Systems and methods for promoting long-term culture of mechanically active cells

Abstract

The systems disclosed herein can include a cell culture surface having a silicon oxide first layer and second layer comprising a plurality of amphiphilic molecules, each amphiphilic molecule having a positively charged portion extending from a silane portion that is bound to the silicon oxide first layer. The cell culture surface can also include extracellular matrix components and water molecules dispersed between and bonded to positively charged portions of the amphiphilic molecules. Methods of using the systems can include plating mechanically active cells onto the cell culture surface, forming a mechanically active cell culture, and maintaining the mechanically active cell culture in a mechanically active state and adhered to the cell culture surface for at least 14 days. The mechanically active cell culture can be exposed to an agent and responses of the mechanically active cell culture to the agent can be detected.

Description

SYSTEMS AND METHODS FOR PROMOTING LONG-TERMCULTURE OF MECHANICALLY ACTIVE CELLSRELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 526,792, filed July 14, 2023, which is hereby incorporated by reference in its entirety.FIELD

[0002] This disclosure relates to cell culture devices, and more particularly to cell culture devices for mechanically active cells.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with Government Support under Grant Nos. R01NS050452 and R44AG059511 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0004] Functional in vitro tissue models provide invaluable opportunities to test new treatment candidates, allowing researchers to observe drug effects in a controlled environment that mimics human physiology. These models are crucial for preclinical testing, helping to predict the efficacy and safety of new therapies before clinical trials. However, developing robust muscle cell cultures poses significant challenges due to the unique properties and requirements of muscle cells.

[0005] Muscle cells have complex nutritional and environmental needs that must be meticulously managed to sustain cell health and functionality. The high metabolic demands require a steady supply of nutrients and oxygen. In vitro culture systems must be designed to deliver these nutrients uniformly, which can be challenging. Furthermore, accumulation of metabolic waste products can inhibit cell growth and function. Efficient removal of these wastes is crucial for maintaining a healthy culture environment.

[0006] Inducing differentiation and maintaining maturation can also be challenging. Muscle cells, or myoblasts, must differentiate into mature myotubes to form functional muscle tissue. Achieving and maintaining this differentiation in vitro requires precise control of the culture conditions, including the composition of the growth medium, the presence of specific growth factors, and mechanical stimuli. Even after differentiation, ensuring that myotubes mature to a state where they exhibit the functional characteristics of muscle tissue, such as synchronized contractions and proper alignment, is complex. This process often necessitates long culture periods and carefully timed interventions.

[0007] Another major challenge is the contractile nature of muscle cells. When muscle cells contract, they generate significant force, which can cause the cells to physically detach from the culture surface. This detachment is particularly problematic during long-term experiments, as repeated contractions exacerbate the issue, leading to loss of cell integrity and functionality. Ensuring that muscle cells remain adhered to the culture substrate while maintaining their ability to contract is essential for accurate and reliable data. Advancements are needed to improve the stability and longevity of muscle cell cultures, enabling more accurate testing of new treatment candidates and providing deeper insights into muscle physiology and pathology.SUMMARY

[0008] Microcantilever culture is an emerging model for measuring and studying force dynamics of myotubes [1, 3, 6-11]. However, the contractile force generated by the myotubes can cause them to detach from the cantilevers, especially during long-term experiments, thus impeding chronic investigations of skeletal muscles for drug efficacy and toxicity. The developments disclosed herein improve the long-term survival and adhesion of mechanically active myotubes to glass and silicon microcantilevers, paving the way for long-term chronic experiments on these systems for both drug efficacy and toxicity studies.

[0009] In some aspects, the techniques described herein relate to a system for promoting longterm culture of mechanically active cells. The system can include a cell culture surface. The cell culture surface can include a silicon oxide first layer and a second layer including a plurality of organosilanes. Each organosilane can have a positively charged portion extending from a silane portion, with the silane portion covalently bound to the silicon oxide first layer. The cell culture surface can also include extracellular matrix components and water molecules dispersed between and bonded to positively charged portions of the plurality of organosilanes.

[0010] In some aspects, the cell culture surface is the exterior surface of a silicon substrate or of a glass substrate. The second layer of the cell culture surface can be, for example, a selfassembled monolayer. In some aspects, the organosilane is an amino silane (for example, DETA silane). The extracellular matrix components can include one or more of elastin, collagen, hyaluronic acid, and heparan sulfate proteoglycan. A contact angle of water with the cell culture surface can be less than 37°, in some aspects.

[0011] In some aspects, the system further includes a culture of mechanically active cells (such as, for example, muscle cells) adhered to the cell culture surface and bound to the extracellular matrix components. A fatigue index of the culture of mechanically active cells can reduce over a period of two weeks or more. In some aspects, the culture of mechanically active cells includes a younger state and an older state, wherein the younger state and the older state are separated by a culture duration of at least two weeks and can be distinguished by one or more cellular aging markers. The older state can be characterized by a reduced fatigue index as compared to the younger state.

[0012] In some aspects, the system further includes a microcantilever, and the cell culture surface is an exterior surface of the microcantilever. The microcantilever can optionally include silicon. In some aspects, the system further includes a passivated surface adjacent to a microcantilever.

[0013] In some aspects, the system can further include a contraction detection setup. The contraction detection setup can optionally include a light source, a photodetector, and / or a computing device. In some aspects, the contraction measurement setup includes piezoelectric or piezoresistive microcantilevers.

[0014] Methods disclosed herein include detecting a response of a long-term mechanically active cell culture to an agent. The methods can include a step of plating mechanically active cells onto a cell culture surface (the cell culture surface including: a silicon oxide first layer, a second layer including a plurality of organosilanes, each organosilane having a positively charged portion extending from a silane portion with the silane portion covalently bound to the silicon oxide first layer, and extracellular matrix components and water molecules dispersed between and bonded to positively charged portions of the plurality of organosilanes). The methods can further include steps of forming a mechanically active cell culture, maintaining the mechanically active cell culture in a mechanically active state and adhered to the cell culture surface for at least 14 days, exposing the mechanically active cell culture to an agent, and detecting a response of the mechanically active cell culture to the agent.

[0015] In some aspects, the methods further include a step of maintaining the mechanically active cell culture in a mechanically active state and adhered to the cell culture surface for at least 28 days. The methods can optionally include a step of electrically stimulating the mechanically active cell culture.

[0016] In some aspects of the methods, detecting a response of the agent includes detecting a deflection of a microcantilever.

[0017] In some aspects, the methods further include steps of continuing to maintain the mechanically active cell culture in a mechanically active state for at least 14 days after initially exposing the mechanically active cell culture to the agent, then repeating the steps of exposing the mechanically active cell culture to the agent and detecting the response of the mechanically active cell culture to the agent.

[0018] Methods disclosed herein further include methods of making cell culture surfaces for promoting long-term culture of mechanically active cells. The methods of making include steps of handling a substrate including a silicon oxide first layer and adding a second layer including a plurality of organosilanes, each organosilane having a positively charged portion extending from a silane portion. The methods of making further include steps of covalently binding the silane portion to the silicon oxide first layer, dispersing extracellular matrix components and water molecules between positively charged portions of the plurality of organosilanes, and bonding the extracellular matrix components and the water molecules between the positively charged portions of the organosilanes.

[0019] In some aspects of the methods of making, the substrate including the silicon oxide first layer is a silicon substrate including a microcantilever.

[0020] In some aspects of the methods of making, prior to adding the second layer, one or more of the following steps can be taken: acid-washing the silicon oxide first layer, passivating the silicon oxide first layer, and / or etching away a non-passivated pattern.

[0021] In some aspects of the methods of making, the step of adding a second layer including a plurality of organosilanes includes a step of exposing the silicon oxide first layer to a solution of organosilanes in tolulene. The step of covalently binding the silane portion to the silicon oxide first layer can optionally include a step of heating the substrate to a temperature of from 60° - 100°C.

[0022] In some aspects of the methods of making, the step of dispersing extracellular matrix components and water molecules includes covering the second layer with a solution including extracellular matrix components and water. In some aspects, the solution can include one ormore of elastin, collagen, hyaluronic acid, and heparan sulfate proteoglycan. For example, the steps can include covering the second layer with 0.03-0.09 pg elastin per mm2, covering the second layer with 0.01-0.06 pg collagen per mm2, covering the second layer with 0.0001-0.0007 pg heparan sulfate proteoglycan per mm2, and / or covering the second layer with 0.0007- 0.0013 pg hyaluronic acid per mm2.

[0023] In some aspects, the methods of making further include a step of adhering mechanically active cells to the cell culture surface to create a mechanically active cell culture. The mechanically active cell culture can optionally be maintained for more than two weeks in culture in a mechanically active state.DESCRIPTION OF DRAWINGS

[0024] The device is explained in even greater detail in the following drawings. The drawings are merely exemplary and certain features may be used singularly or in combination with other features. The drawings are not necessarily drawn to scale.

[0025] FIG. 1 shows a mechanically active cell adhered to a cantilever substrate via a cell culture surface as compared to a muscle attached to a bone via a tendon.

[0026] FIG. 2 shows a schematic of a mechanically active cell adhered to a cell culture surface.

[0027] FIG. 3 shows an example contraction detection setup.

[0028] FIG. 4 is a schematic of the overall experimental plan is described where the listed abbreviations represent the following molecules; D: DETA; E: elastin; C: collagen; H: heparan sulfate proteoglycan; Hy: hyaluronic acid. iPSC stands for induced pluripotent stem cells.

[0029] FIG. 5 A shows contact angle measurements of DETA-silane-modified surfaces upon protein adsorption, indicating static contact angle values and representative droplet images for DETA, DEH, DEC, and DECHHy. Advancing and receding contact angle values along with hysteresis are noted to characterize the wettability of the surfaces. FIG. 5B shows a schematic demonstrating how contact angle is measured.

[0030] FIG. 6 shows XPS survey spectrum showing relative elemental characteristics of DETA, DEH, DEC, and DECHHy substrates.

[0031] FIGS. 7A-7B show XPS high-resolution spectra indicating the change in peak characteristics after deposition of protein for DETA, DEH, DEC, and DECHHy. The high- resolution spectra are shown for the elements: in FIG. 7A: silicon (Si2p), carbon (Cl s), and in FIG. 7B: nitrogen (Nls), oxygen (Ols).

[0032] FIG. 8 shows representative phase images of human primary skeletal muscle myotubes on days 14 and 28 cultured on DETA, DEH, and DEC substrates. These cultures were stimulated on days 14, 17, 21, and 28. The representative images are collected from three different experiments (n = 3). The scale bar shown in the figure is 100 pm.

[0033] FIG. 9 shows phase images of iPSC-derived human skeletal muscle myotubes: DEH, DEC, and DECHHy on days 14 and 28 indicated a stable platform. These cultures were stimulated repeatedly on days 14, 17, 21, and 28. Scale bar = 20 pm.

[0034] FIGS. 10A-10C show fatigue index measurements for human primary skeletal muscle myotubes upon repeat stimulation and testing on days 14, 17, 21, and 28. FIG. 10A shows stimulation (bottom trace) and response measurements (top trace) on DEH. FIG. 10B shows stimulation (bottom trace) and response measurements (top trace) on DEC. FIG. 10C shows the fatigue index results over time.

