Patent Application
20230243812
Embodiments of the disclosure relate to engineered cardiac tissue structures, methods for forming the engineered cardiac tissue structures, and methods of use for the engineered cardiac tissue structures.
The current need for organ and tissue replacement, tissue repair and regeneration for patients is continually growing such that supply is not meeting the high demand primarily due to a paucity of donors as well as biocompatibility issues that lead to immune rejection of the transplant. In an effort to overcome these drawbacks, scientists working in the field of tissue engineering and regenerative medicine have investigated the use of scaffolds as an alternative to transplantation. In addition to use for tissue replacement, tissue repair and regeneration, engineered tissues that recapitulate biological tissues are also useful for in vitro testing of pharmacological agents and for in vitro disease models. However, creating fully matured and functional cardiac tissue in vitro is still challenging. For example, current engineered tissues or organs lack of capabilities of autonomous homeostatic regulation and adaptive behavioral control (e.g., autonomous self-pacing and independent motion control).
Accordingly, there is a need for improved methods and engineered tissue structures for creating autonomous systems capable of homeostatic regulation and adaptive behavioral control and providing a more granular analysis of structure-function relationship in cardiovascular physiology.
Some embodiments of the present invention include engineered cardiac tissue structures (e.g., engineered cardiac tissue structures having geometrically insulated cardiac tissue nodes and multilayer engineered cardiac tissue structures) and methods for forming the engineered cardiac tissue structures.
In a first aspect, the present invention provides an engineered cardiac tissue structure that includes a support having a first side and a first engineered cardiac tissue layer disposed at the first side of the support. The first engineered cardiac tissue layer includes a first plurality of cardiomyocytes. The first engineered cardiac tissue layer further includes a first region having a first area, a second region having a second area smaller than the first area, and a third region forming a conduction pathway for propagation of action potentials between the second region and the first region. The second region forms a geometrically insulated cardiac tissue node.
In one embodiment, a minimum width of the third region, as measured perpendicular to the conduction pathway, is less than a maximum width of the second region.
In one embodiment, more cardiomyocytes of the first plurality of cardiomyocytes are in the first region than in the second region.
In one embodiment, the first region, the second region and the third region collectively form a cardiac cell region having a cardiac cell region perimeter, and wherein the first plurality of cardiomyocytes do not contact any cardiac cells beyond the cardiac cell region perimeter.
In one embodiment, the cardiac cell region perimeter around the first region has one or more corners, and the cardiac cell region perimeter is rounded at each of the one or more corners.
In one embodiment, the first region, the second region, and the third region are sized and shaped for spontaneous contraction of the first engineered cardiac tissue layer to initiate in the second region more often than in the first region.
In one embodiment, the first region, second region, and third region are sized and shaped for a higher probability of initiation of spontaneous contraction of the first engineered cardiac tissue layer in the second region than in the first region.
In one embodiment, the support comprises a hydrogel layer.
In one embodiment, the hydrogel layer comprises gelatin.
In one embodiment, the second region acts as a cardiac pacemaker for the engineered cardiac tissue structure.
In one embodiment, the engineered cardiac tissue structure further includes a second engineered cardiac tissue layer disposed at a second side of the support opposite the first side of the support. The second engineered cardiac tissue layer has an anisotropic tissue orientation and includes a second plurality of cardiomyocytes. The first engineered cardiac tissue layer is physically separated from the second engineered cardiac tissue layer by a thickness of the support.
In one embodiment, excitation and contraction of the first engineered cardiac tissue layer bends the support and strains the second engineered cardiac tissue layer inducing excitation and contraction of the second engineered cardiac tissue layer. The excitation and contraction of the second engineered cardiac tissue layer bends the support and strains the first engineered cardiac tissue layer inducing excitation and contraction of the first engineered cardiac tissue layer thereby producing antagonistic cyclic contractions.
In one embodiment, the antagonistic cyclic contractions are self-sustaining cyclic contractions.
In one embodiment, the antagonistic cyclic contractions are spontaneous, self-sustaining cyclic contractions.
In one embodiment, a self-propelled swimming structure includes the engineered cardiac tissue structure of any one of the above described embodiments of the first aspect, a front body portion coupled to or attached to a first end of the engineered cardiac tissue structure, and a rear body portion coupled to or attached to a second end of the engineered cardiac tissue structure.
In one embodiment, a young's modulus of the front body portion is greater than a young's modulus of the support. A young's modulus of the rear body portion is greater than the young's modulus of the support.
In one embodiment, the self-propelled swimming structure further includes at least one buoyancy element attached to or coupled to the front body portion, the rear body portion, or both. The at least one buoyancy element is selected for the self-propelled swimming structure to maintain neutral buoyancy in a culture medium.
In one embodiment, the at least one buoyancy element is shaped to maintain directional stability of the self-propelled swimming structure.
In a second aspect, the present invention provides an engineered cardiac tissue structure that includes a support, a first engineered cardiac tissue layer, and a second engineered cardiac tissue layer. The support has a first side and a second side opposite the first side, the first side being patterned to promote anisotropic tissue formation, and the second side being patterned to promote anisotropic tissue formation. The first engineered cardiac tissue layer is disposed at the first side of the support. The first engineered cardiac tissue layer has an anisotropic tissue orientation and includes a first plurality of cardiomyocytes. The second engineered cardiac tissue layer is disposed at the second side of the support. The second engineered cardiac tissue layer has an anisotropic tissue orientation and includes a second plurality of cardiomyocytes. The first engineered cardiac tissue layer is physically separated from the second engineered cardiac tissue layer by a thickness of the support. Excitation and contraction of the first engineered cardiac tissue layer bends the support and strains the second engineered cardiac tissue layer inducing excitation and contraction of the second engineered cardiac tissue layer. The excitation and contraction of the second engineered cardiac tissue layer bends the support and strains the first engineered cardiac tissue layer inducing excitation and contraction of the first engineered cardiac tissue layer thereby producing antagonistic cyclic contractions.
In one embodiment, the antagonistic cyclic contractions are self-sustaining.
In one embodiment, the antagonistic cyclic contractions are spontaneous.
In one embodiment, the support has a bending stress in a range of 5 kPa to 15 kPa.
In one embodiment, the support comprises a hydrogel layer.
In one embodiment, the hydrogel layer includes gelatin.
In one embodiment, the support has a thickness in a range of 100 microns (μm) to 300 μm.
In one embodiment, the first engineered cardiac tissue layer includes a first region having a first area, a second region having a second area smaller than the first area, and a third region forming a conduction pathway for propagation of action potentials between the second region and the first region. The second region forms a geometrically insulated cardiac tissue node.
In one embodiment, a self-propelled swimming structure includes the engineered cardiac tissue structure of any one of the above described embodiments of the second aspect, a front body portion coupled to or attached to a first end of the engineered cardiac tissue structure, and a rear body portion coupled to or attached to a second end of the engineered cardiac tissue structure.
In one embodiment, a young's modulus of the front body portion is greater than a young's modulus of the support. A young's modulus of the rear body portion is greater than the young's modulus of the support.
In one embodiment, the self-propelled swimming structure further includes at least one buoyancy element attached to or coupled to the front body portion, the rear body portion, or both. The at least one buoyancy element is selected for the self-propelled swimming structure to maintain neutral buoyancy in a culture medium.
In one embodiment, the at least one buoyancy element is shaped to maintain directional stability of the self-propelled swimming structure.
In a third aspect, the present invention provides a method of forming a functional cardiac tissue structure. The method includes providing or obtaining a support having a first side and a second side opposite the first side, the first side being patterned and configured to promote anisotropic tissue formation, and the second side being patterned and configured to promote anisotropic tissue formation. The method further includes seeding the first side of the support and the second side of the support with cardiomyocytes, and growing a first cardiac tissue layer on the first side of the support and growing a second cardiac tissue layer on the second side of the support, the first cardiac tissue layer physically separated from the second cardiac tissue layer by a thickness of the support, thereby forming a functional cardiac tissue structure.
In one embodiment, during growth of the first cardiac tissue layer on the first side of the support and growth of the second cardiac tissue layer on the second side of the support, excitation and contraction of the first cardiac tissue layer bends the support and strains the second cardiac tissue layer inducing excitation and contraction of the second cardiac tissue layer, and the excitation and contraction of the second cardiac tissue layer bends the support and strains the first cardiac tissue layer inducing excitation and contraction of the first cardiac tissue layer producing antagonistic cyclic contractions.
In one embodiment, the antagonistic cyclic contractions are self-sustaining cyclic contractions.
In one embodiment, the antagonistic cyclic contractions are spontaneous, self-sustaining cyclic contractions.
