Sartorius, UNIVESSELMU) in 1 L of medium. WTC-11 hiPSCs were seeded on day 0 as single cells in 1 L NutriStem hPSC XF media supplemented with 40 ng/mL bFGF and 10 pm Y-27632 dihydrochloride. pH was controlled at pH 7.3 ± 0.2, which was the optimum pH for cell culture and maintaining media components in solution. pH control was regulated by a real-time feedback loop initiated by measurements from the internal pH probe, and responses by addition of 1 m sodium bicarbonate or CO2 gas input. The DO setpoint was set at the equilibrium partial pressure obtained when cell-free media was stirred at 113 RPM at 37 °C with 5.25% oxygen in the headspace. From day 1 of the culture, perfusion medium exchange was started. Medium was extracted via a dip tube with a stainless steel 20 pm pore head at the rate of one vessel volume per day (VVD), and fresh NutriStem media supplemented with 40 ng/mL bFGF was pumped in via another dip tube under a weight control loop.

[0212] Samples were taken via a sampling line during suspension culture. Dissociation and cell counting were performed as described in Section 4.2, except 100 U/mL DNasel (Worthington LS002139) was supplemented during incubation with Accutase, and incubation was 30-60 min at 37 °C on an orbital shaker.

[0213] For differentiation, hiPSC aggregates on day 4 of culture were differentiated into cardiomyocytes via temporal Wnt modulation. Briefly, hAs were treated with 4 pm Gsk3 inhibitor CHIR99021 (BioGems 2520691) for two days followed by 4 pm Wnt inhibitor iWRl (BioGems 1128234) for three days. Beating aggregates were observed from day 7 of the culture. About 20 mb of cardiac aggregates or about 4 billion cells, were obtained from 1 L of culture.

[0214] After differentiation, the cells can be cultured in a scaffold to mimic the 3D cellular pattern and biomechanics of the natural heart. To that end, the differentiated cells can be printed or embedded in a scaffold comprising silicone and, optionally, additional materials, such as extracellular matrix proteins and/or nutrients. Once the printed cultured cells in scaffold are produced, the exogenous force can be applied to mimic the forces experienced by heart cells in a natural beating heart. Thus, the exogeneous forces can be applied in cycles without interruption. In a natural beating heart, in a given cycle of heartbeat, the heart cells contract for about 30% of the time and relax for the remainder of the time. Accordingly, to induce the cultured cells to mimic natural heart cells, exogenous forces can be applied in uninterrupted cycles, where with each cycle, the exogenous force is applied from between 20% to 50% of the cycle and the cells can be allowed to relax for remainder of the cycle. Each cycle can be from about half a second to about two seconds.

[0215] The cells can be cultured under such exogenous force from about 1 week to about 4 weeks. At the end of the culturing period under the influence of the exogenous force, the cultured cells would be induced to mimic the 3D cellular pattern and biomechanics of the natural heart.

Example 3 - Workflow enabling co-printing and 3D mechanostimulation of silicone-tissue constructs

[0216] The ability to co-print silicone and tissue as well as their mechanical coupling is demonstrated in this Example. FIG. 6A illustrates that a multi-material nozzle enabled the fabrication of the silicone-tissue-silicone filament. The mechanical behavior of the co-printed filament under tension was observed. To that end, commercial silicone (Dragon Skin 10, Smooth- On Inc.) and gelbrin, a composite of fibrinogen and gelatin, was used. Then, a specialized coredouble shell nozzle was developed that enables printing of 3D tissue-silicone coaxial tubular constructs (FIG. 6B). The nozzle was designed to extrude the bioink as the outer shell, silicone as the inner shell, and the gelatin microparticle suspension bath material as the core to support the silicone tube formation during the printing process. A 3D printer was modified to enable extrusion of three materials mounted on a single z-axis (FIG. 6C). The coaxial printing occurred in 3 serial segments: a 15 mm segment of silicone and core material, a 20 mm segment of silicone, bioink, and core material, and another 15 mm segment of silicone and core material (FIG. 6C). This core-shell construct was created at a speed of 50 mm/min in the z-axis to minimize disturbance to the supporting bath, resulting in a print time for one tube in approximately one minute. A schematic of the final construct is shown in FIG. 6D.