[0035] FIGS. 11A-11D show fatigue index measurements for iPSC-derived human skeletal muscle myotubes upon repeat stimulation and testing on days 14, 17, 21, and 28. FIG. 11A shows stimulation (bottom trace) and response measurements (top trace) on DEH. FIG. 11B shows stimulation (bottom trace) and response measurements (top trace) on DEC. FIG. 11C shows stimulation (bottom trace) and response measurements (top trace) on DECHHy substrates. FIG. 1 ID shows the fatigue index results over time.

[0036] FIGS. 12A-12F shows various results of integrating skeletal muscle myotubes on silicon cantilevers (DEC), (a) Representative picture of the primary skeletal myotubes on the cantilever at day 14. (b) Representative traces for force measurements upon repeat testing for primary skeletal muscle myotubes on days 14 (blue), 17 (orange), 21 (gray), and 28 (yellow), measured as a factor of the voltage output of the photodetector, (c) Bar chart showing the values of average amplitude on days 14, 17, 21, and 28. (d) Representative picture of the iPSC-derived skeletal myotubes on the cantilever at day 14. (e) Representative force dynamics plot obtained for iPSC- derived skeletal muscle myotubes on silicon cantilevers upon repeat testing; force is expressed as a factor of the voltage output of the photodetector, shown for days 14 (blue), 17 (orange), 21 (gray), and 28 (yellow), (f) Bar chart showing the values of average amplitude at days 14, 17, 21, and 28.

[0037] FIG. 13 shows phase images of iPSC-derived human skeletal muscle at lower magnification, indicating uniform cell coverage. Images are shown from days 14 and 20 for cells cultured on the surfaces DETA-Elastin-HSPG, DETA-Elastin-Collagen, and DETA-Elastin- Collagen-HSPG-Hyaluronic acid. Scale bar = 100 pm

[0038] FIG. 14 is an example computing device.DETAILED DESCRIPTION

[0039] The following description of certain examples of the inventive concepts should not be used to limit the scope of the claims. Other examples, features, aspects, configurations, embodiments, and advantages will become apparent to those skilled in the art from the following description. As will be realized, the device and / or methods are capable of other different and obvious aspects, all without departing from the spirit of the inventive concepts. Accordingly, the drawings and descriptions should be regarded as illustrative in nature and not restrictive.

[0040] For purposes of this description, certain advantages and novel features of the aspects and configurations of this disclosure are described herein. The described methods, systems, and apparatus should not be construed as limiting in any way. Instead, the present disclosure is directed toward all novel and nonobvious features and aspects of the various disclosed aspects, alone and in various combinations and sub-combinations with one another. The disclosed methods, systems, and apparatus are not limited to any specific aspect, feature, or combination thereof, nor do the disclosed methods, systems, and apparatus require that any one or more specific advantages be present or problems be solved.

[0041] Although the operations of exemplary aspects of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that disclosed aspects can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular aspect or implementation are not limited to that aspect or implementation, and may be applied to any aspect or implementation disclosed. It will understood that various changes and additional variations may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention or the inventive concept thereof. Certain aspects and features of any given aspect may be translated to other aspects described herein. In addition, many modifications may be made to adapt a particular situation or device to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular implementations disclosed herein, but that the invention will include all implementations falling within the scope of the appended claims.

[0042] Features, integers, characteristics, compounds, chemical moieties, or groups described in conjunction with a particular aspect, configuration, embodiment or example of the invention are to be understood to be applicable to any other aspect, configuration, embodiment, or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing aspects. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract, and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0043] Throughout this application, various publications and patent applications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this disclosure pertains. However, it should be appreciated that any patent, publication, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material.

[0044] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one nonlimiting aspect the terms are defined to be within 10%. In another non-limiting aspect, the termsare defined to be within 5%. In still another non-limiting aspect, the terms are defined to be within 1%.

[0045] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0046] The terms "coupled," "connected," and the like as used herein mean the joining of two members directly or indirectly to one another. Such joining may be stationary (e.g., permanent) or moveable (e.g., removable or releasable). Such joining may be achieved with the two members or the two members and any additional intermediate members being integrally formed as a single unitary body with one another or with the two members or the two members and any additional intermediate members being attached to one another.

[0047] Certain terminology is used in the following description for convenience only and is not limiting. The words “right,” “left,” “lower,” and “upper” designate direction in the drawings to which reference is made. The words “inner” and “outer” refer to directions toward and away from, respectively, the geometric center of the described feature or device. The terminology includes the above-listed words, derivatives thereof, and words of similar import.

[0048] The term “conjugate” is used to describe a proximate association between two or more chemical components. The components can be covalently bound to one another directly or through a linker moiety, as in a covalent conjugate, or the components can be non-covalently bound to one another, as in a non-covalent conjugate. Non-covalent conjugates have chemical components proximately associated through electrostatic forces (e.g., ionic bonds, dative bonds, hydrogen bonds, van der Waals forces, and the like.) The term “covalently bound” means that a covalent bond exists between entities. Accordingly, the term “covalently bound” refers to a sharing of pairs of electrons between adjacent molecules. “Covalently bound” can encompass both direct (electrons shared directly between the molecules) and indirect (the molecules are covalently bound via a linking molecule) covalent bonds .

[0049] The term “hydrogen bond” describes an attractive interaction between a hydrogen atom from a molecule or molecular fragment X-H in which X is more electronegative than H, and an atom or a group of atoms in the same or different molecule, in which there is evidence of bond formation. The hydrogen bond donor can be a cation and the hydrogen bond acceptor can be an anion.

[0050] “Homologs” are defined herein as two polypeptides, proteins, proteoglycans, or polysaccharides that have some level of sequence identity. Homologs include molecular variantshaving the same relevant function (e.g., to behave similarly to an extracellular matrix molecule). In some embodiments, homologs have about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92, 91% or 90% homology. In other embodiments, homologs have about 80% or about 85% homology.

[0051] Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps. "Exemplary" means "an example of" and is not intended to convey an indication of a preferred or ideal aspect. "Such as" is not used in a restrictive sense, but for explanatory purposes.

[0052] In vitro defined skeletal muscle developmental models have been established as a paradigm for studying muscle pathologies and other age-related abnormalities [1-6].Quantifying the force generated by the skeletal muscle is a crucial physiological parameter to define muscle health [6,7]. One of the emerging models for studying force dynamics is to integrate the differentiated myotubes on microcantilevers [1,3,6-11], where the contractile force generated by the myotubes deflects the cantilevers. The cantilever’s deflection is then proportional to the force generated by the myotube contractions [7]. This can be used for measurement of contractile force generated by myotubes to investigate various physiological conditions and for understanding the therapeutic effects of drug candidates.

[0053] The cantilever-based technology for the measurement of muscular force dynamics is limited as the contractile myotubes can detach from the substrate with repeated stimulations. So long-term studies of muscle development can be challenging, and this technical deficiency needs to be addressed to exploit the full potential of microcantilever technology. One solution to the problem lies in a better understanding of the in vitro extracellular matrix (ECM) requirements of the myotubes, based on its in vivo environment.

[0054] In the human body, muscle attaches to bone through tendons, as shown in FIG. 1. Tendons include the cell types tenocytes and tenoblasts, which are integrated with bone through specific ECM components. The force generated by the muscle is transmitted through the tendon to the bone. The tendon microenvironment is rich in elastomeric proteins that function as a spring between muscle and bone and help to withstand the muscular force. One of the key elastomeric proteins found in the tendon is elastin which is embedded in the collagenous matrix and bathed in an aqueous proteoglycan jelly [12-17]. A similar elastomeric protein to human elastin, resilin, is found in the insect kingdom and provides elasticity to the mechanically active tissue of many insects [18-23]. Interestingly, elastin and resilin are evolutionarily linked, the former being more hydrophobic and the latter more hydrophilic. It has been theorized that thehydrophobicity has increased to meet the increasing force requirements of higher-order animal species [12-17]. Another aspect of the similarity of resilin and elastin is that in an aqueous environment, both forms swell to rubberlike structures containing 50-60% water but on drying become rigid and glass-like [18-24].

[0055] Disclosed herein is a biomimetic approach to address the issue of muscle attachment to silicon microcantilevers based on the utilization of ECM components present in tendon-to-bone integration present in the human and insect musculoskeletal systems described above. The elastomeric properties of elastin present in the tendon suggested that a surface modification of the skeletal muscle substrates with extracellular matrix proteins, such as elastin, collagen, heparan sulfate proteoglycan, and hyaluronic acid, could improve the survival and function on glass coverslips and silicon-microcantilevers. However, to increase the adherence to the silicon oxide surfaces, they were first modified with a positively charged self-assembled amino silane (N1 -(3 -trimethoxy silylpropyl)-diethylenetriamine) (DETA) monolayer

[0025] . The silane surface offers multiple charge-charge interactions for improved deposition and adhesion of the protein molecules to facilitate muscle attachment. The skeletal muscle was then morphologically and functionally characterized. This approach indicates the successful development of a defined ECM composition for the long-term survival of mechanically active skeletal muscle myotubes on glass and silicon microcantilevers that can be integrated into multiorgan microphysiological platforms.

[0056] FIG. 2 shows an example system for promoting long-term culture of mechanically active cells. The system includes a cell culture surface 102 comprising a silicon oxide first layer 104 and a second layer 106 comprising a plurality of organosilanes. Each organosilane has a positively charged portion 108 extending from a silane portion 110, the silane portion covalently bound to the silicon oxide first layer 104. The cell culture surface 102 further includes extracellular matrix components 112 and water molecules 114 dispersed between and bonded to positively charged portions 108 of the organosilanes. The system can also include one or more cantilevers 116, mechanically active cells 118, and a contraction detection setup 130, as shown in FIG. 3.

[0057] The cell culture surface 102 can be formed, in some implementations, of the exterior surface of a silicon substrate 103, such as those conventionally used in the fabrication of micromechanical electrical systems. The silicon oxide first layer 104 can be a native or nonnative silicon oxide surface of a silicon substrate 103. In some implementations, the cell culture surface 102 is the exterior surface of a glass substrate, such as a coverslip or a glass cell culturematerial, and the silicon oxide first layer 104 is a native or non-native surface of the glass. In some implementations, the cell culture surface 102 is any surface that has been treated to include a silicon oxide first layer 104.

[0058] In some implementations, the second layer 106 is formed of amphiphilic molecules, meaning molecules have both hydrophilic (water-attracting) and hydrophobic (water-repelling) portions. For example, the positively charged portions 108 of in the second layer 106 are hydrophilic. In some implementations, the amphiphilic molecules of second layer 106 are organosilanes, such as amino silanes. In one example, the amino silane is amine- terminated alkylsilane (3 -Trimethoxy silyl propyl) diethylenetriamine (DETA). The amphiphilic molecules can take the form of a self-assembled monolayer, or a single layer of molecules that spontaneously organize on a substrate surface.

[0059] Extracellular matrix components 112 and water molecules 114 are dispersed between and bonded to the positively charged portions 108 of the second layer 106. In some implementations, the extracellular matrix components 112 include naturally or synthetically derived whole extracellular matrix molecules (such as, for example, extracellular matrix proteins, glycoproteins, proteoglycans, and / or polysaccharides). In some implementations, the extracellular matrix components 112 can include fragments of extracellular matrix molecules, such as portions of extracellular matrix molecules that participate in structural, binding, cellular activation, or cell signaling activities. The extracellular matrix components and the positively charged portions 108 can be non-covalently conjugated (e.g., not covalently bound), for example, through electrostatic forces or hydrogen bonding. In other implementations, the extracellular matrix components 112 can be covalently conjugated to the positively charged portions 108 of the second layer 106.