In another aspect, the present invention provides a method for identifying a compound that modulates a cardiac tissue function. The method includes providing an engineered cardiac tissue structure as described herein. The method also includes contacting the cardiac tissue structure with a test compound, and determining the effect of the test compound on a cardiac tissue function in the presence and absence of the test compound. A modulation of the cardiac tissue function in the presence of the test compound as compared to the cardiac tissue function in the absence of the compound indicates that the test compound modulates the cardiac tissue function, thereby identifying a compound that modulates a cardiac tissue function.
In another aspect, the present invention provides a method for identifying a compound useful for treating or preventing a cardiac tissue disease. The method includes providing an engineered cardiac tissue structure as described herein. The method also includes contacting the cardiac tissue structure with a test compound. The methods also includes determining the effect of the test compound on a cardiac tissue function in the presence and absence of the test compound. A modulation of the cardiac tissue function in the presence of the test compound as compared to the cardiac tissue function in the absence of the test compound indicates that the test compound modulates the cardiac tissue function, thereby identifying a compound useful for treating or preventing a cardiac tissue disease.
In some embodiments, the cardiac tissue function is a biomechanical activity. In some embodiments, the biomechanical activity is one or more of contractility, cell stress, cell swelling, and rigidity. In some embodiments, the biomechanical activity is one or more of stem cell activation, stem cell maturation, tissue morphogenesis, and tissue remodeling.
In some embodiments, the tissue function is an electrophysiological activity. In some embodiments, the electrophysiological activity is a voltage parameter selected from the group consisting of action potential, action potential duration (APD), conduction velocity (CV), refractory period, wavelength, restitution, bradycardia, tachycardia, reentrant arrhythmia, and/or a calcium flux parameter, e.g., intracellular calcium transient, transient amplitude, rise time (contraction), decay time (relaxation), total area under the transient (force), restitution, focal and spontaneous calcium release.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
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.
In the following description, it is understood that terms such as “top,” “bottom,” “middle,” “outward,” “inward,” and the like are words of convenience and are not to be construed as limiting terms.
As used herein, excitable cells refer to cells that respond to an electrical stimulation by propagating an action potential (e.g., muscle cells, neurons).
As used herein, activation of excitable cells, with respect to muscle cells, refers to contraction of the muscle cells and can also refer to propagation of an action potential (e.g., as measured by channel opening in the muscle cells). Activation of excitable cells, with respect to neurons, refers to propagation of an action potential (e.g., as measured by channel opening in the neurons).
Reference is made in detail to embodiments of the disclosure, which are illustrated in the accompanying figures and examples. Referring to the drawings in general, it will be understood that the illustrations are for the purpose of describing particular embodiments of the disclosure and are not intended to limit the same.
Whenever a particular embodiment of the disclosure is said to comprise or consist of at least one element of a group and combinations thereof, it is understood that the embodiment may comprise or consist of any of the elements of the group, either individually or in combination with any of the other elements of that group.
These and other aspects of the invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions or rearrangements may be made within the scope of the invention, and the invention includes all such substitutions, modifications, additions or rearrangements.
Some embodiments described herein include engineered tissue structures having geometrically insulated tissue nodes. For example, an engineered cardiac tissue structure can be a tissue structure having one or more features of heart, e.g., mechano-electrical signaling and automaticity, autonomously actuating cardiac muscular features, features of tissue-level cardiac biophysics, and/or features associated with cardiovascular physiology. An engineered cardiac tissue structure includes a support (e.g., a hydrogel layer) having a first side and a first engineered cardiac tissue layer disposed at the first side of the support. The first engineered cardiac tissue layer includes a first plurality of cardiomyocytes. In some embodiments, the first engineered cardiac tissue layer further includes a first region having a first area, a second region having a second area smaller than the first area, and a third region forming a conduction pathway for propagation of action potentials between the second region and the first region. The second region forms a geometrically insulated cardiac tissue node (G-node).
In some embodiments, similar to the engineered cardiac tissue structure, other types of cells (e.g., neurons or skeletal muscle cells) can be used to form corresponding types of engineered tissue structures. For example, an engineered tissue structure includes a support (e.g., a hydrogel layer) having a first side and a first engineered tissue layer disposed at the first side of the support. The first engineered tissue layer includes a first plurality of neurons or skeletal muscle cells. The first engineered tissue layer further includes a first region having a first area, a second region having a second area smaller than the first area, and a third region forming a conduction pathway for propagation of action potentials between the second region and the first region. The second region forms a geometrically insulated tissue node.
Some embodiments described herein include multilayer engineered cardiac tissue structures. For example, an engineered cardiac tissue structure can include a support (e.g., a hydrogel layer), a first engineered cardiac tissue layer, and a second engineered cardiac tissue layer in some embodiments. The support has a first side and a second side opposite the first side. The first side is patterned to promote anisotropic tissue formation, and the second side is patterned to promote anisotropic tissue formation. The first engineered cardiac tissue layer is disposed at the first side of the support. The first engineered cardiac tissue layer has an anisotropic tissue orientation and includes a first plurality of cardiomyocytes. The second engineered cardiac tissue layer is disposed at the second side of the support. The second engineered cardiac tissue layer has an anisotropic tissue orientation and includes a second plurality of cells (e.g., cardiomyocytes, neurons or skeletal muscle cells). The first engineered cardiac tissue layer is physically separated from the second engineered cardiac tissue layer by a thickness of the support. Excitation and contraction of the first engineered cardiac tissue layer bends the support and strains the second engineered cardiac tissue layer inducing excitation and contraction of the second engineered cardiac tissue layer. The excitation and contraction of the second engineered cardiac tissue layer bends the support and strains the first engineered cardiac tissue layer inducing excitation and contraction of the first engineered cardiac tissue layer thereby producing antagonistic cyclic contractions.
In some embodiments, similar to the engineered cardiac tissue structure, other types of cells (e.g., neurons or skeletal muscle cells) can be used to form corresponding types of engineered tissue structures. For example, an engineered tissue structure can include a support (e.g., a hydrogel layer), a first engineered tissue layer, and a second engineered tissue layer. The support has a first side and a second side opposite the first side. The first side is patterned to promote anisotropic tissue formation, and the second side is patterned to promote anisotropic tissue formation. The first engineered tissue layer is disposed at the first side of the support. The first engineered tissue layer has an anisotropic tissue orientation and includes a first plurality of neurons or skeletal muscle cells. The second engineered tissue layer is disposed at the second side of the support. The second engineered tissue layer has an anisotropic tissue orientation and includes a second plurality of neurons or skeletal muscle cells. The first engineered tissue layer is physically separated from and mechanically coupled to the second engineered tissue layer by a thickness of the support. Excitation and contraction of the first engineered tissue layer bends the support and strains the second engineered tissue layer inducing excitation and contraction of the second engineered tissue layer. The excitation and contraction of the second engineered tissue layer bends the support and strains the first engineered tissue layer inducing excitation and contraction of the first engineered tissue layer thereby producing antagonistic cyclic contractions.
Some embodiments described herein include methods of forming functional cardiac tissue structures. A method includes providing or obtaining a support (e.g., a hydrogel layer) having a first side and a second side opposite the first side. The first side is patterned and configured to promote anisotropic tissue formation, and the second side is patterned and configured to promote anisotropic tissue formation. The method further includes seeding the first side of the support and the second side of the support with cells (e.g., cardiomyocytes, neurons or skeletal muscle cells), and growing a first cardiac tissue layer on the first side of the support and growing a second cardiac tissue layer on the second side of the support. The first cardiac tissue layer is physically separated from the second cardiac tissue layer by a thickness of the support, thereby forming a functional cardiac tissue structure.
In some embodiments, similar to forming functional cardiac tissue structures, the provided methods can form other types of engineered tissue structures using other types of cells (e.g., neurons or skeletal muscle cells). For example, a method for forming functional tissue structures includes providing or obtaining a support (e.g., a hydrogel layer) having a first side and a second side opposite the first side. The first side is patterned and configured to promote anisotropic tissue formation, and the second side is patterned and configured to promote anisotropic tissue formation. The method further includes seeding the first side of the support and the second side of the support with neurons or skeletal muscle cells, and growing a first tissue layer on the first side of the support and growing a second tissue layer on the second side of the support. The first tissue layer is physically separated from the second tissue layer by a thickness of the support.
Some embodiments described herein include methods for identifying a compound that modulates a cardiac tissue function. A method includes providing an engineered cardiac tissue structure and contacting the engineered cardiac tissue structure with a test compound. The method also includes determining the effect of the test compound on a cardiac tissue function in the presence and absence of the test compound. A modulation of the cardiac tissue function in the presence of the test compound as compared to the cardiac tissue function in the absence of the test compound indicates that the test compound modulates the cardiac tissue function, thereby identifying a compound that modulates a cardiac tissue function.