[0217] Exogenous forces were applied to tissue through the silicone robot to drive complex patterns of cellular and tissue alignment (FIG. 6E). Specifically, torsion and expansion forces were applied to the tissue in an active, dynamic, and cyclical manner, and visualized cellular elongation and alignment via actin staining and confocal microscopy. Alignment resulting from various regimes of 3D mechanostimulation was characterized, including expansion only, twisting only, and both expansion and twisting. Results indicated that tissues subjected to active dynamic stimulation exhibited pronounced cell alignment and anisotropy compared to unstimulated tissue (FIG. 6F) and that the direction of alignment can be programmed by varying the mechanostimulation modality. [0218] FIG. 6A shows a schematic and photography of silicone-bioink coprinting, highlighting mechanical adhesion between the two materials. FIG. 6B shows core-double shell nozzle for 3D printing of concentric and adherent silicone and tissue tubes. FIG. 6C provides a photograph of 3D printer and lapses of silicone-tissue co-printing. FIG. 6D shows schematic highlighting concentric silicone and tissue tubes after co-printing. FIG. 6E illustrates twisting and expansion regimes of 3D mechanostimulation enabled by silicone robot. FIG. 6F (scale bars = 50 micons) shows examples of unstimulated and stimulated tissue alignment on confocal imaging, highlighting anisotropy resulting from 3D mechanostimulation.

Example 4 - Mechanical reactor design and characterization [0219] A bioink made of neonatal human dermal fibroblasts (NHDF) was used and referenced herein to as fibroblasts (FBs), in a gelbrin ink. FIG. 7A outlines the bioprinting pipeline. Bioink preparation involved preheating the gelatin to 37 °C, prior to adding fibrinogen to the centrifuged cell pellet. Gelatin was added to the fibrinogen-FB mixture and transferred to a syringe. To induce gelling and achieve rheological properties compatible with the surrounding FRESH support bath while also preventing cell settling, the syringe was cooled on ice. Frequent rotation of the syringe during this step ensured a homogenous cell distribution. Extrusion of the bioink into the FRESH (freeform reversible embedding of suspended hydrogels bioprinting) support bath containing thrombin (10 units/ml) prompted the polymerization of fibrinogen into fibrin. The printed construct was finally incubated at 37 °C to allow the silicone to cure and melt the gelatin, resulting in a fibrin- cell construct co-printed around the silicone tube. High-throughput co-printing was achieved by replacing the syringes with new silicone and bioink materials. Switching to an acellular bioink in the syringe allowed us to print the residual bioink in the nozzle’s dead volume (FIG. 7B).

[0220] The mechanical reactor was designed to provide 3D mechanical stimulation to the coprinted tissue-silicone constructs (FIGS. 7C-7D). A pressure port enables expansion of the constructs, while a rotary mechanism, consisting of a servo motor and two interconnected gears, allows for twisting of the samples via rotation of the rod onto which the samples are mounted. The pressure port was connected to a pressure regulator and solenoid valves that control the pressurization of the constructs. Graphite rods parallel to the tissue enabled electrical stimulation of the sample, if required. The reactor includes three ports; two for continuous perfusion and media exchange, and one for maintaining atmospheric pressure in the chamber, regardless of the pressurization state of the silicone tube. Pressurization (1-2 psi) and twisting (30°) of the tissue are controlled by a custom- made printed circuit board. The samples were actuated cyclically at a frequency of 1 Hz and with a duty cycle of 33% (FIG. 7E).

[0221] Schematics of all actuation modalities are shown in FIG. 7F. The strains induced by each different regime and varying actuation levels using 3D digital image correlation (DIC) were characterized, reporting changes in the first principal strains during actuation. FIG. 7G illustrates representative graphs for peak expansion pressures of 3 psi and twisting angles of 30°. At these levels, first principal strains of 0.0433 ± 0.0145, 0.0585 ± 0.0148, and 0.0812 ± 0.0197 were achieved for the pressure-only, twist-only, and pressure-twist conditions, respectively. First principal strain measurements for 0, 3, and 4 psi and 0, 20, 30, and 45°, and all combined conditions were tested. Maximum strains of 0.01436 ± 0.014 were recorded during simultaneous expansion and twisting at 3 psi and 45°.