[0060] Exemplary extracellular matrix components include, as shown in FIG. 2, elastin 120, collagen 122, heparan sulfate proteoglycan 124, and hyaluronic acid 126. This could encompass the varying types of elastin, collagen, hyaluronic acid, and / or heparan sulfate proteoglycan. In some implementations, exemplary extracellular matrix components include homologs or fragments of elastin, collagen, hyaluronic acid, and / or heparan sulfate proteoglycan. In some implementations, exemplary extracellular matrix components include chimeric or fusion molecules that include elastin, collagen, hyaluronic acid, and / or heparan sulfate proteoglycan, or homologs or fragments thereof. In some embodiments, extracellular matrix components can include, for example, chimeric or fusion molecules (or homologs or fragments thereof, of: collagen types I-XXIX, elastin, fibronectin, laminins (including Laminin-111, Laminin-211,Laminin-221, Laminin-332, Laminin-411, Laminin-421, Laminin-511, Laminin-521), vitronectin, tenascins (Tenascin-C, Tenascin-R, Tenascin-X), osteonectin (SPARC), thrombospondins 1-5, sulfate proteoglycans (inclusive of heparan, chondroitin, dermatan, and keratan sulfate proteoglycans, including syndecans, glypicans, perlecan, aggrecan, versican, neurocan, brevican, decorin, biglycan, lumican, keratocan, mimecan (osteoglycin)), hyaluronan (hyaluronic acid), fibrillin- 1 , fibrillin-2, fibulins 1 -5, osteopontin, CYR61 , CTGF, MAGP- 1 , MAGP-2, integrins, selectins, cadherins, matrix metalloproteinases (MMP-1 to MMP-28), and tissue inhibitors of metalloproteinases (TIMPs 1-4).

[0061] The inclusion of the extracellular matrix components 112 increases the hydrophilicity of the cell culture surface 102, which is demonstrated by a lowering of the contact angle a water droplet makes with the cell culture surface 102. This phenomenon can be seen in FIG. 5A, where the inclusion of extracellular matrix components elastin, collagen, hyaluronic acid, and / or heparan sulfate proteoglycan to a DETA layer caused a lowering of the contact angle as the droplet spread more thinly over the more hydrophilic surface. FIG. 5B demonstrates how contact angle is measured, with angle 0 measured between the surface and the contacting edge of the water droplet. In some implementations, a contact angle of water with the cell culture surface is less than 37°, including less than 35°, less than 30°, less than 25°, less than 20°, and less than 15°.

[0062] The system further comprises a culture of mechanically active cells 118 adhered to the cell culture surface. Mechanically active cells can include, for example, any type of muscle cell, including but not limited to skeletal muscle cells, cardiomyocytes, and smooth muscle cells. However, the disclosure is not intended to be limited to muscle cell culture. In actuality many cells are mechanically active and may challenge traditional cell culture systems. Thus, the systems and methods disclosed herein might be useful in culture of other mechanically active cell types, such as, but not limited to, fibroblasts, osteoblasts, osteoclasts, tenocytes, tenoblasts, endothelial cells, chondrocytes, epithelial cells, myofibroblasts, and hepatocytes.

[0063] The system promotes long term culture of mechanically active cells 118. One way this can be demonstrated is the measure of fatigue index. In muscle cell culture studies, the fatigue index typically represents a measure of how well the muscle cells maintain their contractile function over repeated stimulations or cycles of activity. It is often used to assess the fatigue resistance of muscle tissue in vitro, mimicking the endurance capacity of muscles in vivo. The systems disclosed herein promote the reduction of fatigue index in the culture of mechanically active cells over time, indicating increased efficiency and improved performance. For example,the systems show a reduction in fatigue index of the culture of mechanically active cells over a period of two weeks or more (including over a period of more than 3 weeks, more than 4 weeks, more than 6 weeks, more than 2 months, more than 4 months, more than 8 months, and more than 1 year).

[0064] The system at various times includes a culture of mechanically active cells 118 in a younger state and in an older state. The younger state and the older state can be distinguished by one or more cellular aging markers (such as, but not limited to, DNA damage, histone modification, telomere attrition, cell cycle arrest, SA-PGal activity, senescence-associated secretory phenotype (SASP) activation, changes in lamina, and nuclear morphology)). The older state can also be characterized by a reduced fatigue index as compared to the younger state, as described above. The younger state and the older state are separated by a culture duration of at least two weeks (including at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 2 months, at least 4 months, at least 8 months and at least 12 months).

[0065] In some implementations, the system includes as a substrate one or more microcantilevers 116. The cell culture surface 102 is formed on an exterior surface of the microcantilever 116. In some implementations, a plurality of microcantilevers 116 are arranged into a microcantilever array, upon which the mechanically active cells are cultured. The microcantilevers 116 can comprise, or can be formed of, silicon. The outermost surface of microcantilevers 116 includes a native or non-native silicon oxide first layer 104. The system can also include a passivated surface adjacent to the one or more microcantilevers 116. For example, the passivated surface can include a PEG coating to prevent cell adherence to unintended surfaces.

[0066] The system can further include a contraction detection setup 130. An exemplary setup is shown in FIG. 3. As noted above, mechanically active cells 118 can be cultured on the surface of one or more microcantilevers 116. A light source 128 directs a light beam (such as a laser beam) at the tip of one or more microcantilevers 116. The light beam is reflected back from the tip of the microcantilever 116 and toward a photodetector 132, which registers a voltage. Movement of the tip causes a change in voltage registered at the photodetector 132. A computing device 134 controls movement and activity of the light source 128, the photodetector 132, a pulse generator 136 (used for stimulating the mechanically active cells 118), and a microscope objective 138. The computing device 134 can further receive and record measurements from the photodetector 132 and perform analyses on the measurements (for example, to calculate fatigue index). The computing device 134 can run one or more cell culturestimulation protocols, over culture durations lasting at least two weeks (including at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 2 months, at least 4 months, at least 8 months and at least 12 months). The computing device can provide a user interface for setting and / or altering culture settings, or for performing analyses. Certain exemplary processes and analyses are described in U.S. Patent No. U.S. 11,022,605, which is incorporated herein by reference.

[0067] In another embodiment, the contraction detection setup 130 can include piezoelectric or piezoresistive cantilevers, as described in U.S. Patent No. 11,614,437, which is incorporated herein by reference.

[0068] In another implementation, the systems and methods disclosed herein can be utilized as part of a larger organ-on-a-chip containing multiple organ components, as described in U.S. Patent Application Publication No. 2021 / 0003554, which is incorporated herein by reference.

[0069] With the basic structure of the system for promoting long-term culture of mechanically active cells 118 being thusly disclosed, a greater appreciation of the construction and benefits may be gained from the following discussion of methods of making the cell culture surface 102. It is to be noted that this discussion is provided for illustrative purposes only.

[0070] The methods include adding a second layer 106 of amphiphilic molecules to a silicon oxide first layer 104. The silicon oxide first layer 104 can be, in some implementations, an exterior surface of a silicon substrate 103 comprising a microcantilever 116. The amphiphilic molecules include positively charged portions 108 extending from silane portions 110. In some implementations, the amphiphilic molecules are organosilanes, as described above. The methods further include covalently binding the silane portion 110 to the silicon oxide first layer 104.

[0071] Some implementations include preliminary steps before adding the second layer. For example, the silicon oxide first layer 104 can be acid- washed and / or passivated prior to adding the second layer. If passivated, a non-passivated pattern is etched away prior to adding the second layer 106.

[0072] In some implementations, the process for adding the second layer 106 includes exposing the silicon oxide first layer 104 to a solution of organosilanes in tolulene. As part of the process of covalently binding the silane portion 110 of the organosilanes to the silicon oxide first layer 104, the substrate 103 and solution can be heated to a temperature of from about 60° to about 100°C (including about 70 °C to about 90 °C, or about 80 °C). The heating step can last from about 10 minutes to about 60 minutes (including from about 20 minutes to about 50 minutes, or about 30 minutes). In some implementations, after the heating step, the substrate 103 can be rinsed again with tolulene (as many as five rinses), and then substrate can be heated a secondtime but with a solution of distilled tolulene (heating temperature and duration may vary as compared to the initial heating step).

[0073] Extracellular matrix components 112 and water molecules 114 are dispersed and bound between positively charged portions 108 of the amphiphilic molecules (e.g., organosilanes). This process can include a step of covering the second layer with a solution of extracellular matrix components and water, or phosphate buffered saline (with magnesium and calcium removed, in some embodiments). The extracellular matrix components 112 in the solution could encompass the varying types, homologs, fragments, chimeras or hybrids discussed above. Particularly, the solution could include varying types, homologs, fragments, chimeras or hybrid molecules of elastin, collagen, hyaluronic acid, and / or heparan sulfate proteoglycan. In one implementation, the methods can include covering the second layer 106 with a solution depositing elastin at about 0.03 pg per mm2to about 0.09 pg per mm2(including about 0.03 pg elastin per mm2, about 0.04 pg elastin per mm2, about 0.05 pg elastin per mm2, about 0.06 pg elastin per mm2, about 0.07 pg elastin per mm2, about 0.08 pg elastin per mm2, and about 0.09 pg elastin per mm2). In one implementation, the methods can include covering the second layer 106 with a solution depositing collagen at about 0.01 pg per mm2to about 0.06 pg per mm2(including about 0.01 pg collagen per mm2, about 0.02 pg collagen per mm2, about 0.03 pg collagen per mm2, about 0.04 pg collagen per mm2, about 0.05 pg collagen per mm2, about 0.06 pg collagen per mm2). In one implementation, the methods can include covering the second layer 106 with a solution depositing heparan sulfate proteoglycan at about 0.0001 pg per mm2to about 0.0007 pg per mm2(including about 0.0001 pg heparan sulfate proteoglycan per mm2, about 0.0002 pg heparan sulfate proteoglycan per mm2, about 0.0003 pg heparan sulfate proteoglycan per mm2, about 0.0004 pg heparan sulfate proteoglycan per mm2, about 0.0005 pg heparan sulfate proteoglycan per mm2, about 0.0006 pg heparan sulfate proteoglycan per mm2, and about 0.0007 pg heparan sulfate proteoglycan per mm2). In one implementation, the methods can include covering the second layer 106 with a solution depositing hyaluronic acid at about 0.0007 pg per mm2to about 0. 0013 pg per mm2(including about 0.0007 pg hyaluronic acid per mm2, about 0.0008 pg hyaluronic acid per mm2, about 0.0009 pg hyaluronic acid per mm2, about 0.0010 pg hyaluronic acid per mm2, about 0.0011 pg hyaluronic acid per mm2, about 0.0012 pg hyaluronic acid per mm2, and about 0.0013 pg hyaluronic acid per mm2).

[0074] The methods further include adhering mechanically active cells 118 to the cell culture surface 102 to create a mechanically active cell culture and maintaining the mechanically active cell culture for more than two weeks in culture in a mechanically active state (including over aperiod of more than 3 weeks, more than 4 weeks, more than 6 weeks, more than 2 months, more than 4 months, more than 8 months, and more than 1 year). Indications that the cell culture is mechanically active can include contractions of muscle cell types, which can be detected utilizing contraction detection setups disclosed herein.