Some embodiments described herein include methods for identifying a compound that modulates a tissue function. For example, where the tissue includes neurons or skeletal muscle cells. A method includes providing an engineered tissue structure (e.g., an engineered tissue structure including neurons or skeletal muscle cells) and contacting the engineered tissue with a test compound. The method also includes determining the effect of the test compound on the tissue function in the presence and absence of the test compound. A modulation of the tissue function in the presence of the test compound as compared to the tissue function in the absence of the test compound indicates that the test compound modulates the tissue function, thereby identifying a compound that modulates a tissue function.
Some embodiments described herein include methods for identifying a compound useful for treating or preventing a cardiac tissue disease. A method for identifying a compound useful for treating or preventing a cardiac tissue includes providing an engineered cardiac tissue structure, and contacting the engineered cardiac tissue structure with a test compound. The method also includes determining the effect of the test compound on the cardiac tissue function in the presence and absence of the test compound. A modulation of the cardiac tissue function in the presence of the test compound as compared to the cardiac tissue function in the absence of the test compound indicates that the test compound modulates the cardiac tissue function, thereby identifying a compound useful for treating or preventing a cardiac tissue disease.
Some embodiments described herein include methods for identifying a compound useful for treating or preventing a tissue disease. For example, where the disease affects neurons or skeletal muscle. A method for identifying a compound useful for treating or preventing a tissue disease includes providing an engineered tissue structure (e.g., an engineered tissue structure including neurons or skeletal muscle cells) and contacting the engineered tissue structure with a test compound In some embodiments, the engineered tissue structure includes neurons or skeletal muscle cells. The method also includes determining the effect of the test compound on the tissue function in the presence and absence of the test compound. A modulation of the tissue function in the presence of the test compound as compared to the tissue function in the absence of the test compound indicates that the test compound modulates the tissue function, thereby identifying a compound useful for treating or preventing a tissue disease.
Turning to the drawings,
In some embodiments, the second region 24 forms a geometrically insulated tissue node (G-node). In some embodiments, the first region 22, the second region 24, and the third region 26 are sized and shaped for initiation of spontaneous activation of excitable cells (e.g., contraction of CMs or skeletal muscle cells, propagation of APs in neurons), or initiation of a spontaneous propagating wave of activation of excitable cells, to more often occur in the second region than in the first region due to geometry of the second region 24, of the first region 22, and of the third region. In some embodiments, the first region 22, the second region 24, and the third region 26 are sized and shaped for initiation of spontaneous activation of excitable cells (e.g., contraction of CMs or skeletal muscle cells, propagation of APs in neurons), or initiation of a spontaneous propagating wave of activation of excitable cells, to have a higher probability of occurring in the second region 24 than in the first region 22. In some embodiments, the excitable cells are CMs and the second region acts as a cardiac pacemaker for the engineered tissue structure. Various factors that affect the probability of spontaneous activation (e.g. spontaneous contraction) initiating in the second region rather than the first region, or of a spontaneous propagating wave of activation initiating in the second region rather than the first region are described below and in the Example.
In some embodiments width 26w (e.g., a minimum width) of the third region 26 as measured perpendicular to the conduction pathway, is less than a width 24w (e.g., a maximum width) of the second region 24 providing increased geometric isolation for the second region 24 from the first region 22. As described below with respect to the Example, this increase geometric isolation can result in increased frequency for spontaneously initiated waves of activation and more synchronized activation, due, in part, to the reflection of intracellular currents at the tissue edges (tissue perimeter) in the smaller second region. In some embodiments, the cell region perimeter 34 around the first region 22 has corners one or more corners that are rounded, or includes only rounded corners 34a-34d. Rounding corners in the first region 22 significantly decreases the probability of spontaneous initiation of activation in the first region 22. In other embodiments, the cell region perimeter 34 around the first region 22 or the second region 24 can have at least one corner that is not rounded as further described below. As explained below with respect to the Example, whether the second region 24 has rounded corners or sharp corners may not significant affect a probability of spontaneous initiation of activation in the second region 24.
In engineered tissue structure 50, 60 and 70, the respective first region is rectangular in shape. In some embodiments, the first region is square in shape. In some embodiments, the first has a shape other than square or rectangular. In some embodiments, the first region may have one or more corners with internal angles different from 90 degrees. An engineered tissue structure 80 includes a first region 82 having a trapezoidal shape with sharp corners and a second region 84 having rounded corners in accordance with another embodiment. In some embodiments, a corner in the first region may have an acute internal angle of less than 90 degree 83a, and/or a corner in the second region may have an obtuse internal angle of more 90 degree 83b.
In some embodiments, a corner in the second region may have an acute internal angle of less than 90 degree 83a, and/or a corner in the second region may have an obtuse internal angle of more 90 degree 83b.
Although the respective second regions in each of engineered tissue structures 50, 60, 70 and 80 each have four corners and four sides, in some embodiments, an engineered tissue structure has more than four corners and more than four sides. In some embodiments, an engineered tissue structure has less than four corners and four sides.
As described in the Example below with respect to
For example, as shown and described below in the Example with respect to
In some embodiments, the support 110 is or includes a hydrogel layer. In some embodiments, the hydrogel layer includes a gelatin hydrogel. In some embodiments, the thickness and stiffness or Young's modulus of the support is selected such that contraction of one of the engineered tissue layers causes sufficient tensile stress in the other engineered tissue on the opposite side of the support to produce contraction of the other layer. In some embodiments, the support has a thickness in a range of 100 μm to 300 μm. In some embodiments, the support has a bending stress in a range of 5 kPa to 15 kPa. In some embodiments, the support has a bending stress of about 10 kPa.
In some embodiments, at least one of the first engineered tissue layer 120 and the second engineered tissue layer 130 can have a geometrically insulated tissue node.
In some embodiments, a self-propelled swimming structure includes a multilayer engineered tissue structure that exhibits antagonistic cyclic contractions as described herein.
In some embodiments, a young's modulus of the front body portion is greater than a young's modulus of the support 110. In some embodiments, a young's modulus of the rear body portion is greater than the young's modulus of the support 110. In some embodiments, the self-propelled swimming structure further includes at least one buoyancy element 184 (e.g., a plastic floater fin of
Some embodiments provide a method of forming a functional engineered tissue structure (e.g., a functional cardiac tissue structure, a functional muscle tissue structure, a functional neural tissue structure). The method includes providing or obtaining a support having a first side and a second side opposite the first side, the first side being patterned and configured to promote anisotropic tissue formation, and the second side being patterned and configured to promote anisotropic tissue formation. Further description of patterning and configuring to promote anisotropic tissue function can be found in the section below entitled Seeding and Culturing of Engineered Tissue Layers and in the Example. The method further includes seeding the first side of the support and the second side of the support with excitable and contractile cells (e.g., cardiomyocytes, skeletal muscle cells), and growing a first tissue layer (e.g. a cardiac tissue layer, a skeletal muscle tissue layer) on the first side of the support and growing a second tissue layer (e.g. a cardiac tissue layer, a skeletal muscle tissue layer) on the second side of the support, the first cardiac tissue layer physically separated from the second cardiac tissue layer by a thickness of the support. Further description of seeding the cells and growing the tissue layers can be found in the section below entitled Seeding and Culturing of Engineered Tissue Layers and in the Example.
In some embodiments, during growth of the first tissue layer 176 on the first side of the support 174 and growth of the second tissue layer 178 on the second side of the support 174, excitation and contraction of the first tissue layer 176 bends the support 174 and strains the second tissue layer 178 inducing excitation and contraction of the second tissue layer 178, and the excitation and contraction of the second tissue layer 178 bends the support 174 and strains the first tissue layer 176 inducing excitation and contraction of the first tissue layer producing antagonistic cyclic contractions. In some embodiments, the antagonistic cyclic contractions are self-sustaining cyclic contractions. In some embodiments, the antagonistic cyclic contractions are spontaneous, self-sustaining cyclic contractions. In some embodiments, forces exerted by the self-sustaining cyclic contractions help to develop and sustain functional cell growth in the first tissue layer and the second tissue layer. Additional description of self-sustaining cyclic contractions sustaining functional cell development and growth over extended times is provided in the Example.
The supports are seeded with a population of cells to fabricate the first engineered tissue layer and the second engineered tissue layer in engineered tissue structures in some embodiments.
Accordingly, in some embodiments, a support is seeded with a plurality of cells and cultured in an incubator under physiologic conditions (e.g., at 37° C.) until the cells form a functional tissue engineered tissue layer.
A functional tissue structure is an in vitro tissue that recapitulates one or more interactions that occur between cells and their surrounding tissue in vivo. For example, the sarcomeres in the muscle cells and/or the cell themselves of a functional muscle tissue layer may be anisotropically aligned and/or the tissue is electrically functional and actively contractile.
