CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/733,362 filed Sep. 19, 2018, the contents of which are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. R01 NS094388, awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

The technology generally relates to the field of cell growth and tissue engineering. The technology described herein relates to devices, systems and assays comprising electrode arrays for at least two types of electroconductive cells.

BACKGROUND

Peripheral neuropathies are a clinically and genetically heterogeneous set of severely debilitating neurological conditions. It has been suggested that the neuromuscular junction (NMJ) is an early target for pathological onset in a number of peripheral nerve diseases. For example, patients with amyotrophic lateral sclerosis (ALS) commonly possess defects in the maturation of their NMJs. These defects typically precede the onset of progressive muscular degeneration to the point that impairments in pre- and post-synaptic development in lower motor neuron connectivity to muscle is characteristic of early stages of the disease. It is therefore likely that defective NMJ maturation contributes to the subsequent onset of complete denervation and associated muscle weakness. Based on these data, it is clear that the NMJ is an important site of early, selective pathology in peripheral neuropathies, such as ALS. In order to prevent the degradation of synaptic structures, and improve patient mobility, it is vital to understand the upstream effects that lead to NMJ breakdown. Recent advances in human induced pluripotent stem cell (hiPSC) technology now makes phenotypic comparison of individual patient mutations a viable possibility. However, to evaluate the effect of different mutations, e.g., ALS mutations, on NMJ development requires a suitable high-throughput screening platform with which to evaluate synaptic function and breakdown.

In vitro models of NMJ physiology and function are currently available. They typically focus on dual patch analysis (see e.g., Umbach et al., PLoS One. 2012, 7: e36049) or assessment of muscle twitch responses to neuronal activation (see e.g., Smith et al., Technology 2013, 1:37-48; Guo et al., Biomaterials. 2011, 32:9602-11; Santhanam et al., Biomaterials. 2018, 166:64-78.). However, such systems are extremely low throughput and time consuming, preventing side by side comparison of multiple disease mutations as well as simultaneous analysis of multiple dose responses to novel therapeutics. A multiplexed, high-throughput NMJ functional screening platform is thus needed for the study of peripheral neuropathies.

Moreover, the culture of electroconductive cells, e.g. neurons and muscle cells, in a manner that permits measurement of their electroconductive behavior is important for understanding the biology of these cells, determining if candidate therapeutics perturb this behavior (possibly leading to dangerous side effects in patients), and identifying agents that can therapeutically modify electroconductive behaviors, e.g. for the treatment of arrhythmia. However, the existing technologies for culturing these cells do not discern the electrical properties from each cell type if more than one cell type is used, and suffer from suboptimal spatial accuracy, low-throughput, and alter the natural physiology and/or behavior of electroconductive cells due the characteristics of the culture environment.

In addition, culture systems and devices that permits the measurement of the electroconductive behavior are typically constructed for the evaluation of a single cell type at a time. While in some instances, the devices can be used for a spatially separated culture of two or more cell types, the different cell types are cultured together on the same surface, therefore preventing systematic analysis of the effect of one cell type on the other. A culture system that can serve as a functional screening platform for the analysis of the electroconductive behavior each cell type individually is needed for the study of peripheral neuropathies.

SUMMARY

The technology described herein is directed to cell culture devices integrating electrode-based detection of neuronal firing and muscle contraction, particularly by compartmentalization of neuronal and muscle populations, and comprises bioinspired topographic substrates.

More particularly, the technology described herein relates to microelectrode array (MEA) devices, systems, methods, and assays comprising electrode arrays for electroconductive cells. In particular, the technology generally relates to MEA devices comprising both field potential (FP) electrodes and impedance electrodes (IE) that are spatially separated for the functional analysis of the electrical connectivity between at least two cell populations. Accordingly, the compositions and methods described herein relate to MEA devices for culturing and assessing at least two populations of electroconductive cells on a substrate, where the two cell populations are spatially separated. That is, each cell population attaches and adheres to a spatially distinct cell growth area on the substrate, and one or both cell types of each population can extend axons or other cellular projections into, and in some embodiments through, an intermediate area referred to herein as an “axon outgrowth area”, which is located between each of the cell growth areas such that cells of each cell population can connect electrically, e.g., make synaptic connections. That is, while the cell bodies of each cell population are spatially separated or distinct, cells from the different populations can connect or contact each other via axonal, dendritic or other projections from one cell type to the other cell type.

Therefore, by having the at least two cell populations spatially separated, yet permitting them to make synaptic connections to allow for electrical communication between the two cell types, it permits a flexible system in terms of both electrical stimulation and/or data collection capabilities. For example, one can electrically stimulate one cell type, and record the effect on the other cell type. Additionally, the MEA devices can be used in assays to see the effect of an agent on the electrical connectivity between the two cell types, when the agent is applied to one cell type, or both cell types. Moreover, the MEA devices disclosed herein can be used in assays and in vitro models for disease and disorders, e.g., neurodegenerative diseases, neuromuscular disorders and myopathies, to see the effect of a gene mutation on the electrical conductivity between the two cell types.