[0075] With the basic structure of the system and methods of making the long-term culture of mechanically active cells being thusly disclosed, a greater appreciation of the construction and benefits may be gained from the following discussion of methods of using the system for promoting long-term cell culture. It is to be noted that this discussion is provided for illustrative purposes only.

[0076] The methods of use include a method of detecting a response of a long-term mechanically active cell culture to an agent, the method comprising plating mechanically active cells onto a cell culture surface 102, and forming a mechanically active cell culture. The methods further include maintaining the mechanically active cell culture in a mechanically active state and adhered to the cell culture surface for at least 14 days. Finally, the methods include exposing the mechanically active cell culture to an agent, and detecting a response of the mechanically active cell culture to the agent. Some implementations include a step of electrically stimulating the mechanically active cell culture.

[0077] In some implementations, the process of detecting a response to the agent comprises detecting a deflection of a microcantilever. The response to the agent can be also or alternatively be a characteristic of cell contractions, including, but not limited to, contraction force, contraction frequency, and / or fatigue index. The response can also or alternatively be a biomarker.

[0078] In some implementations, the methods include maintaining the mechanically active cell culture in a mechanically active state and adhered to the cell culture surface for at least two weeks (including at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 2 months, at least 4 months, at least 8 months and at least 12 months). The mechanically active cell culture may be maintained after exposure to the agent, and the step of exposing the culture to the agent can be repeated after an additional culture duration (of at least two weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 2 months, at least 4 months, at least 8 months and at least 12 months). Certain methods from U.S. Patent No. 10,160,953, which is incorporated by reference, may be utilized in the long-term culture of mechanically active cells.

[0079] The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims below are intended to include any structure, material, or act forperforming the function in combination with other claimed elements as specifically claimed. The description of the present invention has been presented for purposes of illustration and description, but is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. The implementation was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various implementations with various modifications as are suited to the particular use contemplated.EXAMPLE 1: Study of Skeletal muscle adhesion and longevity in a functional microcantilever platform

[0080] To improve the integration of myotubes with microcantilevers, we drew inspiration from the elastomeric proteins, elastin and resilin, that are present in the animal and insect worlds, respectively. The spring action of these proteins plays a critical role in force dampening in vivo. In animals, elastin is present in the collagenous matrix of the tendon which is the attachment point of muscles to bones. The tendon microenvironment consists of elastin, collagen, and an aqueous jelly-like mass of proteoglycans. In an attempt to mimic this tendon microenvironment, elastin, collagen, heparan sulfate proteoglycan, and hyaluronic acid were deposited on a positively charged silane substrate. This enabled the long-term survival of mechanically active myotubes on glass and silicon microcantilevers for over 28 days. The skeletal muscle cultures were derived from both primary and induced pluripotent stem cell (iPSC)-derived human skeletal muscles. Both types of myoblasts formed myotubes which survived for five weeks. Primary skeletal muscles and iPSC-derived skeletal muscles also showed a similar trend in fatigue index values. Upon integration with the microcantilever system, the primary muscle and iPSC-derived myotubes were tested successively over a one-month period, thus paving the way for long-term chronic experiments on these systems for both drug efficacy and toxicity studies.

[0081] MATERIALS AND METHODS

[0082] Surface Preparation

[0083] Modification o f glass cover slip with DETA silane: A three-step process was employed to coat glass coverslips with DETA silane. Initially, the glass coverslips (22 x 22 mm, VWR) underwent acid cleaning as outlined in previous publications. Briefly, they were immersed in a 1: 1 hydrochloric acid and methanol solution for 30 minutes, followed by rinsing with deionizedwater. Subsequently, they were submerged in concentrated sulfuric acid for 30 minutes, rinsed in deionized water, then cleaned in boiling deionized water for an additional 30 minutes. After rinsing in ethyl alcohol to remove the water content, the coverslips were allowed to air dry. Activation of the coverslips was performed using oxygen plasma for 20 minutes at 750 mTorr with a PDC-32G plasma cleaner from Harrick Plasma [1-3, 6].

[0084] Prior to coating, a reaction mixture containing 0.1 % DETA-silane (United Chemical Laboratories, Inc., T2910) in distilled toluene (VWR, BDH1151-4LG) was prepared in an inert glovebox (MBraun, Stratham, NH) to prevent moisture contamination. The plasma-treated coverslips were immersed in the reaction mixture and heated to 80 °C for 30 minutes. After cooling to room temperature, the surfaces were rinsed thrice in toluene. The coverslips were then reheated in distilled toluene to 80 °C for another 30 minutes and cured overnight at 100 °C in an oven. They were subsequently stored in a desiccator until further use [1-3, 6].

[0085] Surface coating with biomolecules on DETA-modified surfaces: Prior to protein coating, the DETA-modified surfaces were sterilized in 100% ethanol and dried in a laminar flow hood. Sterilized DETA-coated coverslips were placed in a 6-well plate. Elastin from human skin (E7402), heparan sulfate proteoglycan (H4777), and hyaluronic acid sodium salt from bovine vitreous humor (H7630) were purchased from Sigma-Aldrich. Rat tail collagen I (A1048301) was acquired from Thermo Fisher Scientific. Stock solutions of individual ECM components were prepared in phosphate buffer saline (PBS) with no calcium or magnesium. For elastin-heparan sulfate (DEH) proteoglycan coating, 0.056 pg / mm2of elastin and 0.00043 pg / mm2of heparan sulfate proteoglycan were used to cover the DETA-coated coverslip. For elastin-collagen (DEC) coating, 0.056 pg / mm2of elastin and 0.031 pg / mm2of collagen were coated on the DETA coverslip. For elastin-collagen-heparan sulfate proteoglycan-hyaluronic acid coating, 0.056 pg / mm2of elastin, 0.00043 pg / mm2of heparan sulfate proteoglycan, 0.031 pg / mm2of collagen, and 0.001 pg / mm2of hyaluronic acid were coated (DECHHy). The coatings were performed by soaking DETA coverslips in ECM components as described above and storing them overnight at 4 °C. Before cell seeding, excess ECM component solution was aspirated.

[0086] Surface Characterization

[0087] Surface contact a .it I e and hysteresis measurements: A rame-hart standard goniometer (model number: 250-00-115) was used for contact angle measurements. Contact angle values were measured by placing a 5 pL water droplet on each surface. For hysteresis calculations, the advancing angle (0A) was measured by adding a 10 pL water drop on each surface, and thereceding angle (0R) was measured by removing 4 pL from the previously added droplet.Hysteresis was calculated as COS(0R)-COS(0A). Contact angle values are reported in degrees as average ± standard error, n = 3.

[0088] X-ray photoeleclron spectroscopy (XPS} XPS analysis was conducted using a Thermo Scientific K-alpha XPS spectrophotometer. Three points were analyzed for each sample. Survey spectra and high-resolution spectra for silicon (Si 2p), carbon (C I s), nitrogen (N I s), and oxygen (O Is) were obtained. The pass energy for high-resolution spectra was set at 50 eV for silicon, nitrogen, and oxygen, and 25 eV for carbon.

[0089] Cell culture

[0090] Human primary skeletal muscle cell culture: The human primary skeletal muscle cell line was obtained from Lonza, Allendale, NJ, USA. A frozen vial of myoblasts was thawed in a water bath set at 37 °C, and 9 mL of PBS was added. The mixture was spun in a centrifuge at 280g, 4 °C for 5 minutes. The supernatant was aspirated, and the cell pellet was resuspended in 1 mL of proliferation medium (adult growth medium). Cells were plated on the aforementioned surface-coated coverslips at a density of 150 cells / mm2and incubated for 45 minutes before adding 2 mL of proliferation medium. Upon reaching 80% confluence, the culture medium was switched to differentiation medium NbActiv4 (BrainBits) supplemented with an antibiotic and antimycotic (Thermo Fisher Scientific, 15240062). Components of the adult growth medium are detailed in Table 1.TABLE 1: Components of Adult Growth Medium

[0091] Human-induced pluripotent stem cell (iPSC)-derived skeletal muscle cell cultures: The human iPSC line (ND41865) was purchased from the Coriell Institute Stem Cell Biobank at NIH, Camden, New Jersey 08103, USA. These cells were differentiated into human-induced pluripotent stem cell (iPSC) skeletal muscle myoblasts following procedures described in previous publications

[0026] . Myoblasts were thawed in a 37 °C water bath. Subsequently, 9 mL of proliferation medium (MyoCult medium) was slowly added. Cells were pelleted at 280g for 5 minutes at 4 °C, supernatant was discarded, and the pellet was resuspended in 1 mL of proliferation medium. Cells were seeded onto protein- modified surfaces at a density of 100 cells / mm2. After an initial 45 -minute incubation, proliferation medium was added. Once cells reached 50-60% confluence, half of the medium was replaced with priming medium. After 48 hours, half of the medium was switched to differentiation medium, and cultures were maintained in differentiation medium thereafter. MyoCult medium was prepared by adding 10 mL of human MyoCult-SF Expansion 10X Supplement (05982, STEMCELL Technologies) to 90 mL of Dulbecco’s modified Eagle’s medium (DMEM) with 1000 mg / L D-glucose (36253, STEMCELL Technologies). HI medium was used as the priming medium, the constituent components of which are detailed in Table 2. NbActiv4 (BrainBits) supplemented with antibiotic-antimycotic was used as differentiation medium.TABLE 2: Components of HI MediumTABLE 3: Summary of Medium Formulation Used for Human Primary and iPSC-Derived Skeletal Muscle Myoblast Cultures

[0092] Phase-contrast imaging of the cultures: Cultures were maintained in differentiation media with a half medium change every 3 days. Phase images were obtained using a Zeiss inverted microscope. Day 14 images were obtained before cultures were electrically stimulated. Day 28 images were taken after cultures were repeatedly stimulated on days 14, 17, 21, and 28.

[0093] Myotube fatigue index measurements using pixel differential technique'. The culture coverslips were assembled in acrylate single organ housings. For testing, a full media change was performed with Leibovitz’s L-15 medium (11415064, Gibco™, Thermo Fisher Scientific) supplemented with serum-free B-27 supplement (17504044, Gibco™, Thermo Fisher Scientific), antibiotic, and anti-mycotic. Chlorinated silver wire electrodes dipped in the medium were used to stimulate human skeletal muscle culture using a pulse stimulator (model 2100, A-M Systems). For the study, stimulation frequencies of 1.0 and 2.0 Hz were used for 15 s using pulses of 2 V as described in an earlier study

[0027] . The systems were maintained at 37 °C throughout the testing. To record, an upright microscope was connected to a camera (Hamamatsu, model C8484-05G) capturing 50 frames / s while the myotubes contracted upon stimulation. LabVIEW (National Instruments, Austin, TX, USA) software was used as a platform to image, stimulate, and record skeletal muscle myotubes and analyzed using OpenCV-Python. Upon analysis, the fatigue index was calculated at frequencies of 1.0 and 2.0 Hz. For fatigue index calculation, the area under the curve was subtracted from the area of a rectangle with sides as time and peak force; this has been described in detail in previous studies [27, 28]. Fatigue index values range between 0 and 1 , and it is a measurement of physical fatigue or muscle activity at high frequencyleading to induction of tetanus-like behavior. This equation is commonly utilized in clinical studies

[0028] . A fatigue index value of 0 indicates ideal behavior where the muscle shows no sign of tiring, whereas myotubes with a fatigue index value of 1 show signs of extreme weakness wherein tetanus cannot be introduced.