Any appropriate cell culture method may be used. The seeding density of the cells will vary depending on the cell size and cell type, but can easily be determined by methods known in the art. In one embodiment, cells are seeded at a density of between about 1×105 to about 6×105 cells/cm2, or at a density of about 1×104, about 2×104, about 3×104, about 4×104, about 5×104, about 6×104, about 7×104, about 8×104, about 9×104, about 1×105, about 1.5×105, about 2×105, about 2.5×105, about 3×105, about 3.5×105, about 4×105, about 4.5×105, about 5×105, about 5.5×105, about 6×105, about 6.5×105, about 7×105, about 7.5×105, about 8×105, about 8.5×105, about 9×105, about 9.5×105, about 1×106, about 1.5×106, about 2×106, about 2.5×106, about 3×106, about 3.5×106, about 4×106, about 4.5×106, about 5×106, about 5.5×106, about 6×106, about 6.5×106, about 7×106, about 7.5×106, about 8×106, about 8.5×106, about 9×106, or about 9.5×106. Values and ranges intermediate to the above-recited values and ranges are also contemplated by the present invention.
In some embodiments, the support is contacted with living cells during the fabrication process such that engineered tissue layers populated with cells are produced. The support may also be contacted with additional agents, such as proteins, nucleotides, lipids, drugs, pharmaceutically active agents, biocidal and antimicrobial agents during the fabrication process.
Suitable cells for use in the invention may be normal cells, abnormal cells (e.g., those derived from a diseased tissue, or those that are physically or genetically altered to achieve an abnormal or pathological phenotype or function), normal or diseased muscle cells, stem cells (e.g., embryonic stem cells), or induced pluripotent stem cells. Suitable cells include vascular smooth muscle cells, cardiac myocytes, skeletal muscle cells, and cells that will differentiate into muscle cells. Such cells may be seeded on the support and cultured to form a functional tissue.
Cells for seeding can be cultured in vitro, derived from a natural source, genetically engineered, or produced by any other means. Any natural source of cells may be used.
The term “progenitor cell” is used herein to refer to cells that have a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell that it can give rise to by differentiation. Often, progenitor cells also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.
The term “progenitor cell” is used herein synonymously with “stem cell.”
The term “stem cell” as used herein, refers to an undifferentiated cell that is capable of proliferation and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiable daughter cells.
The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. The term “stem cell” refers to a subset of progenitors that have the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and that retains the capacity, under certain circumstances, to proliferate without substantially differentiating. In one embodiment, the term stem cell refers generally to a naturally occurring mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cellular differentiation is a complex process typically occurring through many cell divisions. A differentiated cell may derive from a multipotent cell which itself is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors. In many biological instances, stem cells are also “multipotent” because they can produce progeny of more than one distinct cell type, but this is not required for “stem-ness.” Self-renewal is the other classical part of the stem cell definition. In theory, self-renewal can occur by either of two major mechanisms. Stem cells may divide asymmetrically, with one daughter retaining the stem state and the other daughter expressing some distinct other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only. Formally, it is possible that cells that begin as stem cells might proceed toward a differentiated phenotype, but then “reverse” and re-express the stem cell phenotype, a term often referred to as “dedifferentiation” or “reprogramming” or “retrodifferentiation”.
The term “embryonic stem cell” is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see U.S. Pat. Nos. 5,843,780, 6,200,806, the contents of which are incorporated herein by reference). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, U.S. Pat. Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein by reference). The distinguishing characteristics of an embryonic stem cell define an embryonic stem cell phenotype. Accordingly, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell such that that cell can be distinguished from other cells. Exemplary distinguishing embryonic stem cell characteristics include, without limitation, gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like.
The term “adult stem cell” or “ASC” is used to refer to any multipotent stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue. Stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells.
Hydrogels suitable for use in the engineered tissue structures of the invention include, for example, polyacrylamide gels, poly(N-isopropylacrylamide), poly(hydroxyethyl) methacrylate (pHEMA), collagen, fibrin, gelatin, alginate, and dextran. In some embodiment the hydrogel includes gelatin. In some embodiment, the hydrogel includes alginate. In some embodiments, the stiffness of the hydrogel is tuned to mimic the mechanical properties of healthy muscle tissue, e.g., cardiac tissue in vivo, e.g., to have a Young's modulus of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 kPa. In some embodiments, the stiffness of the hydrogel is tuned to mimic the mechanical properties of diseased muscle tissue, e.g., cardiac tissue in vivo, e.g., to have a Young's modulus of greater than about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or about 55 kPa.
A surface of a hydrogel layer may be uniformly or selectively patterned with engineered surface chemistry to elicit, e.g., an extracellular matrix protein, (or inhibit, e.g., a Pluronic) specific cell growth and function. The engineered surface chemistry can be provided via contact printing, exposure to ultraviolet radiation or ozone or via acid or base wash or plasma treatment to increase the hydrophilicity of the surface. In some embodiments, the hydrogel is micro-molded to have selective spatially patterned dimensions to elicit (or inhibit) specific cell growth and function. In some embodiments, a surface of the support is patterned to form anisotropic tissue.
A specific biopolymer (or combination of biopolymers) may be selected to recruit different integrins, or an engineered surface chemistry may be fabricated on the flexible polymer layer and/or hydrogel layer to enhance or inhibit cell and/or protein adhesion. The specific type of biopolymer used and geometric spacing of the patterning will vary with the application. In one embodiment, the engineered surface chemistry comprises a biopolymer, such as an ECM protein, to pattern specific cell types. The ECM may comprise fibronectin, laminin, one or more collagens, fibrin, fibrinogen, or combinations thereof.
In some embodiments, prior to uniformly or selectively patterning a hydrogel with an engineered surface chemistry to elicit, e.g., an extracellular matrix protein, (or inhibit, e.g., a Pluronic) specific cell growth and function, the hydrogel is coated with a compound to permit covalent attachment of an engineered surface chemistry, such as an extracellular matrix protein.
In one embodiment, the ECM is not uniformly distributed on the surface of the flexible polymer and/or hydrogel, but rather is patterned spatially using techniques including, but not limited to, soft lithography, self-assembly, printed on the solid support structure with a polydimethylsiloxane stamp, vapor deposition, and photolithography. Methods of forming an anisotropic tissue layer are describe in U.S. Patent Application Publication No. 2014/0342394, entitled “Muscle Chips and Methods of Use”, which is incorporated by reference herein in its entirety. Additional methods of forming anisotropic tissue layers on a support are described below with respect to the Example.
To attach cells, a support is placed in culture with a cell suspension allowing the cells to settle and adhere to the surface. In the case of an adhesive surface treatment, cells bind to the material in a manner dictated by the surface chemistry. For patterned chemistry, cells respond to patterning in terms of maturation, growth and function. Examples of cell types that may be used include contractile cells, such as, but not limited to, vascular smooth muscle cells, vascular endothelial cells, myocytes (e.g., cardiac myocytes), neurons, skeletal muscle, and cells that will differentiate into contractile cells (e.g., stem cells, e.g., embryonic stem cells or adult stem cells, progenitor cells or satellite cells).
In one embodiment, cardiac myocytes are co-cultured with neurons to prepare innervated engineered tissue comprising pacemaking cells, and/or to accelerate the maturation of the cultured cells as described in U.S. Patent Application Publication No. 20130046134-A1, filed Oct. 31, 2012, the entire contents of which are incorporated herein by reference.
Methods for Identifying a Compound that Modulates a Tissue Function and Methods for Identifying a Compound Useful for Treating or Preventing a Cardiac Tissue Disease
Some embodiments described herein include methods for identifying a compound that modulates a tissue function (e.g., a cardiac tissue function, a muscle tissue function, a neural tissue function). A method includes providing an engineered tissue structure as described herein and contacting the engineered tissue structure with a test compound. In some embodiments, the engineered tissue structure includes a first engineered tissue layer (e.g., a first engineered cardiac tissue layer, a first engineered muscle tissue layer, a first engineered neural tissue layer) on a first side of a support including a geometrically isolated node. In some embodiments, the engineered tissue structure is a multilayer tissue structure including a first engineered tissue layer (e.g., a first engineered cardiac tissue layer, a first engineered muscle tissue layer) on a first side of a support and second engineered tissue layer (e.g., a second engineered cardiac tissue layer, a second muscle tissue layer) on a second side of the support. The method also includes determining the effect of the test compound on the tissue function (e.g., cardiac tissue function, muscle tissue function) in the presence and absence of the test compound, wherein a modulation of the tissue function (e.g., cardiac tissue function, muscle tissue function) in the presence of the test compound as compared to the tissue function in the absence of the test compound indicates that the test compound modulates the cardiac tissue function, thereby identifying a compound that modulates a cardiac tissue function.