[0222] FIG. 7A provides an illustration of the 3D bioprinting steps in FRESH using core-double shell nozzle. FIG. 7B provides a schematic of high-throughput printing of silicone-tissue constructs. FIG. 7C describes a schematic of mechanical reactor for 3D mechanostimulation. Pressure port and rotary mechanism enable simultaneous expansion of twisting of the tissue construct via the silicone robot. FIG. 7D provides an exemplary image of the reactor with the bioink-silicone tube mounted. FIG. 7E provides input signals for the expansion and twisting of the constructs over five consecutive cycles. FIG. 7F provides an illustration of 3D mechanostimulation modalities (control, twist, pressure, twist-pressure). FIG. 7G provides corresponding first principal strain measured via DIC. DIC graphs obtained at 3 psi expansion and 30° rotation.

Example 5 - Mechanical reactor design and characterization

[0223] Evaluation of tissue alignment showed pronounced tissue anisotropy due to 3D mechanostimulation compared to the unstimulated control samples. Representative images from (n = 2) samples, each under four stimulation conditions (control, pressure, twist, twist-pressure) are shown in FIG. 8A. Qualitative analysis of F-actin and DAPI staining images, highlighting the actin cytoskeletal protein and cell nuclei, shows that both the control samples exhibit random patterns of cell orientation. Conversely, all three stimulated conditions show anisotropy, whereby the preferred cell orientation varies depending on the stimulation regime.

[0224] Quantitatively, polar histograms of the actin orientation obtained from these images highlight differences in the alignment angle (FIG. 8B). In all images, the 0° angle corresponds to the longitudinal axis of the tubular constructs. The average orientation angle between the two samples under pressure only was 75.2 ± 19.9°. This suggests that, in tubular structures under hoop stress, cells align circumferentially, which is consistent with alignment of smooth muscle cells in blood vessels. Under twist-only mechanostimulation, the average alignment angle was 21.6 ± 5.1°. The samples subjected to both pressure and twisting exhibited an alignment angle of 52.7 ± 13.5°. Combination of these two actuation modalities resulted in helical stress patterns, which could be relevant to mimicking physiologic alignment of cardiomyocytes for cardiac tissue engineering applications. In fact, in the native heart, cardiomyocytes are oriented along 3D helical patterns at an angle of about 50-60°. Preliminary data suggest that these patterns of cellular orientation could be recapitulated using the proposed system. Evaluation of the coherency anisotropy further validates these findings (FIG. 8C). The lowest coherency was found to be associated with the unstimulated controls (0.0875 ± 0.0191), whereas combination of twisting and pressure resulted in the highest value (0.3650 ± 0.0665).

[0225] FIG. 8A (scale bars = 50 micron) shows F-actin and DAPI stains of confocal images of tissues subjected to no mechanical stimulation (control, C), pressure only (P), twist only (T), and both twist and pressure (TP) from n = 2 biological replicates. FIG. 8B provides polar plots of cell alignment angle across all samples. FIG. 8C provides coherency plot of cell anisotropy.

Example 6 - Materials and Methods

[0226] Mechanical reactor design:

[0227] The mechanical reactor was designed to enable simultaneous mechanostimulation modalities of the 3D printed constructs. The base of the reactor and the rotating rod where the constructs are mounted were 3D printed on a Form 3+ stereolithography printer with biocompatible Biomed Black Resin (FP-F3P-01, Formlabs). The motor holder was 3D printed on a MarkTwo Carbon Fiber 3D printer (MF-M2-00, Markforged). All the 3D-printed components were designed in SolidWorks (2022, Dassault Sysf ernes). The reactor lid was cut out of 1/8 inch-thick clear cast acrylic (8560K239, McMaster-Carr). A silicone o-ring (9319K158, McMaster-Carr) was added to create a watertight seal between the base of the reactor and the acrylic sheet.