[0094] Integrating myotubes on a silicon microcantilever system to study the force dynamics: There are 32 silicon cantilevers in a silicon chip of 2.25 cm2. The dimension of each cantilever is 100 pm x 750 pm. The cantilevers are acid washed before coating them with DETA silane. After acid washing, the cantilever surface is passivated with a solution of 0.15% polyethylene glycol (PEG) prepared in toluene. A mask of 50 pm pattern width per cantilever is used to pattern the PEG-modified cantilevers with a laser of wavelength 93 nm, frequency 10 Hz, and pulse intensity 200 mJ / pulse for 45 s. These etched cantilevers are then coated with 0.1% DETA-silane in distilled toluene for 30 min at 80 °C. Once the reaction mixture cools to room temperature, the cantilevers are washed thrice with distilled toluene and finally heated in distilled toluene to 80 °C for 30 min. The cantilevers are oven-dried overnight and stored in a desiccator. The cantilevers are sterilized in 100% ethanol in a laminar flow hood and coated with DETA-elastin-collagen (DEC) as described above. Skeletal muscle cells are plated on cantilevers at a density of 300 cells / mm2.

[0095] To study the force dynamics, each cantilever chip is assembled in acrylate housings and set up on the testing platform. The skeletal muscle myotubes are stimulated at a frequency of 0.5 Hz with a pulse stimulator (model 2100, A-M Systems) connected to the housing using a printed circuit board. Two chlorinated silver electrodes are dipped in the media to stimulate the system. When stimulated, the myotubes exert force on the cantilever. The laser focused on the tip of the cantilever is used to measure the deflection for 10 s per cantilever (as a factor of the voltage of the photodetector, recorded using the Lab VIEW software and analyzed using a script written in Python as described in previous publications). Primary and iPSC-derived skeletal muscle myotubes cultured on silicon cantilevers are repeatedly stimulated on days 14, 17, 21, and 28, and force measurements are taken. The measurements of the voltage of the photodetector have been shown as average amplitudes + standard error in microvolts [29, 30].

[0096] RESULTS

[0097] The overall experimental layout and sequence of figures is summarized in FIG. 4. The four surface types (DETA, DEC, DEH, DECHHy) are characterized using contact angle and XPS techniques. Upon understanding the chemical nature of each surface type and confirming the integration of ECM proteins, the adhesion of primary and iPSC-derived skeletal muscle cellson these surfaces is characterized. Since these surfaces are designed to improve the longevity of skeletal muscle in in vitro systems, the myotubes are functionally characterized by repeated stimulation to mimic their physiological activity and further integration on the microcantilever system to measure physiologically relevant force readouts.

[0098] Surface Preparation

[0099] Glass coverslips were acid-washed to eliminate impurities from the surface, and subsequent oxygen plasma treatment activated the glass surface before DETA silane modification for uniform reactivity. DETA silane is a positively charged tri-amino silane that has previously been shown to bind ECM protein.31The DETA silane rendered the surface positively charged, aiding an entropically favored charge-charge interaction between the modified glass coverslips and cantilevers with the deposited proteins. The proteins deposited on the surface were elastin (0.056 pg / mm2), collagen (0.031 pg / mm2), heparan sulfate proteoglycan (0.00043 pg / mm2), and hyaluronic acid (0.001 pg / mm2).

[0100] Contact angle measurements: Contact angle measurements are shown in FIG. 5A with values for DETA of 41.40° ± 0.84, DETA-elastin-HSPG (DEH) of 33.80° ± 2.08, DEC of 16.48° ± 1.73, and DETA-elastin-collagen-HSPG-hyaluronic acid (DECHHy) of 17.94° ± 2.14, expressed as average ± standard error, n = 3. All of the surfaces are hydrophilic, and their degree of hydrophilicity increased with protein adsorption. It is interesting to note that the DEC and DECHHy surfaces retained water for a longer duration after rinsing, indicating slower drying kinetics as compared to DETA and DEH. To explain this wetting property, hysteresis measurements were conducted, since contact angle hysteresis, the differences in horizontal components of advancing and receding angles, indicates the amount of movement caused to remove unit volume and the corresponding energy required to move the drop on the surface. The higher the interaction between surfaces, the higher the energy required to move the drop, and for unit volume, the lower the movement of the drop. Therefore, lower hysteresis values for DEC and DECHHy indicated enhanced wettability of the surfaces. The DECHHy surface had the lowest contact angle with significant hysteresis, indicating the surface most likely to retain significant water and enable the tightest binding of the ECM components.

[0101] XPS measurements: XPS survey spectra for DETA, DEH, DEC, and DECHHy show the presence of silicon, carbon, nitrogen, and oxygen, as presented in FIG. 6. High-resolution XPS spectra for silicon (Si 2p), carbon (C Is), nitrogen (N Is), and oxygen (O Is) are shown for DETA, DEH, DEC, and DECHHy in FIGS. 7A-7B. A prominent carbonyl peak is observedupon the addition of the protein ECM components on DETA, indicating the incorporation of these molecules on the DETA-modified surfaces.

[0102] Cell culture

[0103] Phase images for human primary skeletal muscle cultures after days 14 and 28 are shown in FIG. 8. Days were counted after cultures were switched to differentiation medium. Day 14 images are before electrical stimulation, and day 28 images are after cultures had been repeatedly exposed to electrical stimulations on days 14, 17, 21, and 28. These repeat stimulations were performed to test the efficacy of the surface modifications when myotubes were functionally active. These results indicate that physiologically active myotubes were maintained in vitro with the DEH and DEC substrates for more than a month.

[0104] Upon integrating the primary and iPSC skeletal muscle on the microcantilever systems, their physiologically relevant parameters were tested successively over a one-month period. These results indicate the suitability of these surface compositions for long-term chronic experiments in vitro. Both primary and iPSC skeletal muscle cells were cultured in the absence of serum in chemically defined medium to ensure that the test beds were adaptable to test compounds in integrated multiorgan systems and to enable pharmacokinetic and pharmacodynamic studies. This platform would also be useful to understand the temporal expression of different biomarkers from the skeletal muscle system. The medium compositions are shown in Tables 1 and 2 and summarized for both cell types in Table 3.

[0105] Phase images for human-iPSC-derived skeletal muscle cultures for days 14 and 28 after the cultures were switched to differentiation medium are shown in FIG. 9. Phase images indicate that the mechanically active myotubes on the modified surfaces were stable in this platform for 28 days with repeat stimulations on days 14, 17, 21, and 28. Phase images at lower magnification showing uniform skeletal muscle myotube coverage on day 14 and 20 for muscle cells cultured on three different surface conditions are shown in FIG. 13.

[0106] Myotube fatigue index measurements using pixel differential imaging Fatigue index values for the primary skeletal muscle grown on the DEH- and DEC-modified surface were measured for repeat stimulations on the same cultures on day 14, day 17, day 21, and day 28 as indicated in Table 4.TABLE 4: Fatigue Index Values for Human Primary Skeletal Muscle Myotubes

[0107] The cultured primary skeletal muscle myotubes were functionally active, and a representative curve per condition has been shown for the two surface modifications used during the study (FIGS. 10A-10C).

[0108] Fatigue index values for frequencies 1 and 2 Hz on days 14, 17, 21, and 28 when iPSC-derived skeletal muscle myotubes were cultured on DEH, DEC, and DECHHy substrates are shown in Table 5.TABLE 5: Fatigue Index Values for iPSC Skeletal Muscle Myotubes on Modified Surfaces over28 Days

[0109] The iPSC-derived skeletal muscle myotubes maintained structural and functional integrity for four weeks. Representative traces and fatigue index plots for various substrates are shown in FIGS. 11A-11D.

[0110] Inleyratiny myotubes on a silicon-based microcantilever system to study the force dynamics'. Primary skeletal muscle myotubes were repeat stimulated, and corresponding contractions were measured on days 14, 17, 21, and 28. The average amplitudes of the voltage of the photodetector were measured in microvolts as 5.61 ± 0.87, 2.46 ± 0.36, 4.41 ± 1.97, and 2.59 ± 0.62 on days 14, 17, 21, and 28, respectively. Stimulation-induced contractions were measured for iPSC-derived skeletal muscle myotubes on days 14, 17, 21 , and 28. On days 14, 17, 21 , and 28, the average voltage of the photodetector (measured in pV) was 39.40 ± 8.67, 30.41 ± 6.19, 23.12 ± 3.56, and 10.83 ± 1.21 on days 14, 17, 21, and 28, respectively. FIGS. 12A-12F show the results of this study.

[0111] DISCUSSION

[0112] The results showed a long-term survival of the myotubes on the DETA-ECM protein- modified substrates and microcantilever systems. After repeated stimulation and testing, the myotubes remain viable for more than four weeks. The fatigue index reduces over time, and the myotubes stabilize and contract on the microcantilever systems.

[0113] upon modification with DETA, the surface could be considered a “porous substrate” similar to the surface of bone. While the Si-O-Si network offers a firm attachment on the glass substrate to form a monolayer, the amino-terminated chain offers flexibility and a wide charge distribution to form stable complexes with the proteins, as shown in the XPS data [32-39]. The gap between the flexible amino-terminated chains functions as a porous gap where proteins and water molecules get confined. The decreased contact angle with protein deposition on DETA and significantly higher wettability of DEC and DECHHy surfaces indicate the proposed water confinement hypothesis in the DETA-protein matrix. The confinement of water is of direct significance from the biological perspective of cell adhesion. In a way, the DETA-protein surface functions like a jelly matrix which supports long-term functional survival of mechanically active tissue, viz., contractile skeletal muscle. This complex remains relatively stable since the Si-O-Si network offers a firm anchorage to the substrate. This unique feature of the DETA-ECM protein complex and the supporting evidence led us to explain the “tendonmimicking” feature of the novel substrate (FIG. 1). The proposed surface modification protocol will allow for long-term muscle cultures under stimulation which is more demanding on cell attachment than spontaneous contraction [29, 40].

[0114] In terms of the physiological significance of the present study, an emerging trend in the fatigue index (a measure of resistance to fatigue) was observed. It was noted that the fatigue index reduces over time upon repeated stimulation of both primary and iPSC-derived skeletalmuscle. Thus, these myotubes become more efficient and exhibit improved performance with repeated exercise, provided the initial ECM is optimal. This observation correlates with findings that a regular exercise regime helps reduce the fatigue index in vivo. The study warrants further exploration as it represents the first consideration of muscle and tendon in one common system. This opens up the possibility that injecting tendon-mimicking skeletal muscle ECM proteins at the site of muscular injury could be a potential approach to promote muscle repair [41-45]. This area requires further investigation due to its significant implications in sports nutrition, medicine, and strategic performances. Additionally, it has been observed in other mechanically active cells such as cardiomyocytes and smooth muscle cells that pathology and repair lead to ECM remodeling [46-48]. The present model presents a defined ECM that offers opportunities for exploring disease pathologies and repair mechanisms in skeletal muscle cells with greater control.