Some embodiments described herein include methods for identifying a compound useful for treating or preventing a tissue disease (e.g., a cardiac tissue disease, a muscular tissue disease, a neural tissue disease). A method for identifying a compound useful for treating or preventing a tissue disease includes providing an engineered tissue structure (e.g., an engineered cardiac tissue, an engineered muscle tissue, an engineered neural tissue), and contacting the engineered tissue structure with a test compound. In some embodiments, the engineered tissue structure includes a first engineered tissue layer (e.g., a first engineered cardiac tissue layer, a first engineered muscle tissue layer, a first engineered neural tissue layer) on a first side of a support including a geometrically isolated node. In some embodiments, the engineered tissue structure is a multilayer tissue structure including a first engineered tissue layer (e.g., a first engineered cardiac tissue layer, a first engineered muscle tissue layer) on a first side of a support and second engineered tissue layer (e.g., a second engineered cardiac tissue layer, a second muscle tissue layer) on a second side of the support. The method also includes determining the effect of the test compound on a tissue function (e.g., a cardiac tissue function, a muscle tissue function, a neural tissue function) in the presence and absence of the test compound, where a modulation of the tissue function in the presence of the test compound as compared to the tissue function in the absence of the test compound indicates that the test compound modulates the tissue function, thereby identifying a compound useful for treating or preventing a tissue disease.
In some embodiments in which the cells are contractile cells, a deflection, a movement, or a change in curvature of the support due to contraction of the first engineering tissue layer and the second engineering tissue layer may be used to measure the tissue function. In some embodiments in which the cells are contractile cells, a magnitude of a deflection, a movement, or a change in curvature of the support may be used to measure the tissue function. In some embodiments in which the cells are contractile cells, a frequency of deflections, movements, or changes in curvature of the support may be used to measure the tissue function. In some embodiments, both a magnitude of a frequency of deflections, movements, or changes in curvature of the support may be used to measure the tissue function. Additional description of measurements of tissue function are described in U.S. Patent Application Publication No. 2014/0342394, entitled “Muscle Chips and Methods of Use”, which is incorporated by reference herein in its entirety. Additional description of measurements of tissue function are provided in the Example below.
In some embodiments, the tissue function may be a biomechanical activity. The biomechanical activity may be one or more of contractility, cell stress, cell swelling, and rigidity. The biomechanical activity may also be one or more of stem cell activation, stem cell maturation, tissue morphogenesis, and tissue remodeling. In other embodiments, the tissue function may be an electrophysiological activity. The electrophysiological activity may be a voltage parameter selected from the group including action potential, action potential duration (APD), conduction velocity (CV), refractory period, wavelength, restitution, bradycardia, tachycardia, reentrant arrhythmia, and/or a calcium flux parameter, e.g., intracellular calcium transient, transient amplitude, rise time (contraction), decay time (relaxation), total area under the transient (force), restitution, focal and spontaneous calcium release.
As used herein, the various forms of the term “modulate” are intended to include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).
As used herein, the term “contacting” (e.g., contacting a scaffold including pancreatic islet cells or adipocytes with a test compound) is intended to include any form of interaction (e.g., direct or indirect interaction) of a test compound and a scaffold or cells. The term contacting includes incubating a compound and scaffold or tissue together (e.g., adding the test compound to scaffold including pancreatic islet cells or adipocytes in culture).
Test compounds can be any agents including chemical agents (such as toxins), small molecules, pharmaceuticals, peptides, proteins (such as antibodies, cytokines, enzymes, and the like), nanoparticles, and nucleic acids, including gene medicines and introduced genes, which may encode therapeutic agents, such as proteins, antisense agents (i.e., nucleic acids comprising a sequence complementary to a target RNA expressed in a target cell type, such as RNAi or siRNA), ribozymes, and the like.
The engineered tissue structure and/or the population(s) of cells can be contacted with a test compound by any suitable means. For example, the test compound can be added drop-wise onto the surface structure and allowed to diffuse into or otherwise enter the structure, or it can be added to the nutrient medium and allowed to diffuse through the medium.
This invention is further illustrated by the following Example, which should not be construed as limiting.
The circulatory system of organisms is intricately designed to transport blood throughout the body. Its most basic function is fluid transport, and a diversity of similar fluid pumping mechanisms and designs are found throughout nature. Fluid pumps in vertebrates, considered broadly, range from a human circulatory system with closed vessels within which fluid moves, to oscillatory fluid mechanisms in aquatic species in which fluid is transported along the body to generate propulsive thrust. Inspired by these distinct but similar natural processes, biohybrid analogs of an external fluid pump capable of mimicking the locomotion of aquatic species have been developed (J. C. Nawroth et al., Nat Biotechnol 30, 792-797 (2012); S. J. Park et al., Science (New York, N.Y.) 353, 158-162 (2016); L. Ricotti et al., Science Robotics 2, eaaq0495 (2017)). The underlying motivation for developing biohybrid systems capable of reproducing biological behaviors has been to better understand the design principles and coordination mechanisms of biological systems, although their performance has been lacking in comparison to natural fluid transport pumps (L. Ricotti et al., Science Robotics 2, eaaq0495 (2017)).
A key feature of aquatic species is closed-loop actuation of antagonistic musculature that provides control over the direction of momentum transfer from the body muscles to the fluid enabling efficient locomotion. Similarly, in the circulatory system, muscles of the heart dynamically respond to physiological demands through internal feedback systems and impart momentum to drive fluid motion. Mechano-electrical signaling and cardiac automaticity play an essential role in regulating the contractile pace and strength in a closed-loop control system (
Using principles of cardiac control systems to design a biohybrid platform could result in a fluid pumping system with comparable efficiencies to natural fish-like fluid pumping systems. Leveraging fundamental features of cardiac function allows for autonomous self-pacing and independent motion control, while providing the basis for a closed-loop design that mimics aquatic swimming systems. A biohybrid fish (
A muscular bilayer construct was developed by modifying hydrogel-based muscular thin films (S. J. Park et al., Circulation 140, 390-404 (2019); M. L. McCain et al., Biomaterials 35, 5462-5471 (2014); materials and methods are described below). The double-sided micromolded gelatin thin film (200 μm thick) was engineered by sandwiching a gelatin and crosslinker (microbial transglutaminase) mixture between two polydimethylsiloxane (PDMS) stamps with line groove features (25 μm ridge width, 4 μm groove width, and 5 μm groove depth). Then, CMs were seeded onto both sides of the micromolded gelatin so that CMs could self-assemble as laminar, anisotropic muscle with engineered cellular alignment, characteristic of the ventricular myocardium (
To demonstrate independent activation between the muscular bilayer tissues, blue-light-sensitive (ChR2 (E. S. Boyden et al., Nat Neurosci 8, 1263-1268 (2005)) and red-light-sensitive (ChrimsonR (N. C. Klapoetke et al., Nature methods 11, 338-346 (2014)) ion channels were expressed in each muscle layer using lentiviral transduction (
B. Integration of Muscular Bilayer into Biohybrid Fish
The muscular bilayer construct was integrated into the biohybrid fish (as descried below) using tissue engineering techniques (
To characterize the system-level kinematics of the muscular bilayer, antagonistic muscle contractions in the biohybrid fish was controlled by external optogenetic stimulation (
Antagonistic muscle contractions of the biohybrid fish generated a hydrodynamic signature like those of wild-type BCF swimmers—specifically the water flow in the wake and around the fish bodies which were visualized using particle image velocimetry (PIV) (
The inclusion of a muscular bilayer architecture improved the high-frequency swimming of the biohybrid fish. The biohybrid fish (6.4 mm long muscle tissue body) was optogenetically controlled responded up to 3-4 Hz (
Whether reconstructing antagonistic muscle contractions with CMs could sustain spontaneous rhythmic contractions via mechano-electrical signaling was tested (
Whether reconstructing a geometrically distinct and electrically insulated node could initiate spontaneous electrical activity due to the automaticity of CMs in the absence of an external stimulus was tested. Inspired by the partial electrical insulation of a sinoatrial node (R. W. Joyner et al., Biophysical journal 50, 1157-1164 (1986)), the G-node (
To decouple the effect of antagonistic muscle contractions to muscle activation from the G-node, muscle movement with laboratory tape on a glass slide was mechanically restricted and muscle activation was determined using calcium imaging (Code and scripts. Zenodo (2021); http://doi.org/10.5281/zenodo.5618323/). CMs in the G-node and four corners (Anterior ventral corner: AV, Anterior dorsal corner: AD, Posterior ventral corner: PV, and Posterior dorsal corner: PD and
However, upon removing the restrictions on muscle movement, the G-node primarily acted as a secondary mechanism of controlling contractions. Only when the antagonistic muscle contractions were minimal, would the G-node initiate sequential local muscle activation and contraction, leading to undulatory locomotion (
Although these G-node-entrained, mechano-electrical signaling-sustained, cyclic antagonistic muscle contractions are autonomous, optogenetic stimulation can be used for on-demand locomotion control. Antagonistic muscle contractions became coupled with optical pacing within less than three sequential light pulses. Further, optogenetic stimulation can also be used to inhibit autonomous locomotion—pausing right after a pulsed stimulation can stop muscle contractions for an extended period (e.g., 50 secs). Prolonged continuous optogenetic stimulation stops muscle contractions and autonomous locomotion. External stimulation reinitiates autonomous, antagonistic muscle contractions via activating mechano-electrical signaling.