[0228] The reactor features three ports; two for continuous-perfusion media exchange and one to maintain the pressure of the chamber to atmospheric level. All ports are connected to soft polyvinyl chloride tubing (5233K93, McMaster-Carr) via 3D-printed barbed connectors integrated with the base of the reactor. Two 1/4-inches conductive graphite rods (9121K85, McMaster-Carr) are inserted bilaterally into the respective slots in the reactor and at an equal distance of about 2.5 cm from the edge of the tissue. The conductive rods allow for electrical stimulation of the tissue if needed. Alligator clips and conductive M2.5 screws are used to transmit current to the graphite rods. [0229] Twisting is achieved by torque transmission from a servo motor (DS6125E, MSK servos USA) to the rotating rod via two gears; a 33-mm diameter gear (28106401, Maedler North America) attached to the motor and an 11-mm diameter gear attached to the rod (28102001, Maedler North America). A plastic bearing (6455K8, McMaster-Carr) and two o-rings (1173N01, McMaster-Carr) are attached to the rod to facilitate rotation while preventing leakage of the media out of the reactor. [0230] Pneumatic pressure is delivered to the silicone-tissue constructs via a pressure port and through the rotating rod. The pressure level is adjusted through a digital pressure regulator (PCD- 5PSIG-D-PCV1O.3O/5P, Alicat) and the airflow is controlled by a solenoid pressure manifold (VV5Q21-08N9FU0, SMC). Pressurization via the solenoid manifold and the twisting are synchronized via a custom printed circuit board (PCB).

[0231] Board design and implementation:

[0232] The PCB was designed to power and synchronize the modalities of mechanical stimulation of up to 12 mechanical reactors. The board allows for simultaneous actuation and independent control of each reactor, enabling dynamic stimulation under various conditions. The circuit consists of five ESP32 development boards (YEJMKJ): one for servo motor actuation, one for the solenoid valve, one for the cooling fans, and a master board. An additional board is used to enable biphasic electrical stimulation if needed. The board is connected to a single 32V-power supply (LW- K3010D, Longwei), drawing about 1 A at any given time. Each board was programmed on Arduino IDE (version 2.3.2).

[0233] In vitro evaluation of construct mechanics:

[0234] DIC was carried out on analogous cellular constructs under the same stimulation dynamics as used for mechanical training. The deformation of the tubes was measured for n = 1-3 tubes for a total of 11 distinct configurations, combining maximum angular rotation magnitudes of 0°, 30°, 45°, and 60° with inflation pressures of 0, 3, and 4 psi. 3D deformation was evaluated by measuring the first principal strain via DuoDIC, an open-source stereo DIC MATLAB toolbox [38], 480 fps videos of samples mounted on a modified reactor were acquired simultaneously using two digital cameras (ZV-1, Sony) approximately at a 60° angle. The 3D space was then calibrated on the MATLAB’s Stereo Camera Calibrator App, using the frames acquired by the two cameras, and the 3D strains of the tissue were then reconstructed. The quality of the reconstruction was evaluated through the normalized least squares correlation criterion.

[0235] Bioink preparation:

[0236] The bioink was made of normal human dermal fibroblasts (NHDF), fibrinogen and gelatin. FBs were cultured in 2D flasks with high-glucose DMEM (11965126, Thermo Fisher Scientific) with 10% Fetal Bovine Serum (F4135, Sigma-Aldrich).

[0237] To prepare the bioink, the FBs were detached via a 3 minute 37 °C incubation in TrypLE express enzyme (12605010, Thermo Fisher Scientific). The enzyme was then inhibited by adding FB medium (see above) at a 1 : 1 ratio with the enzyme. The FB flask contents were then transferred to 15 mL conical centrifuge tubes for centrifugation and cell counting via Trypan Blue assay (T10282, Invitrogen). The cell content was mixed in a 5 mL plastic syringe with final concentrations of 50 mg/mL of bovine fibrinogen (F8630, Sigma-Aldrich) and 7.5% type B gelatin (G6650, Sigma-Aldrich) in PBS++ with Calcium Chloride and Magnesium (14040117, Thermo Fisher Scientific) used for the dilutions. A volume of 2.5 mL was used for printing of four tubes, one per experimental group. The syringe with the bioink was cooled on ice for 5-10 minutes to allow it to gel before printing.