[0115] The microcantilever results exhibit long-term growth and stabilization of the myotubes with time. The average amplitude of the voltage is higher in the iPSC compared to the primary skeletal muscle. This could be partly because iPSC-derived myotubes have higher plasticity than the primary skeletal muscle. Further, primary skeletal muscle cells are obtained from adult individuals. The average voltage decreases with age, as observed from the data.EXAMPLE 2: Additional information on microcantilever fabrication and contraction detection systems

[0116] Spontaneous or electrical stimulation-evoked force generation of muscle cells can be measured based on optical detection or electrical detection of cantilever-bending. Optical detection of displacement of the end of the cantilever is based on the principle routinely used in atomic force microscopes (AFM) in which a laser beam is reflected from the cantilever to a sensor. The sensor detects the displacement of the laser beam caused by changes in the position of the cantilever. In this optical detection method, an automated electrical shutter is placed in the beam path. This placement minimizes the exposure of the cells to the reflected laser light. Electrical detection of the displacement is based on the piezoelectric principle that requires special design and materials for the fabrication of the cantilevers. Both detection methods enable automation and high-throughput screening on myocyte contraction force. Peak contraction force and force-contraction velocity relationships can be calculated based on the geometry of the cantilevers and the thickness of cultured muscle cells (which can determined from immunostaining data, for example). Test compounds can be applied using traditional methods tothe medium and possible changes in force or force- velocity relationship can be detected and analyzed using standard statistical methods.

[0117] In some implementations, microcantilevers are fabricated as follows, as described in U.S. Patent. No 10,386,360, which is incorporated herein by reference. The layout for the microcantilevers was generated using AutoCAD 2004. The patterns were written to chrome coated 4-5 inch soda-lime glass masks for front and back side photolithography.Microcantilevers were fabricated from 6 inch double-sided polished silicon-on-insulator (SOI) wafers with a 5 mm crystalline silicon layer (front side) and a 500 mm silicon dioxide layer (back side). The front side was primed with a 10 nm layer of hexamethyldisilazane (HMDS) to promote resist adhesion. A 5 mm layer of the photoresist AZ 5214 E (Clariant, Muttenz, Switzerland) was spun onto the device layer followed by softbake, alignment, exposure, and development. The device layer was etched using the deep reactive ion etch (DRIE) process at a rate of 2 mm / min. Resist was stripped and a 0.5 mm thick layer of silicon dioxide was deposited via Plasma Enhanced Chemical Vapor Deposition (PECVD) process to protect the device layer during subsequent processing. The wafer was then flipped over and was primed with a 100 A layer of HMDS and spun with 4.15 mm layer of AZ 9245 photoresist (Clariant, Muttenz, Switzerland). Coating was followed by softbake, front-back alignment, development, and DRIE etch at 4 mm / min until the bulk of the back side had been etched through leaving only the buried native oxide layer. The devices were then immersed in a buffered HF dip to remove the buried native oxide layer as well as the protective silicon dioxide that had been deposited onto the device layer. Individual devices were separated by breaking connecting tabs that were incorporated into the device design. Microcantilever dimensions were measured using a JEOL 6400 scanning electron microscope (SEM) at a take-off angle of 50° off normal.

[0118] Prior to cell culture experiments the microcantilevers were coated with the amine- terminated alkylsilane (3-Trimethoxysilyl propyl) diethylenetriamine (DETA). Prior to coating microcantilevers were cleaned using serial acid baths. Substrates were arranged in a porcelain coverslip holder (Thomas Scientific, Swedesboro, N.J.). The substrates were then immersed in a 1 :1 (vokvol) solution of methanol and concentrated HC1 for at least 1 hour. This step removed surface contaminants. After 1 hour the substrates were rinsed 3x in diH2O and transferred to a solution of concentrated sulfuric acid for at least 1 hour. This step oxidized the surface of the microcantilevers leaving a hydrophilic surface suitable for reaction of the silane derivatives. After at least one hour in sulfuric acid the substrates were washed 3x in diH2O. The rinsed substrates were then boiled in diH2O for 30 minutes. After boiling the samples were place in a120° C. oven for at least 3 hours. The resulting surfaces were analyzed using contact angle goniometry and XPS to verify hydrophilicity of the surfaces (CA<5.0°) and the elemental composition of the surfaces respectively. Surfaces with a CA of less that 5.0° and an elemental carbon content of approximately 5.0% were considered suitable for derivitization.

[0119] After cleaning the microcantilevers, fresh distilled toluene was transferred into a Pyrex bottle that had been dried in an 120° C. oven to dry off excess surface water. Dry nitrogen was used to replace the air in the remaining volume of the bottle to minimize free oxygen. The bottle was sealed and placed in the antechamber of an MBraun glovebox, which was evacuated and refilled with dry nitrogen 3 times. The toluene was transferred into the main chamber. DETA was added to the toluene to a final concentration of 0.1% (vokvol). The DETA-toluene solution was removed from the glove box and transferred to a pyrex beaker and the samples were immersed in the solution. To drive the reaction forward the solution was gently heated to no more than 65° C. Optimal reaction time was analyzed for these conditions by incubating the samples 10, 20, and 30 minutes. After reaction with DETA the samples were allowed to cool to room temperature, washed 3 time with dry tolune and heated too 65° C. for 30 more minutes. The resulting samples were analyzed by XPS and contact angle goniometry. Cell cultures then could be added as described in Example 1, above.

[0120] For optical detection systems, a detection system similar to those used in atomic force microscope (AFM) systems can be designed for measuring deflection of the cantilevers during contraction. The entire system can be assembled around an upright Olympus BX51WI electrophysiology microscope (Olympus Inc., Center Valley, Pa.). This detection system consists of a class 2 red photodiode laser (Newport, Irvine, Calif.), a stimulation chamber, a 4-quadrant photodetector (Noah Industries, Melbourne, Fla.), and a computer with pClamp 10.0 data acquisition software (Molecular Devices, Union City, Calif.). The laser and photodetector (PD) are mounted on x-y-z-h translators (Newport, Irvine, Calif.), which are mounted on the underside of the microscope stage. The stimulation chamber is fabricated from a 5 mm thick polycarbonate sheet. An approximately 15 mmxl5 mm square chamber is milled out of the sheet and fitted with silver wires (0.015 inch diameter) for field stimulation. The silver wires are mounted parallel to each other with a separation of 15 mm. The bottom of the chamber is sealed using a 22 mmx22 mm glass coverslip. This creates a transparent base through which the laser beam can easily pass. The silver wires are connected to an external pulse generator (A-M systems, Sequim, Wash.) capable of producing field stimulation pulses of varying intensity, frequency, andwaveform. Both the pulse generator and PD are connected to an Axon Instruments series 1440 digitizer (Molecular Devices, Union City, Calif.), which is interfaced with the computer.

[0121] For electrical detection systems, piezoelectricity is the ability of certain materials (crystals and certain ceramics) to generate an electric potential in response to applied mechanical stress. The piezoelectric effect is used in various sensors to measure stresses or geometrical deformations in various mechanical devices. The reverse piezoelectric effect turns piezoelectric material into actuators, when an external voltage is applied to the crystal. Piezoelectric materials are known to the art and include, but are not limited to, the following: quartz, bone, sodium tungstate, zinc oxide, or lead zirconate titanate (PZT). A similar effect is the piezoresistive phenomenon. When subjected to mechanical stress, these materials change their resistivity.

[0122] Silicon wafers with silicon on insulator can serve as base material in the fabrication of piezoelectric cantilevers. An additional layer of 100-200 nm SiOz can be deposited onto the base material to insulate conductive materials from the semi-conductive silicon. Metal layers can be fabricated to connect the piezoelectric components with microelectronics. Layers of piezoelectric materials, such as ZnO and PTZ sol-gel, can be deposited exactly in those areas where microcantilevers remain after the etching process. Another conductive layer contacts the piezoelectric components from top to apply voltages for actuation or current read out during sensor mode. An insulation layer of silicon-ONO-stacks (oxide-nitride-oxide) can protect conductive elements from aqueous solutions during cell culture. Alternatively, piezoelectric elements can be replaced by piezoresistive materials. This alternative approach can offer a higher sensitivity during readout; however, piezoresistive materials do not provide the usage of the cantilevers as actuators and a field stimulator would be needed.EXAMPLE 3: Additional Information on Computing Devices

[0123] It should be appreciated that the logical operations described herein with respect to the various figures may be implemented (1) as a sequence of computer implemented acts or program modules (i.e., software) running on a computing device (e.g., the computing device described in FIG. 14), (2) as interconnected machine logic circuits or circuit modules (i.e., hardware) within the computing device and / or (3) a combination of software and hardware of the computing device. Thus, the logical operations discussed herein are not limited to any specific combination of hardware and software. The implementation is a matter of choice dependent on the performance and other requirements of the computing device. Accordingly, the logical operations described herein are referred to variously as operations, structural devices, acts, ormodules. These operations, structural devices, acts and modules may be implemented in software, in firmware, in special purpose digital logic, and any combination thereof. It should also be appreciated that more or fewer operations may be performed than shown in the figures and described herein. These operations may also be performed in a different order than those described herein.

[0124] Referring to FIG. 14, an example computing device 1 100 upon which embodiments of the invention may be implemented is illustrated. It should be understood that the example computing device 1100 is only one example of a suitable computing environment upon which embodiments of the invention may be implemented. Optionally, the computing device 1100 can be a well-known computing system including, but not limited to, personal computers, servers, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, network personal computers (PCs), minicomputers, mainframe computers, embedded systems, and / or distributed computing environments including a plurality of any of the above systems or devices. Distributed computing environments enable remote computing devices, which are connected to a communication network or other data transmission medium, to perform various tasks. In the distributed computing environment, the program modules, applications, and other data may be stored on local and / or remote computer storage media.

[0125] In its most basic configuration, computing device 1100 typically includes at least one processing unit 1106 and system memory 1104. Depending on the exact configuration and type of computing device, system memory 1104 may be volatile (such as random access memory (RAM)), non-volatile (such as read-only memory (ROM), flash memory, etc.), or some combination of the two. This most basic configuration is illustrated in FIG. 14 by dashed line 1102. The processing unit 1106 may be a standard programmable processor that performs arithmetic and logic operations necessary for operation of the computing device 1100. The computing device 1100 may also include a bus or other communication mechanism for communicating information among various components of the computing device 1100.

[0126] Computing device 1100 may have additional features / functionality. For example, computing device 1100 may include additional storage such as removable storage 1108 and nonremovable storage 1110 including, but not limited to, magnetic or optical disks or tapes. Computing device 1100 may also contain network connection(s) 1116 that allow the device to communicate with other devices. Computing device 1100 may also have input device(s) 1114 such as a keyboard, mouse, touch screen, etc. Output device(s) 1112 such as a display, speakers, printer, etc. may also be included. The additional devices may be connected to the bus in orderto facilitate communication of data among the components of the computing device 1100. All these devices are well known in the art and need not be discussed at length here.

[0127] The processing unit 1106 may be configured to execute program code encoded in tangible, computer-readable media. Tangible, computer-readable media refers to any media that is capable of providing data that causes the computing device 1100 (i.e., a machine) to operate in a particular fashion. Various computer-readable media may be utilized to provide instructions to the processing unit 1106 for execution. Example tangible, computer-readable media may include, but is not limited to, volatile media, non-volatile media, removable media and nonremovable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. System memory 1104, removable storage 1108, and non-removable storage 1110 are all examples of tangible, computer storage media. Example tangible, computer-readable recording media include, but are not limited to, an integrated circuit (e.g., field-programmable gate array or application-specific IC), a hard disk, an optical disk, a magneto-optical disk, a floppy disk, a magnetic tape, a holographic storage medium, a solid-state device, RAM, ROM, electrically erasable program read-only memory (EEPROM), flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices.