The autonomously swimming biohybrid fish (e.g., swimming at 15.0 mm/s) outperformed the locomotory speed of prior biohybrid muscular systems (J. C. Nawroth et al., Nat Biotechnol 30, 792-797 (2012); S. J. Park et al., Science (New York, N.Y.) 353, 158-162 (2016); A. W. Feinberg et al., Science (New York, N.Y.) 317, 1366-1370 (2007); J. Xi, J. J. Schmidt et al., Nature materials 4, 180-184 (2005); C. Cvetkovic et al., Proceedings of the National Academy of Sciences of the United States of America 111, 10125-10130 (2014); V. Chan et al., Scientific reports 2, 857 (2012); J. Kim et al., Lab on a chip 7, 1504-1508 (2007); Y. Akiyama et al., PloS one 7, e38274 (2012); Y. Akiyama et al., Biomed Microdevices 14, 979-986 (2012); G. J. Pagan-Diaz et al., Adv. Funct. Mater. 28, 13 (2018); R. Raman et al., Proceedings of the National Academy of Sciences of the United States of America 113, 3497-3502 (2016); B. J. Williams et al., Nature communications 5, 3081 (2014); O. Aydin et al., Proceedings of the National Academy of Sciences of the United States of America 116, 19841-19847 (2019)) (5-27× the speed of the biohybrid stingray (S. J. Park et al., Science (New York, N.Y.) 353, 158-162 (2016)) and the biohybrid skeletal muscle biorobot (G. J. Pagan-Diaz et al., Adv. Funct. Mater. 28, 13 (2018)) (
The biohybrid fish resembles the swimming performances of wild-type BCF swimmers with a similar body length (juvenile zebrafish, juvenile white molly, and Micro-devario kubotai) (
To analyze the efficiency of the biohybrid fish, scaling and dimensional analysis was used. Wild-type swimmers achieved energetically favorable locomotion via convergent evolution and are found to hew to the two scaling relationships, St˜Re−1/4 and Re˜Sw−1/4 in the low Re and high St flow regime (M. Gazzola et al., Nat. Phys. 10, 758-761 (2014)) (
The performance of the biohybrid fish is very sensitive to muscle kinematics and coordination. Some biohybrid fish accelerated by increasing tail-beat amplitude (
Given the autonomous antagonistic muscle contractions of the biohybrid fish, whether this spontaneous activity would improve its long-term performance was analyzed. The biohybrid fish maintained spontaneous activities for 108 days (16-18× over the biohybrid stingray—6 days (S. J. Park et al., Science (New York, N.Y.) 353, 158-162 (2016)) or skeletal muscle-based biohybrid actuator—7 days (Y. Morimoto et al., Science Robotics 3, 10 (2018))), equivalent to 38 million beats (
As described in this example, two functional design features of the heart—mechano-electrical signaling and automaticity—were integrated into a biohybrid platform and an autonomously actuating cardiac muscular system was created in a biohybrid fish. This biohybrid fish is a closed-loop system where the muscle contraction-induced bending is used as a feedback input to the endogenous mechanosensors—stretch-activated ion channels in the muscles. These channels respond to this feedback input and induce muscle activation and contraction, producing self-sustainable rhythmic BCF propulsion. The self-driven spontaneous contractions in the muscular bilayer induced coordinated global tissue-level contractions with comparable efficiencies to wild-type fish. Alternatively, integrated optogenetic control enabled overriding internal control mechanisms to stop and control asynchronous muscle contractions. There are few, if any, closed-loop mechanical fish robots that are free-swimming, and fish robots typically require numerous actuators and sensors to control fin movements that are difficult to engineer at smaller size scale (mm to cm scale) (R. Du, Z. Li et al., Eds. (Springer Berlin Heidelberg, Berlin, Heidelberg, 2015), pp. 1-24). However, by integrating the cardiac activation system as an embedded mechanism of both sensing and control enabled the generation of fish-like locomotion at the smaller size scales (G. V. Lauder et al., Eds. (Springer Berlin Heidelberg, Berlin, Heidelberg, 2015), pp. 25-49). The use of biological muscle actuators with intrinsic closed-loop control simplifies the construction compared with current mechanical robotic systems and provides control beyond existing biohybrid systems.
Additionally, the muscular bilayer construct of the Example provides a platform for studying tissue-level cardiac biophysics. It demonstrated that dynamic axial stretching can induce excitations and contractions on a beat-by-beat basis in engineered human stem cells-derived CMs tissues by contributing to antagonistic muscle contractions. It was found that antagonistic contractions are streptomycin and Gd3+-sensitive, which indicates that mechano-electrical signaling via stretch-activated ion channels is one of the essential mechanisms that mediate antagonistic contractions. Interestingly, in normal myocardium where CMs are mechanically and electrically coupled, mechano-electrical signaling contributes to synchronizing local ventricular repolarization and protects against cell-to-cell repolarizations and contractile heterogeneities across the heart (T. Opthof et al., Cardiovascular research 108, 181-187 (2015)). In contrast, in the muscular bilayer where antagonistic muscle pairs are mechanically coupled yet electrically decoupled across sides, mechano-electrical signaling generated stretch-induced depolarizations on a beat-by-beat basis. The stretch-induced excitations and contractions were also observed in quiescent single CMs and in a resting ventricular myocardium (T. A. Quinn et al., Circulation. Arrhythmia and electrophysiology 10, (2017)), but these observations were restricted to the ectopic responses of CMs to acute mechanical stimulation, which induced reentrant arrhythmias. The muscular bilayer construct of the Example is the first to demonstrate that the mechano-electrical signaling of CMs could induce self-sustaining muscle excitations and contractions for extended periods (108 days, equivalent to 38 million beats). These findings are aligned with the growing appreciation for cardiac stretch-activated channels and mechano-electrical signaling mechanism as targets of heart rhythm management (T. A. Quinn et al., Circulation. Arrhythmia and electrophysiology 10, (2017); T. A. Quinn et al., Cardiovascular research 108, 1-3 (2015)). The longevity of the autonomously moving fish system also raises the question of whether a feedback between repetitive electrical and mechanical activity and the regulation of its molecular elements via altered gene expression or other basic cellular processes is correlated.
The G-node, an isolated cluster of cells connected through a single conducting exit pathway, initiated spontaneous activation waves by reducing the impedance between source and sink. G-node integration improved locomotion speeds by enhancing the pacing frequency. This increased frequency in the presence of the G-node is reminiscent of entrainment in re-entry cycles where the focus shortens the re-entry cycle (J. Almendral et al., Pacing Clin Electrophysiol 36, 508-532 (2013)). Another possible underlying mechanism of the increased frequency is that the G-node produced regular contractions and consequently induced stronger and more rapid contractions of the muscular bilayer, which could enhance the dynamics of antagonistic, asynchronous muscle contractions. The G-node functionality as a node of automaticity in the biohybrid fish indicates that functionally, a pacemaker may be defined by its geometry and source-sink relationships as well as its ion channel expression.
Taken together, the technology described here represents foundational work for the goal of creating autonomous systems capable of homeostatic regulation and adaptive behavioral control. More importantly, the results suggest an opportunity to revisit long standing assumptions of how the heart works in biomimetic systems that allow a more granular analysis of structure-function relationship in cardiovascular physiology.
The following Materials and Methods were used in the Example.
Fabrication of the tissue-engineered fish (
The laser-cut laboratory tape was placed on the polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Midland, Mich.) stamp with line groove features (25 μm ridge width, 4 μm groove width, and 5 μm groove depth) to make a micropatterned gelatin body parallel to the biohybrid fish's longitudinal axis (
Commercial human stem cell-derived CMs (human stem cell-derived CMs, Axiogenesis, Ncardia, Belgium) were transduced with Channelrhodopsin-2 (ChR2) and ChrimsonR lentivirus at a multiplicity of infection of 5 for 24 hours. ChR2-expressing human stem cell-derived CMs were seeded onto one side at first (
The chip was designed within the vector graphics editing software CorelDraw (Corel, Canada). 6.35-millimeter-thick acrylic was then laser cut with a CO2 laser engraver (VersaLaser 2.0, Universal Laser Systems, Scottsdale, Ariz.). Half of the cut outs were made to be bottoms of a whole chip by introducing threads into the cut acrylic. The acrylic chips were placed in a 70% ethanol bath along with screws and placed in a sonicator for sterilization and cleaning.