[0238] Bath preparation:

[0239] FRESH was prepared according to the published protocol [40], A 50% v/v solution of ethanol and distilled water was heated to 45°C and stirred at 300 rpm. 2.0 wt% of gelatin type B (G7, Fisher Scientific), 0.2 wt% of pluronic F-127 (P2443, Millipore Sigma), and 0.1 wt% of gum arabic (G9752, Sigma-Aldrich) were added to the mix and dissolved at 45°C for 1 hr. The pH was then balanced to optimize for sphere dimensions (60-80 pm). The pH level ranged between 5.2 and 7 based on the lot number of gelatin used. To achieve appropriate spherical formation of the gelatin, the mix was poured into 500 mL bottles and stirred at 250 rpm with an 8 cm stir bar overnight. The mix was then centrifuged at 500 g for three minutes and three additional PBS washes at 2000 g for two minutes before use or storage. FRESH was stored at 4°C in 50 mL conicals. 1% antibiotic- antimyotic (15240062, Thermo Fisher Scientific) was added for storage.

[0240] Before use, FRESH was centrifuged at 1500 g for 2 minutes. After the supernatant was removed, FRESH was poured into 80 mL flacktek cups and 10 units/mL bovine thrombin were added before mixing at 500 rpm for one minute. Subsequently, FRESH was transferred into a clear acrylic container for printing. A 5 mL syringe was loaded with FRESH to be extruded as the core material in core-shell-shell nozzle for printing.

[0241] Bioprinting and mounting on mechanical reactor:

[0242] Three 5 mL syringes were loaded for printing and attached to the nozzle, containing FRESH as the core, silicone as the inner shell and bioink as the outer shell. Dragonskin 10 fast (Smooth-on) was used as the silicone, with the addition of 2 w% (of part A + B) of thinner and thickener agents (Smooth-on). The silicone syringe was centrifuged at 2000 g for 90 seconds for degassing.

[0243] A six-axis low-cost printer was adapted to function with 3 extruders and one single z-axis. Pronterface (Printrun version 2.0.1) was used as the user interface to control the printer. The code involved co-extruding the silicone (19.5 pL/mm of z-axis movement), the tissue (25.8 pL/mm), and the bath core (11.4 pL/mm), while moving up with the z-axis to form a patent silicone-tissue construct. All motors moved at a rate of 50 mm/min. The constructs were printed to have a final length of 50 mm, consisting of a 20-mm tissue-silicone region flanked by two 15-mm silicone-only regions.

[0244] The printed tubes were left in the bath for 1 hour to ensure complete conversion of fibrinogen to fibrin by thrombin, before being transferred to individual 15 mL conical containers with medium and incubated overnight (Heracell Vios 250i, Thermo Fisher Scientific). Each tube was then transferred to the reactor. The ends of the silicone tube were attached to the barbed connectors of the base of the reactor and the rod. Tissue adhesive (Vetbond, 3M) was added to secure the attachments. The tissue was kept hydrated by adding media drops via a transfer pipette through the mounting process every 30-60 seconds. After the tissue was mounted, the reactor was manually filled with medium. A hydrophilic filter was added at the inlet, and a hydrophobic filter was added at the air vent. The outlet port was connected via tubing to a drain bottle. The o-ring was then added into the reactor base and the acrylic lid was screwed on. The motor was then mounted on and secured with screws to the mechanical reactor and the pressure line of the solenoid valve was connected to the free end of the rod.

[0245] Stimulation:

[0246] Tissues were then dynamically stimulated for a total of seven days. The same parameters were used throughout the study: 30° torsion and 1-2 psi. A total of 8 constructs were divided into four groups and two biological replicates were carried out. The groups were defined by the modality of mechanical stimulation applied, namely pressure only, twisting only, both twisting and pressure, and none (control). All modalities of stimulation followed the physiologic time-varying elastance model, whereby actuation peaks at approximately 33% of the cardiac cycle. In this work, a constant stimulation frequency of 1 Hz or 60 bpm was used.