[0128] In an example implementation, the processing unit 1106 may execute program code stored in the system memory 1104. For example, the bus may carry data to the system memory 1104, from which the processing unit 1106 receives and executes instructions. The data received by the system memory 1104 may optionally be stored on the removable storage 1108 or the nonremovable storage 1110 before or after execution by the processing unit 1106.

[0129] It should be understood that the various techniques described herein may be implemented in connection with hardware or software or, where appropriate, with a combination thereof. Thus, the methods and apparatuses of the presently disclosed subject matter, or certain aspects or portions thereof, may take the form of program code (i.e., instructions) embodied in tangible media, such as floppy diskettes, CD-ROMs, hard drives, or any other machine-readable storage medium wherein, when the program code is loaded into and executed by a machine, such as a computing device, the machine becomes an apparatus for practicing the presently disclosed subject matter. In the case of program code execution on programmable computers, the computing device generally includes a processor, a storage medium readable by the processor (including volatile and non-volatile memory and / or storage elements), at least one input device,and at least one output device. One or more programs may implement or utilize the processes described in connection with the presently disclosed subject matter, e.g., through the use of an application programming interface (API), reusable controls, or the like. Such programs may be implemented in a high level procedural or object-oriented programming language to communicate with a computer system. However, the program(s) can be implemented in assembly or machine language, if desired. Tn any case, the language may be a compiled or interpreted language and it may be combined with hardware implementations.REFERENCES(1) Das, M.; Gregory, C. A.; Molnar, P.; Riedel, L. M.; Wilson, K.; Hickman, J. J. A Defined System to Allow Skeletal Muscle Differentiation and Subsequent Integration with Silicon Microstructures. Biomaterials 2006, 27, 4374-4380.(2) Das, M.; Rumsey, J. W.; Bhargava, N.; Stancescu, M.; Hickman, 1. 1. Skeletal Muscle Tissue Engineering: A Maturation Model Promoting Long-Term Survival of Myotubes, Structural Development of the Excitation-Contraction Coupling Apparatus and Neonatal Myosin Heavy Chain Expression. Biomaterials 2009, 30, 5392-5402.(3) Das, M.; Wilson, K.; Molnar, P.; Hickman, J. J. Differentiation of Skeletal Muscle and Integration of Myotubes with Silicon Microstructures Using Serum-Free Medium and a Synthetic Silane Substrate. Nat. Protoc. 2007, 2, 1795-1801.(4) McAleer, C. W.; Rumsey, I. W.; Stancescu, M.; Hickman, J. J. Functional Myotube Formation from Adult Rat Satellite Cells in a Defined Serum-Free System. Biotechnol. Prog. 2015, 31, 997-1003.(5) Rumsey, J. W.; Das, M.; Kang, J. F.; Wagner, R.; Molnar, P.; Hickman, J. J. Tissue Engineering Intrafusal Fibers: Dose and Time Dependent Differentiation of Nuclear Bag Fibers in a Defined in Vitro System Using Neuregulin 1-B-l. Biomaterials 2008, 29, 994-1004.(6) Wilson, K.; Das, M.; Wahl, K. J.; Colton, R. J.; Hickman, J. J. Measurement of Contractile Stress Generated by Cultured Rat Muscle on Silicon Cantilevers for Toxin Detection and Muscle Performance Enhancement. PLoS One 2010, 5, No. el 1042.(7) Pirozzi, K.; Long, C. J.; McAleer, C. W.; Smith, A. S. T.; Hickman, J. J. Correlation of Embryonic Skeletal Muscle Myotube Physical Characteristics with Contractile Force Generation on an Atomic Force Microscope-Based Biomicroelectromechanical Systems Device. Appl. Phys. Lett. 2013, 103, 083108.(8) Hoshino, T.; Morishima, K. Muscle-Powered Cantilever for Microtweezers with an Artificial Micro Skeleton and Rat Primary Myotubes. J. Biomech. Sci. Eng. 2010, 5, 245-251.(9) Morimoto, Y.; Onoe, H.; Takeuchi, S. Biohybrid Robot with Skeletal Muscle Tissue Covered with a Collagen Structure for Moving in Air. APL Bioeng. 2020, 4, 026101.(10) Stancescu, M.; Molnar, P.; McAleer, C. W.; McLamb, W.; Long, C. J.; Oleaga, C.; Prot, J.- M.; Hickman, J. J. A Phenotypic in Vitro Model for the Main Determinants of Human Whole Heart Function. Biomaterials 2015, 60, 20-30.(11) Truskey, G. A. Development and Application of Human Skeletal Muscle Microphysiological Systems. Lab Chip 2018, 18, 3061-3073.(12) Kannus, P. Structure of the Tendon Connective Tissue. Scand. J. Med. Sci. Sports 2000, 10, 312-320. (13) Kristensen, J. H.; Thorlacius-Ussing, J.; Rpnnow, S. R.; Karsdal, M. A. Elastin. In Biochemistry of Collagens, Laminins and Elastin. Structure, Function and Biomarkers; Karsdel, M. A., Ed.; Elsevier Science, 2016; pp 197-201.(13) Kristensen, J. H.; Thorlacius-Ussing, J.; Rpnnow, S. R.; Karsdal, M. A. Elastin. In Biochemistry of Collagens, Laminins and Elastin. Structure, Function and Biomarkers; Karsdel, M. A., Ed.; Elsevier Science, 2016; pp 197-201.(14) Kumashiro, K. K.; Ho, J. P.; Niemczura, W. P.; Keeley, F. W. Cooperativity between the Hydrophobic and Cross-Linking Domains of Elastin. I. Biol. Chem. 2006, 281, 23757-23765.(15) Murphy, S.; Ohlendieck, K. The Extracellular Matrix Complexome from Skeletal Muscle, Composition and Function of the Extracellular Matrix in the Human Body. In Composition and Function of the Extracellular Matrix in the Human Body; Travascio, F., Ed.; IntechOpen Limited: London, UK, 2016.(16) Urry, D.W.; Hugel, T.; Seitz, M.; Gaub, H. E.; Sheiba, L.; Dea, J.; Xu, J.; Parker, T. Elastin: A Representative Ideal Protein Elastomer. Philos. Trans. R. Soc. London, Ser. B 2002, 357, 169-184.(17) Wu, W. I.; Vrhovski, B.; Weiss, A. Glycosaminoglycans Mediate the Coacervation of Human Tropoelastin through Dominant Charge Interactions Involving Lysine Side Chains. Biol. Chem. 1999, 274, 21719-21724.(18) Andersen, S. O.; Weis-Fogh, T. Resilin. A Rubberlike Protein in Arthropod Cuticle. In Advances in Insect Physiology; Beament, J. W. L., Treheme, J. E.,Wigglesworth, V. B., Eds.; Academic Press, 1964; Vol. 2, pp 1-65.(19) Burrows, M.; Shaw, S. R.; Sutton, G. P. Resilin and Chitinous Cuticle Form a Composite Structure for Energy Storage in Jumping by Froghopper Insects. BMC Biol. 2008, 6, 41.(20) Elvin, C.M.; Carr, A. G.; Huson, M. G.; Maxwell, J.M.; Pearson, R. D.; Vuocolo, T.; Liyou, N. E.; Wong, D. C.; Merritt, D. J.; Dixon, N. E. Synthesis and Properties of Crosslinked Recombinant Pro-Resilin. Nature 2005, 437, 999-1002.(21) Gosline, J.; Lillie, M.; Carrington, E.; Guerette, P.; Ortlepp, C.; Savage, K. Elastic Proteins: Biological Roles and Mechanical Properties. Philos. Trans. R. Soc. London, Ser. B 2002, 357, 121-132.(22) Li, L.; Kiick, K. L. Resilin-Based Materials for Biomedical Applications. ACS Macro Lett.2013, 2, 635-640.(23) Weis-Fogh, T. Thermodynamic Properties of Resilin, a Rubber-Like Protein. J. Mol. Biol. 1961, 3, 520-531.(24) Smith, C. M.; Roy, T. D.; Bhalkikar, A.; Li, B.; Hickman, J. J.; Church, K.H. Engineering a Titanium and Polycaprolactone Construct for a Biocompatible Interface between the Body and Artificial Limb. Tissue Eng., Part A 2010, 16, 717-724.(25) Wilson, K.; Stancescu, M.; Das, M.; Rumsey, J.W.; Hickman, J. J. Direct Patterning of Coplanar Polyethylene Glycol Alkylsilane Monolayers by Deep-Ultraviolet Photolithography as a General Method for High Fidelity, Long-Term Cell Patterning and Culture. J. Vac. Sci. Technol., B 2011, 29, 021020.(26) Guo, X.; Badu-Mensah, A.; Thomas, M. C.; McAleer, C. W.; Hickman, J. J. Characterization of Functional Human Skeletal Myotubes and Neuromuscular Junction Derived from the Same Induced Pluripotent Stem Cell Source. Bioengineering 2020, 7, 133.(27) Guo, X.; Smith, V.; Jackson, M.; Tran, M.; Thomas, M.; Patel, A.; Lorusso, E.; Nimbalkar, S.; Cai, Y.; McAleer, C.W.; Wang, Y.; Long, C. J.; Hickman, J. J. A Human-Based Functional NMJ System for Personalized ALS Modeling and Drug Testing. Adv. Ther. 2020, 3, 2000133.(28) Lou, J.-S. Approaching Fatigue in Neuromuscular Diseases. Phys. Med. Rehabil. Clin. N. Am. 2005, 16, 1063-1079.(29) Oleaga, C.; Lavado, A.; Riu, A.; Rothemund, S.; Carmona-Moran, C. A.; Persaud, K.; Yurko, A.; Lear, J.; Narasimhan, N. S.; Long, C. J.; Sommerhage, F.; Bridges, L. R.; Cai, Y.; Martin, C.; Schnepper, M. T.; Goswami, A.; Note, R.; Langer, J.; Teissier, S.; Cotovio, J.; Hickman, J. J. Long-Term Electrical and Mechanical Function Monitoring of a Human-on-a- Chip System. Adv. Funct. Mater. 2018, 29, 1805792.(30) Oleaga, C.; Legters, G.; Bridges, L. R.; Kumanchik, L.; Martin, C.; Cai, Y.; Schnepper, M.; McAleer, C. W.; Long, C. J.; Hickman, J. J. Contractile Force Readout of HESC- Cardiomyocytes. In Stem Cell-Derived Models in Toxicology, Methods in Pharmacology and Toxicology; Clements, M., Roquemore, L., Eds.; Humana Press: New York, 2017; pp 229-246.(31) Wilson, K. A.; Finch, C. A.; Anderson, P.; Vollmer, F.; Hickman, J. J. Whispering Gallery Mode Biosensor Quantification of Fibronectin Adsorption Kinetics onto Alkylsilane Monolayers and Interpretation of Resultant Cellular Response. Biomaterials 2012, 33, 225-236.(32) Hickman, J. J.; Stenger, D. A. Interactions of Cultured Neurons with Defined Surfaces. In Enabling Technologies for Cultured Neural Networks, 1st ed.; Stenger, D. A., McKenna, T. M., Eds.; Academic Press, Inc.: San Diego, 1994; p 51.(33) Hickman, J. J.; Stenger, D. A. Lithographic Definition of Neuronal Microcircuits. In Enabling Technologies for Cultured Neural Networks; Stenger, D. A., McKenna, T. M., Eds.; Academic Press, Inc.: San Diego, 1994; p 51.(34) Ravenscroft, M. S.; Bateman, K. E.; Shaffer, K. M.; Schessler, H. M.; Jung, D. R.; Schneider, T. W.; Montgomery, C. B.; Custer, T. L.; Schaffner, A. E.; Liu, Q. Y.; Li, Y. X.; Barker, J. L.; Hickman, J. J. Developmental Neurobiology Implications from Fabrication and Analysis of Hippocampal Neuronal Networks on Patterned Silane-Modified Surfaces. J. Am. Chem. Soc. 1998, 120, 12169-12177.(35) Schaffner, A. E.; Barker, J. L.; Stenger, D. A.; Hickman, J. J. Investigation of the Factors Necessary for Growth of Hippocampal Neurons in a Defined System. J. Neurosci. Methods 1995, 62, 111-119.(36) Spargo, B. J.; Testoff, M. A.; Nielsen, T. B.; Stenger, D. A.; Hickman, J. J.; Rudolph, A. S. Spatially Controlled Adhesion, Spreading, and Differentiation of Endothelial Cells on Self- Assembled Molecular Monolayers. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 11070-11074.(37) Stenger, D. A.; Georger, J. H.; Dulcey, C. S.; Hickman, J. J.; Rudolph, A. S.; Nielsen, T. B.; McCort, S. M.; Calvert, J. M. Coplanar molecular assemblies of amino- and perfluorinated alkylsilanes: characterization and geometric definition of mammalian cell adhesion and growth. J. Am. Chem. Soc. 1992, 114, 8435-8442.(38) Stenger, D. A.; Hickman, J. J.; Bateman, K. E.; Ravenscroft, M. S.; Ma, W.; Pancrazio, J. J.; Shaffer, K.; Schaffner, A. E.; Cribbs, D. H.; Cotman, C. W. Microlithographic Determination of Axonal / Dendritic Polarity in Cultured Hippocampal Neurons. J. Neurosci. Methods 1998, 82, 167-173.(39) Stenger, D. A.; Pike, C. J.; Hickman, J. J.; Cotman, C.W. Surface Determinants of Neuronal Survival and Growth on Self-Assembled Monolayers in Culture. Brain Res. 1993, 630, 136-147.(40) McAleer, C.W.; Smith, A. S. T.; Najjar, S.; Pirozzi, K.; Long, C. J.; Hickman, J. J. Mechanistic Investigation of Adult Myotube Response to Exercise and Drug Treatment in Vitro Using a Multiplexed Functional Assay System. J. Appl. Physiol. 2014, 117, 1398-1405.(41) Holst, J.; Watson, S.; Lord, M. S.; Eamegdool, S. S.; Bax, D. V.; Nivison-Smith, L. B.; Kondyurin, A.; Ma, L.; Oberhauser, A. F.; Weiss, A. S.; Rasko, J. E. Substrate Elasticity Provides Mechanical Signals for the Expansion of Hemopoietic Stem and Progenitor Cells. Nat. Biotechnol. 2010, 28, 1123-1128.(42) Kuraitis, D.; Ebadi, D.; Zhang, P.; Rizzuto, E.; Vulesevic, B.; Padavan, D. T.; Al Madhoun, A.; McEwan, K. A.; Sofrenovic, T.; Nicholson, K.; Whitman, S. C.; Mesana, T. G.; Skerjanc, I. S.; Musarb, A.; Ruel, M.; Suuronen, E. J. Injected Matrix Stimulates Myogenesis and Regeneration of Mouse Skeletal Muscle after Ischaemic Injury. Eur. Cells Mater. 2012, 24, 175-195.(43) Lv, S.; Dudek, D. M.; Cao, Y.; Balamurali, M. M.; Gosline, J.; Li, H. Designed Biomaterials to Mimic the Mechanical Properties of Muscles. Nature 2010, 465, 69-73.(44) Molnar, P.; Wang, W.; Natarajan, A.; Rumsey, J.W.; Hickman, J. J. Photolithographic Patterning of C2cl2 Myotubes Using Vitronectin as Growth Substrate in Serum-Free Medium. Biotechnol. Prog. 2007, 23, 265-268.(45) Riso, E.M.; Kaasik, P.; Seene, T. Remodelling of Skeletal Muscle Extracellular Matrix: Effect of Unloading and Reloading. In Composition and Function of the Extracellular Matrix in the Human Body; Travascio, F., Ed.; IntechOpen Limited: London, UK, 2016.(46) Steucke, K. E.; Win, Z.; Stemler, T. R.; Walsh, E. E.; Hall, J. L.; Alford, P. W. Empirically Determined Vascular Smooth Muscle Cell Mechano-Adaptation Law. J. Biomech. Eng. 2017, 139, 0710051-0710059.(47) Stegemann, J. P.; Hong, H.; Nerem, R. M. Mechanical, hiochemical, and extracellular matrix effects on vascular smooth muscle cell phenotype. J. Appl. Physiol. 2005, 98, 2321-2327.(48) Steucke, K. E.; Tracy, P. V.; Hald, E. S.; Hall, J. L.; Alford, P. W. Vascular smooth muscle cell functional contractility depends on extracellular mechanical properties. J. Biomech. 2015, 48, 3044-3051.