Human stem cell-derived cardiomyocytes (husk-CMs) were purchased from Axiogenesis and Ncardia (Leiden, Netherlands) and cultured using the manufacturer's protocols. Briefly, 500 μl of culture media (Ncardia, Leiden, Netherlands) was added to each frozen vial of cells and thawed at 37° C. Thawed cells were resuspended in 9 mL of complete culture media (Axiogenesis, Ncardia, Leiden, Netherlands) and incubated in a fibronectin (0.01 μg/mL, BD Biosciences, Bedford, Mass.)-coated T25 flask in a 37° C. incubator for 4 hours, after which puromycin was supplemented to a final concentration of 2 μg/ml. The culture media was replaced with fresh media after an additional 24 hours, and then replaced every 48 hours for cell maintenance until use. For cell seeding on the biohybrid fish, human cardiomyocytes were dissociated with 0.25% trypsin-EDTA (Life Technologies, CA) and resuspended in puromycin free media.
Animal procedures were performed under protocols approved by Harvard University's Institutional Animal Care and Use Committee. Neonatal rat ventricular myocytes (NRVMs) (2-day old) were isolated as previously published (A. W. Feinberg et al., Science (New York, N.Y.) 317, 1366-1370 (2007)). Briefly, ventricles were removed from 2-day old Sprague Dawley rat pups (Charles River Laboratories, MA). Then, the manually minced tissues were placed in a 0.1% trypsin (Sigma Aldrich, St. Louis, Mo.) Hanks' Balanced Salt solution (HBSS) solution at 4° C. for approximately 14 hours. For additional enzymatic digestion, a 0.1% type II collagenase (Worthington Biochem, NJ) solution at room temperature was used to isolate ventricular myocytes. NRVMs were further dissociated by centrifuging, resuspending with HBSS, and passing the isolated cell solution through a 40 μm cell strainer. The solution was pre-plated twice for 50 minutes each at 37° C. in a M199 cell media (Life Technologies) supplemented with 10% heat-inactivated FBS (Life Technologies, CA) to remove fibroblasts and endothelial cells. Resulting NRVMs were seeded onto the biohybrid fish in M199 cell media (Life Technologies, CA) supplemented with 10% heat-inactivated FBS (Life Technologies, CA).
pLenti-Synapsin-hChR2(H134R)-EYFP-WPRE (E. S. Boyden et al., Nat Neurosci 8, 1263-1268 (2005)) and FCK-ChrimsonR-GFP (N. C. Klapoetke et al., Nature methods 11, 338-346 (2014)) was a gift from Karl Deisseroth (Addgene plasmid #20945; RRID:Addgene_20945) and Edward Boyden (Addgene plasmid #59049; RRID:Addgene_59049), respectively. hChR2 and ChrimsonR lentiviral vectors (cTnT-ChR2-eYFP and cTnT-ChrimsonR-mCherry) were constructed by cloning inserts (hChR2(H134R) and ChrimsonR) to lentiviral vectors with a cardiac-specific promotor (the cardiac troponin T, cTnT) and a fluorescent tag (enhanced yellow fluorescent protein, eYFP or red fluorescent protein, mCherry). The lentivirus was produced and purchased from VectorBuilder Inc (Chicago, Ill.) and used to infect both human-CMs and NRVMs with lentivirus (5×106 TU/ml) per 1 million CMs.
Light-emitting diode sources (LED) were used to independently activate ChR2 and ChrimsonR transduced cardiac tissues. Fiber-coupled LED light source (Prizmatix, Israel) at 450 nm and 630 nm were mounted through mono fiber optic cannulas (flat end, 400 μm diameter, NA 0.48, Doric Lenses Inc, Canada). To change the pacing frequency and duration, each LED source was independently controlled by analog signals that were synthesized with an analog output module (NI 9264, National Instruments, Austin, Tex.) by a custom software written (Code and scripts. Zenodo (2021); http://doi.org/10.5281/zenodo.5618323/) in LabVIEW (National Instruments, Austin, Tex.), as previously published (S. J. Park et al., Science (New York, N.Y.) 353, 158-162 (2016)). In addition, the analog output module was triggered by digital trigger signals that were generated by two push button switches through a digital board (USB-6501, National Instruments, Austin, Tex.), allowing the digital signals to change the frequency without time-delay, as previously published (S. J. Park et al., Science (New York, N.Y.) 353, 158-162 (2016)).
The biohybrid fish was placed in Tyrode's solution (1.8 mM CaCl2, 5 mM glucose, 5 mM Hepes, 1 mM MgCl2, 5.4 mM KCl, 135 mM NaCl, and 0.33 mM NaH2PO4 in deionized water, pH 7.4, at 37° C.; Sigma Aldrich, St. Louis, Mo.) in a 22 cm square chamber (VWR International, Radnor, Pa.) on a hotplate (VWR International, Radnor, Pa.) and temperature was maintained at 35° C. to 37° C. The recording of swimming performance of biohybrid fish was filmed with a sCMOS camera (Pco.edge, PCO AG, Germany) coupled with a zoom camera lens (Thorlabs Inc, Newton, N.J.) at 50 frames per second or a single-lens recorder (AXP55 4K Handycam, Sony, Japan) with a 26.8 mm wide-angle lens at 50 frames per second.
For the longevity measurements, the biohybrid fish were detached after 3 days in culture and incubated in complete culture medium (Ncardia, Leiden, Netherlands), allowing them for self-paced swimming. Every one or two weeks, the performance measurement was conducted in a 37° C. Tyrode's physiological salt solution. After each measurement, the biohybrid fish were placed back and incubated in complete culture medium. The culture medium was exchanged with 12 ml of fresh medium for each biohybrid fish every 4 days for cell maintenance.
To check the effect of mechanical signaling on the antagonistic contraction of muscular bilayer tissues, the biohybrid fish were treated with 250 μM streptomycin or 100 μM Gd3+ (both from Sigma-Aldrich, St. Louis, Mo.) before recording, streptomycin or Gd3+ for at least 24 hours.
Digital videos recorded during locomotion experiments were converted to image stacks using a custom-made Matlab program (24) (R2020b, Mathworks, Natick, Mass.). The head, body, and tail positions of the biohybrid fish were tracked using an image processing software (ImageJ, NIH, Bethesda, Md.). The moving distance of the biohybrid fish was measured during each tail-beat cycle, and the swimming speed was calculated by dividing the cumulative distance travelled by the total time. Tail-beat amplitude was measured as the distance between maximum right and left excursions. Tail-beat angle (θ) was calculated as the body angle difference between chord lines of its anterior body and caudal fin at the maximum right and left excursions. Antagonistic contraction was determined by checking whether a muscle contraction induced the subsequent contraction of the opposite side muscle with any delay or not. The empirical probability of antagonistic contraction was determined by dividing the number of antagonistic contractions by the total number of contractions.
The stress of the muscular bilayer was estimated by considering the geometric relationship of the curvature (κ), radius of curvature (r), length of muscle tissue (lmuscle), and tail-beat angle (θ) (κ=1/r=θ/lmuscle) (
where tmuscle is thickness of muscle tissue, tgelatin is thickness of gelatin, Egelatin is young's modulus of gelatin and υgelatin is Poisson's ratio of gelatin (Y. Akiyama et al., PloS one 7, e38274 (2012)).
Calcium activities of the muscle circuit were monitored with a calcium indicator, X-Rhod-1 (Invitrogen, Carlsbad, Calif.), using a modified tandem-lens macroscope as previously published (S. J. Park et al., Science (New York, N.Y.) 353, 158-162 (2016); S. J. Park et al., Circulation 140, 390-404 (2019)). Briefly, tandem-lens macroscope (Scimedia, Costa Mesa, Calif.) used in this example was equipped with a high-speed camera (MiCAM Ultima, Scimedia, Costa Mesa, Calif.), a plan APO 1× objective, a collimator (Lumencor, Beaverton, Oreg.) and a 200-mW mercury lamp for epifluorescence illumination (X-Cite exacte, Lumen Dynamics, Canada). A filter set (excitation filter: 580/14 nm, dichroic mirror: 593 nm cut-off, emission filter: 641/75, Semrock, Rochester, N.Y.) was used for X-Rhod-1 imaging.
The biohybrid fish was incubated with 2 μM X-Rhod-1 for 30 min at 37° C., rinsed with culture medium with 2% FBS to remove nonspecifically associated dye, and incubated again for 30 mins for complete de-esterification of the dye. Then, the biohybrid fish was rinsed with Tyrode's solution and mounted on a 37° C. heating stage (Warner Instruments, Hamden, Conn.) of the tandem-lens macroscope.