[0247] Full media changes were performed every 2-3 days using a 12-channel peristaltic pump (MFLX78006-24, Avantor) and 0.38 mm-pump tubing (MFLX95723-14, Avantor). Each set of pump tubes was connected to the mechanical reactor and the media reservoir via a combination of soft polyvinyl chloride tubing (5233K92, McMaster-Carr), 22G blunt needles (Nordson) and barbed connectors (51525K123, McMaster-Carr). The media reservoir was kept in a refrigerator at 4°C for the duration of the experiment. 1% antibiotic-antimyotic (15240062, Thermo Fisher Scientific) was added to the FB media as described above.

[0248] Tissue staining:

[0249] After cessation of the stimulation, the tissue was gently separated from the silicone tube using tweezers, and incubated in 4% paraformaldehyde (PF A) for 20 minutes after two PBS++ washes. Following fixation, samples were washed three times in PBS++ for 5 minutes each time. Prior to snap-freezing, tissues were placed in a 30% wt/v sucrose solution in PBS++ for 48 hours. Samples were then transferred to a 1 :2 mix of 30% sucrose solution and optimal cutting temperature (OCT, 23-730-571, Fisher Scientific) for about 90 minutes. Each sample was then placed into a cryostat tissue mold, filled with 100% OCT, and frozen at -20°C using dry ice. Care was taken to ensure that all sucrose was removed. The samples were stored at -80°C prior to cryosectioning. The frozen tissues were sliced (30 pm thickness) along the long axis using a cryostat (CM1950, Leica). [0250] The tissue slides were thawed for 10-20 minutes at -20°C, followed by hydration in PBS++ for 10 minutes at room temperature. After aspiration of PBS, the tissue was incubated in a 1 : 1000 solution of Triton X (ab286840, Abeam) in animal free buffer AFB (1 :5 with PBS ++) for 30 minutes at room temperature. The slides were washed twice with the AFB stock for 15 minutes each and a hydrophobic barrier was drawn around the sample using a PAP pen. The tissue was then incubated overnight at 4°C in an AFB solution with 1:200 Phalloidin (A12379, Thermo Fisher Scientific) and 1 : 1000 Hoechst 33342 (A3472, ApexBio). The slides were placed in a black-out box to prevent photobleaching. The tissue was then washed twice with the Tween-20 solution for 15 minutes each, followed by a single 15-minute wash with PBS++. Easy index (EI-100-1.52, LifeCanvas Technologies) was then added as a clearing solution. The slide was then covered with 22 x 30 mm #1.5 glass cover slip (CLS-1764-2230, Chemglass life sciences) before imaging.

[0251] Structural evaluation:

[0252] Samples were imaged on a confocal microscope (ZEISS LSM 980) and acquired using the Zen software (Zen 3.8, ZEISS). Lasers with wavelength 405 nm and 488 nm were used. Z-stack images were taken in full-Z stack per track mode, with a frame size of 512 x 512 px, and a frame time of about 2.0 sec and 8x averaging. Images were acquired at 2.5x, lOx, and 20x magnification. Alignment was processed using the Orientation! Image! plug-in (version 1.54g) from 20x images. Alignment plots were graphed in Matlab (R2024a, MathWorks).

[0253] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WE CLAIM:

1. A method for producing a cultured tissue that mimics a natural tissue or organ, the method comprising: a) culturing cells in a three-dimensional (3D) pattern on a scaffold, b) applying an exogenous force to the cells while the cells are cultured in step a), wherein the applied exogenous force induces the cultured cells to form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ.

2. The method of claim 1, wherein the cultured tissue mimics a natural heart.

3. The method of claim 1 or 2, wherein the exogenous force comprises one or more of: expanding force, twisting force, and elongation force.

4. The method of any one of claims 1 to 3, wherein the cultured cells comprise one or more of: cardiomyocytes, muscle cells, epithelial cells, connective tissue cells, blood cells, germ cells, stem cells, endothelial cells, immune cells, and glandular cells.