Claims

WHAT IS CLAIMED IS:

1. A system for promoting long-term culture of mechanically active cells, the system comprising: a cell culture surface comprising; a silicon oxide first layer; a second layer comprising a plurality of organosilanes, each organosilane having a positively charged portion extending from a silane portion, the silane portion covalently bound to the silicon oxide first layer; and extracellular matrix components and water molecules dispersed between and bonded to positively charged portions of the plurality of organosilanes.

2. The system of claim 1 , wherein the cell culture surface is the exterior surface of a silicon substrate.

3. The system of either of claims 1 or 2, wherein the cell culture surface is the exterior surface of a glass substrate.

4. The system of any one of claims 1-3, wherein the organosilane is an amino silane.

5. The system of claim 4, wherein the amino silane is DETA silane.

6. The system of any one of claims 1-5, wherein the second layer is a self-assembled monolayer.

7. The system of any one of claims 1-6, wherein the extracellular matrix components comprise elastin.

8. The system of claim 7, wherein the extracellular matrix components further comprise one or more of collagen, hyaluronic acid, and heparan sulfate proteoglycan.

9. The system of either claim 7 or 8, wherein a contact angle of water with the cell culture surface is less than 37°.

10. The system of any one of claims 1-9, wherein the system further comprises a culture of mechanically active cells adhered to the cell culture surface and bound to the extracellular matrix components.11 . The system of claim 10, wherein the mechanically active cells are muscle cells.

12. The system of either claim 10 or claim 11, wherein a fatigue index of the culture of mechanically active cells reduces over a period of two weeks or more.

13. The system of any one of claims 10-12, wherein the culture of mechanically active cells comprises a younger state and an older state, wherein the younger state and the older state are separated by a culture duration of at least two weeks and are distinguished by one or more cellular aging markers, and wherein the older state is characterized by a reduced fatigue index as compared to the younger state.

14. The system of any one of claims 1-13, wherein the system further comprises a microcantilever, and the cell culture surface is an exterior surface of the microcantilever.

15. The system of claim 14, wherein the microcantilever comprises silicon.

16. The system of either claim 14 or 15, wherein the system further comprises a passivated surface adjacent to the microcantilever.

17. The system of any one of claims 1-16, wherein the system further comprises a contraction detection setup.

18. The system of claim 17, wherein the contraction detection setup comprises a light source and a photodetector.

19. The system of either claim 17 or 18, wherein the contraction measurement setup comprises piezoelectric or piezoresi stive microcantilevers.

20. The system of any one of claims 17-19, wherein the contraction detection setup comprises a computing device.

21. A method of detecting a response of a long-term mechanically active cell culture to an agent, the method comprising: plating mechanically active cells onto a cell culture surface, the cell culture surface comprising: a silicon oxide first layer; a second layer comprising a plurality of organosilanes, each organosilane having a positively charged portion extending from a silane portion, the silane portion covalently bound to the silicon oxide first layer; and extracellular matrix components and water molecules dispersed between and bonded to positively charged portions of the plurality of organosilanes; forming a mechanically active cell culture; maintaining the mechanically active cell culture in a mechanically active state and adhered to the cell culture surface for at least 14 days; exposing the mechanically active cell culture to an agent; and detecting a response of the mechanically active cell culture to the agent.

22. The method of claim 21, further comprising electrically stimulating the mechanically active cell culture.

23. The method of either claim 21 or claim 22, wherein the cell culture surface is an exterior surface of a microcantilever, and detecting a response of the agent comprises detecting a deflection of a microcantilever.

24. The method of any one of claims 21-23, further comprising maintaining the mechanically active cell culture in a mechanically active state and adhered to the cell culture surface for at least 28 days.

25. The method of any one of claims 21-24, further comprising continuing to maintain the mechanically active cell culture in a mechanically active state for at least 14 days after initially exposing the mechanically active cell culture to the agent, then repeating the stepsof exposing the mechanically active cell culture to the agent and detecting the response of the mechanically active cell culture to the agent.

26. A method of making a cell culture surface for promoting long-term culture of mechanically active cells, the method comprising: handling a substrate comprising a silicon oxide first layer; adding a second layer comprising a plurality of organosilanes, each organosilane having a positively charged portion extending from a silane portion; covalently binding the silane portion to the silicon oxide first layer; dispersing extracellular matrix components and water molecules between positively charged portions of the plurality of organosilanes; and bonding the extracellular matrix components and the water molecules between the positively charged portions of the organosilanes.

27. The method of claim 26, wherein the substrate comprising the silicon oxide first layer is a silicon substrate comprising a microcantilever.

28. The method of either claim 26 or 27, further comprising acid-washing the silicon oxide first layer prior to adding the second layer.

29. The method of claim 28, further comprising passivating the silicon oxide first layer prior to adding the second layer.

30. The method of any one of claims 26-29, further comprising etching away a nonpassivated pattern prior to adding the second layer.

31. The method of any one of claims 21-30, wherein adding a second layer comprising a plurality of organosilanes comprises exposing the silicon oxide first layer to a solution of organosilanes in tolulene.

32. The method of claim 31, wherein covalently binding the silane portion to the silicon oxide first layer comprises heating the substrate to a temperature of from 60° - 100°C.

33. The method of any one of claims 21-32, wherein dispersing extracellular matrix components and water molecules comprises covering the second layer with a solution comprising extracellular matrix components and water.

34. The method of claim 33, wherein the solution comprises elastin.

35. The method of either claim 33 or claim 34, wherein the solution further comprises one or more of collagen, hyaluronic acid, and heparan sulfate proteoglycan.

36. The method of any one of claims 26-35, further comprising covering the second layer with 0.03-0.09 pg elastin per mm237. The method of any one of claims 26-36, further comprising covering the second layer with one or more of: 0.01-0.06 pg collagen per mm2, 0.0001-0.0007 pg heparan sulfate proteoglycan per mm2, and 0.0007- 0.0013 pg hyaluronic acid per mm2.

38. The method of any one of claims 26-37, further comprising adhering mechanically active cells to the cell culture surface to create a mechanically active cell culture.

39. The method of claim 38, further comprising maintaining the mechanically active cell culture for more than two weeks in culture in a mechanically active state.

Images