Post-processing of the raw calcium data was conducted with custom software written in MATLAB (24) (R2020b, MathWorks, Natick, Mass.). A spatial filter with 3×3 pixels was applied to improve the signal-noise ratio. Activation time of each pixel was calculated at the average maximum upstroke slope of multiple pulses of X-Rhod-1 signals over a 5 second recording window. The total activation time was determined as the difference between activation times at the last and the first activation sites along the posterior body of biohybrid fish.
Flow fields generated by the biohybrid and animal fish were monitored using Particle imaging velocimetry (PIV).
For the PIV measurement of biohybrid fish, algae particles (10-12 μm diameter, Tetraselmis sp., Reed Mariculture) were seeded in Tyrode's solution in a water chamber. 450 nm and 630 nm LED sources were used to independently activate ChR2 and ChrimsonR transduced cardiac tissues through mono fiber optic cannulas (flat end, 400 μm diameter, NA 0.48, Doric Lenses Inc, Canada). While infrared LED sources (IR30, CMVision, Houston, Tex.) illuminated the particles, the motions of the particles were recorded at 100 frames per second with a sCMOS camera (Pco.edge, PCO AG, Germany).
For the PIV measurement of freely-swimming animal fish, individual larval zebrafish, white molly, and Micro-devario kubotai were placed in a recirculating flow tank at speeds of 1.5 to 3.2 body lengths per second (not including zebrafish: for zebrafish, flow was not circulated). While infrared LED sources (IR30, CMVision, Houston, Tex.) illuminated seeded algae particles, the motions of the particles were recorded at 250-500 frames per second with a high-speed video camera (Photron PCI-2014, San Diego, Calif.).
Flow patterns of both biohybrid and animal fish were analyzed by calculating the velocity distribution within seeded particles between successive video frames using an open-source software (PIVlab 2.02 (W. Thielicke et al., Journal of Open Research Software 2, e30 (2014))) written in MATLAB (R2014b, MathWorks, Natick, Mass.).
Biohybrid fish was fixed for 12 min in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, Pa.) and 0.5% Triton-X (Sigma-Aldrich, St. Louis, Mo.) in PBS at 37° C. and incubated with mouse anti-sarcomeric α-actinin monoclonal primary antibody (Sigma-Aldrich, St. Louis, Mo.), and then stained with DAPI (Sigma-Aldrich) and goat anti-mouse secondary antibody conjugated to Alexa-647 (Invitrogen, Carlsbad, Calif.). The biohybrid were mounted on glass slides with ProLong Gold antifade mountant (Invitrogen, Carlsbad, Calif.). All images of biohybrid fish were acquired with an inverted microscope (Olympus IX83, Tokyo, Japan) with an attached spinning disk confocal system (Andor, Concord, Mass.), and three-dimensional reconstructions of images of muscular bilayer were analyzed and processed using image-processing software (ImageJ 1.52p, NIH, Bethesda, Md.).
The muscle mass of 42-days old biohybrid fish was measured by a laboratory scale (Mettler Toledo, Columbus, Ohio). The muscle mass was measured by subtracting the mass of gelatin-paper substrate (without cell seeding) from the total mass of biohybrid fish. In another way, the muscle mass of biohybrid fish using the single cell mass and the number of seeded cardiomyocytes on the gelatin portions were estimated. To estimate the mass of single cardiomyocyte, the mass of two million of cardiomyocytes was measured and then divided by the number of cardiomyocytes.
In cardiomyocytes, pacemaking arises from an interplay between hyperpolarizing and dominating depolarizing currents during the phase 4 depolarization period (the period between repolarization and the rising phase of the subsequent action potential). In the sinoatrial node, the hyperpolarization-induced inward current (HCN isoforms) of cardiac pacemaker cells plays a major role in pacemaking (W. Liang et al., Stem Cells. 38, 352-368 (2020)). However, in the case of G-node of this example where stem cell-derived CMs and NRVMs supposedly lack the expression of HCNs, the pacemaking potentials are a result of inward currents produced by Ca2+ cycling (driven by rhythmic releases of intracellular Ca2+ from the sarco/endoplasmic reticulum).
The remaining question was how a region of cells initiate coordinated pacemaking and how this relates to electrical cell-to-cell coupling. The geometrical node design played a crucial role here because the current being exchanged between individual cells of different membrane potentials is locally accumulated in the membrane capacitance at the edges and is reflected at the tissue edges (A. G. Kléber et al., Physiol Rev 84, 431-488 (2004); A. G. Kléber et al., Biophysics Reviews 2, 031301 (2021)). The reflection of intracellular currents at the tissue edges synchronizes the spontaneous activity in the structurally isolated small tissues like a G-node and increases their firing rate. The mechanism of reflection at the corners of cultures behave similarly (since downstream impedance is reduced), in particular the anterior corners with acute angles albeit less than in the G-node, and as a result, firing is enhanced in the whole anterior side (AD, AV and G-node).
To test the theoretical considerations, the role of geometric factors of the G-node and sink that determine the leading muscle activation site in human stem cell-derived cardiomyocyte tissue (
1) G-node integration: G-node integrated tissues predominantly activated the muscle construct at the G-node compared to the four corners of the muscle tissue (G-node: 72% vs corners: 5-9% in
2) Sink's corner design: Rounding the sink's corners decreased the corners' activation probability, thus increasing the probability of activation at the G-node activation to 91% (A3 in
3) G-node's corner design: To investigate the contribution of a sharp corner to the probability of initial activation at the G-node, square and diamond G-nodes were compared to the circular G-node design of this example. The square and diamond G-nodes (B2 and B3 in
The contribution of the corner designs in the tissue to the probability of initial activation at the corner was quantified. First, the number of downstream cells to be activated by a spontaneously activated cell at the corner was estimated: the number of cells in the given area was calculated. An area 1-mm away from the corner was chosen, because a 1-mm radius corner in the A3 design (
ζ=P/PG-node.
ζ of various G-node-integrated tissue designs (
All animal procedures were done in accordance with the guidelines of Harvard University's Animal Care and Use Committee. Fishes were anesthetized in neutrally buffered MS222. Fish were either bred in lab or purchased from Uncle Ned's Fish Factory (Millis, Mass.).
Tissue samples of fishes were washed by PBS and then fixed in 4% Paraformaldehyde (SigmaAldrich, St. Louis, Mo.), which were incubated at 4° C. for 24 hours. The samples were decalcificated with Decalcifier-Original (Avantik, Pine Brook, N.J.) for 2.5 h and incubated with 5% sodium sulfate solution for 12 h. Decalcification, paraffin embedding, sectioning, imaging, and staining with Masson Trichrome stain were completed by Applied Pathology Systems (Shrewbury, Mass.).
Statistical analysis was performed using JMP Pro 15 (SAS Institute, Inc.). Functional performances of the biohybrid fish were compared using one-way ANOVA followed by Tukey-Kramer honestly significant difference test. Data represent mean±SEM. The numbers of fish indicate the numbers of independent experiments.
In describing exemplary embodiments, specific terminology is used for the sake of clarity. For purposes of description, each specific term is intended to at least include all technical and functional equivalents that operate in a similar manner to accomplish a similar purpose. Additionally, in some instances where a particular exemplary embodiment includes a plurality of system elements or method steps, those elements or steps may be replaced with a single element or step. Likewise, a single element or step may be replaced with a plurality of elements or steps that serve the same purpose. Further, where parameters for various properties are specified herein for exemplary embodiments, those parameters may be adjusted up or down by 1/20th, 1/10th, ⅕th, ⅓rd, ½, etc., or by rounded-off approximations thereof, unless otherwise specified. Moreover, while exemplary embodiments have been shown and described with references to particular embodiments thereof, those of ordinary skill in the art will understand that various substitutions and alterations in form and details may be made therein without departing from the scope of the invention. Further still, other aspects, functions and advantages are also within the scope of the invention.
The contents of all references, including patents and patent applications, cited throughout this application are hereby incorporated herein by reference in their entirety. The appropriate components and methods of those references may be selected for the invention and embodiments thereof. Still further, the components and methods identified in the Background section are integral to this disclosure and can be used in conjunction with or substituted for components and methods described elsewhere in the disclosure within the scope of the invention.
As may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, numerous changes and modifications may be made to the above-described and other embodiments of the present disclosure without departing from the spirit of the invention as defined in the appended claims. Accordingly, this detailed description of embodiments is to be taken in an illustrative, as opposed to a limiting, sense.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application claims the benefit of priority to U.S. Provisional Application No. 63/299,920, filed on Jan. 15, 2022, the entire contents of which are incorporated herein by reference.
This invention was made with government support under TR000522 awarded by the National Institutes of Health (NIH), and under 2011754 awarded by the National Science Foundation (NSF). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63299920 | Jan 2022 | US |