5. The method of any one of claims 1 to 4, wherein the exogenous force is applied using one or more of: pneumatic actuators, electric actuators, hydraulic actuators, piezoelectric actuators, shape memory alloys, electromagnetic actuators, thermal actuators, and muscle wire, and nitinol actuators.

6. The method of any one of claims 1 to 5, wherein the cultured cells in the 3D pattern are printed with a scaffold material to produce the cultured cells on the scaffold.

7. The method of claim 6, wherein the cultured cells are co-printed with the scaffold material.

8. The method of claim 7, wherein the co-printed is performed using a core-double shell nozzle.

9. The method of claim 6, wherein the cultured cells and the scaffold are separately produced and coupled together to form the 3D pattern comprising the cultured cells on the scaffold.

10. The method of any one of claims 1 to 9, wherein the scaffold material is silicone, silicone derivative, alginate, ovalbumin, titanium, porous polyethylene glycol, polycaprolactone, silk gland fibroin, collagen, elastin, PolyHIPE polymer, chitosan, collagen-coated poly-1 actide-co- glycolide-gelatin/chondroitin/hyaluronate, poly-lactic-co-glycolic acid, hydroxyapatite, bone morphogenetic protein, coralline hydroxyapatite, P-tricalcium phosphate, silk fibroin, gelatin, collagen-glycosaminoglycan, fibrin, gelatin-fibrin blend, xantham gum, or a cellular material.

11. The method of any one of claims 1 to 10, wherein the cultured tissue achieves a desired 3D alignment to optimize the alignment for an arbitrary tissue geometry such as a tubular or spherical organ.

12. The method of any one of claims 1 to 11, further comprising applying an electrical stimulation to the cultured cells.

13. The method of claim 12, wherein applying the electrical stimulation comprises providing biphasic current to a conductive material mounted in the scaffold.

14. The method of any one of claims 1 to 11, further comprising applying hydrodynamic stimulation to the cultured cells.

15. The method of claim 14, wherein the hydrodynamic stimulation comprises a continuous or pulsatile flow.

16. The method of any one of claims 1 to 15, wherein the 3D pattern is a tubular shape.

17. The method of any one of claims 1 to 15, wherein the 3D pattern is a ventricular shape.

18. The method of any one of claims 1 to 15, wherein the 3D pattern is selected from a heart chamber shape, a four-chambered heart, or a patient-specific heart.

19. A bioreactor for producing a cultured tissue that mimics a natural tissue or organ, the bioreactor comprising: a scaffold for culturing cells in a 3 -dimensional (3D) pattern, an exogenous force applicator to exert an exogenous force to the cells cultured in the 3- dimensional pattern.

20. The bioreactor of claim 19, wherein, under the applied exogenous force, the cultured cells form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ.

21. The bioreactor of claim 19 or 20, wherein the scaffold comprising cultured cells in the 3D pattern is in a tubular shape.

22. The bioreactor of claim 19 or 20, wherein the scaffold comprising cultured cells in the 3D pattern is in a ventricular shape.

23. The bioreactor of claim 19, 20, or 22, wherein the cultured tissue mimics a natural heart.

24. The bioreactor of any one of claims 19 to 23, wherein the applied exogenous force comprises one or more of: expanding force, twisting force, and elongation force.

25. The bioreactor of any one of claims 19 to 24, wherein the exogenous force applicator comprises one or more of: pneumatic actuators, electric actuators, hydraulic actuators, piezoelectric actuators, shape memory alloys, electromagnetic actuators, thermal actuators, and muscle wire, and ni tinol actuators.

26. The bioreactor of any one of claims 19 to 25, wherein the cultured cells in the 3D pattern are printed with a scaffold material to produce the scaffold comprising cells cultured in the 3D pattern.

27. The bioreactor of any one of claims 19 to 26, further comprising an electrical stimulator for applying an electrical stimulation to the cultured cells.

28. The bioreactor of claim 27, wherein the electrical stimulation comprises providing biphasic current to a conductive material mounted in the scaffold.

29. The bioreactor of any one of claims 19 to 28, further comprising a hydrodynamic stimulator for applying a hydrodynamic stimulation to the cultured cells.

30. The bioreactor of claim 29, wherein the hydrodynamic stimulation comprises a continuous or pulsatile flow.

31. The bioreactor of any one of claims 19 to 30, wherein the cultured cells in the 3D pattern are printed with a scaffold material to produce the cultured cells on the scaffold.

32. The bioreactor of any one of claim 31, wherein the scaffold material is silicone, silicone derivative, alginate, ovalbumin, titanium, porous polyethylene glycol, polycaprolactone, silk gland fibroin, collagen, elastin, PolyHIPE polymer, chitosan, collagen-coated poly-lactide-co- glycolide-gelatin/chondroitin/hyaluronate, poly-lactic-co-glycolic acid, hydroxyapatite, bone morphogenetic protein, coralline hydroxyapatite, P-tricalcium phosphate, silk fibroin, gelatin, coll agen-glycosaminogly can, fibrin, gelbrin, gelbrin-fibrin blend, xantham gum, or a cellular material.

33. A cultured tissue that mimics a natural tissue or organ as produced from a method of any one of claims 1 to 18.

34. A cultured tissue that mimics a natural tissue or organ, the cultured tissue comprising: a scaffold comprising cells cultured in a three-dimensional (3D) pattern, wherein an exogenous force is applied to the cultured cells, and wherein the applied exogenous force induces the cultured cells to form a cultured tissue that mimics the 3D cellular pattern and biomechanics of the natural tissue or organ.

35. The cultured tissue of claim 31, wherein the cultured tissue mimics a natural heart.

36. The cultured tissue of claim 34 or 35, wherein the scaffold comprises reinforcement fibers.

37. The cultured tissue of any one of claims 34 to 36, wherein the cultured cells comprise one or more of: cardiomyocytes, muscle cells, epithelial cells, connective tissue cells, blood cells, germ cells, stem cells, endothelial cells, immune cells, and glandular cells.

38. The cultured tissue of any one of claims 34 to 37, wherein the cultured cells in the 3D pattern are printed with a scaffold material to produce the cultured cells on the scaffold.

39. The cultured tissue of claim 38, wherein the scaffold material is silicone, silicone derivative, alginate, ovalbumin, titanium, porous polyethylene glycol, polycaprolactone, silk gland fibroin, collagen, elastin, PolyHIPE polymer, chitosan, collagen-coated poly-lactide-co-glycolide- gelatin/chondroitin/hyaluronate, poly-lactic-co-glycolic acid, hydroxyapatite, bone morphogenetic protein, coralline hydroxyapatite, P-tricalcium phosphate, silk fibroin, gelatin, collagenglycosaminoglycan, fibrin, and gelbrin.

40. The cultured tissue of any one of claims 34 to 39, wherein the 3D pattern is a tubular shape.

41. The cultured tissue of any one of claims 34 to 39, wherein the 3D pattern is a ventricular shape.

42. The cultured tissue of any one of claims 34 to 41, wherein the cultured tissue changes its shape when under an exogenous force and reverts to its original shape when the exogenous force is removed.

43. The cultured tissue of claim 42, wherein the exogenous force is a mechanical force or an electrical force.

44. The cultured tissue of claim 42, wherein the cultured tissue mimics a natural heart.

45. The cultured tissue of claim 44, wherein the cultured tissue twists and shrinks when under an exogenous force and reverts to its original shape when the exogenous force is removed.

46. A method of transplanting into a subject a cultured tissue that mimics a natural tissue or organ as produced from a method of any one of claims 1 to 18.

47. The method of claim 46, wherein the subject is a human.

48. The method of claim 46, wherein the subject is a non-human animal.

49. The method of any one of claims 46 to 48, wherein the cells cultured to produce the cultured tissue are obtained from the subject into which the cultured tissue is transplanted.

50. A method of evaluating a test agent, the method comprising contacting the test agent to a cultured tissue that mimics a natural tissue or organ as produced from a method of any one of claims 1 to 18, and analyzing the effect of exposure to the test agent on the cultured tissue.

51. The method of claim 50, comprising evaluating the test agent for the test agent’ s metabolism profile, therapeutic efficacy, toxicity, or interactions with other test agents.