In some embodiments, such devices permit real-time analysis of NMJ development and function across a wide array of healthy and diseased cell lines. In some embodiments, primary human induced pluripotent stem cell-derived motor neurons and primary human muscle cells can be used to establish organized cultures overlying electrode beds for functional analysis. A barrier or wall can separate the cell populations into neuronal and muscle cell compartments. Microchannels in the surface of the device and/or voids in the barrier(s) separating the cells permit neuronal processes to grow from the neuronal cell compartment into the muscle compartment, facilitating synaptic contact. Drug compounds can be analyzed using this system and assessed with regard to their ability to ameliorate in vitro disease phenotypes. Such devices can be used to model human peripheral neuropathies and as a preclinical screening tool for assessing therapeutics.

In some embodiments, the technology described herein provides a platform for assessing neuromuscular junction functionality. The inventors discovered that to facilitate synapse development, rather than a co-culture of neurons cultured directly on top of muscle cells, the spatial separation of the two cell types permitted optimization for each cell type, as well as controlled stimulation of neurons and/or measurement of contractile responses of skeletal muscle to neuronal activation. As such, the inventors developed a cell surface platform integrating field potential electrodes on one side and impedance electrodes on the other, with an intervening cell surface area through which the neuronal axons extend to contact the muscle cells (see e.g., FIG. 1A). In some embodiments, neurons cultured on the field potential electrode side can be monitored for spontaneous activity and/or electrically stimulated to enhance output and strengthen synaptic connections. In some embodiments, a monolayer or multilayered skeletal muscle construct is plated on the surface which comprises impedance electrodes. Small scale electrical impulses can then be run between the impedance electrodes; these pulses can be too small to affect the overlying muscle, but large enough to permit quantification of impedance parameters. Other groups have demonstrated that contractile cells modulate the impedance associated with electrical pulses passing between electrodes that they cover. This modulation can be plotted over time to provide a surrogate measure of contractile ability, with the degree of impedance change correlating directly with contraction magnitude. A barrier (e.g., made from e.g., PDMS, among other materials) between the two compartments can permit isolation of each cell type and prevent migration of either cell type into the other population. Micro-scale channels permit neuritic extension from the neuron compartment to the muscle and thereby facilitate synapse development (see e.g., FIGS. 1B, 1C). Testing of electrode sensitivity demonstrates that the deposited electrodes are sufficiently sensitive to be used as described above (see e.g., FIGS. 1D, 1E) and bright-field imaging confirms the capacity for human iPSC-derived neurons to extend axons from one chamber, through the channels, and out into the second chamber (see e.g., FIG. 1F). The platform can be tested to validate its ability to support both cell types and assay functional connectivity between the muscle and neuronal components.

In some embodiments, tests with cholinergic synaptic agonists and antagonists in terms of their ability to alter synaptic communication between cell populations are used to demonstrate the suitability of this model for assaying NMJ function in vitro (see e.g., Example 1).

In some embodiments, this system incorporates motor neurons carrying different ALS mutations in one of at least three separate genes (e.g., TARDBP, SOD1, and C9ORF72). Spatially separated culture constructs incorporating these cells or cells bearing other ALS-related mutations can be used to model ALS disease pathophysiology in vitro and can be used to highlight phenotypic differences arising between motor neurons derived from different mutant hiPSC lines (see e.g., Example 2). In some embodiments, drug compounds can be analyzed using this system and assessed with regards to their ability to ameliorate in vitro disease phenotypes. (see e.g., Example 3).

In some embodiments, described herein is a device and system for the study of neuropathies such as Charcot-Marie-Tooth disease (CMT; see e.g., Example 4).

Without wishing to be bound by theory, it is proposed that differences in NMJ development and function between skeletal muscle and motor neurons derived from either normal or ALS (or other neuropathic disease-derived) hiPSC lines permits stratification of disease phenotypes that can be successfully assayed and identified using electrode-based, real-time monitoring. Devices and systems as described herein can be used to model human peripheral neuropathies and as a preclinical screening tool for assessing therapeutics.

In one aspect, described herein is a device for monitoring the electrical communication between two different electrically excitable cell types, comprising at least one module on a substrate, each module comprising: (a) a first cell growth area, a second cell growth area, and an axon outgrowth area flanked between the first cell growth area and the second cell growth area, and (b) a plurality of field potential electrodes and a plurality of impedance electrodes on the surface of the substrate, wherein the plurality of field potential electrodes are located on the surface of the first cell growth area and the plurality of impedance electrodes are located on the surface of second cell growth area, and wherein the plurality of field potential electrodes and the plurality of impedance electrodes are connected to an electronic interface.

In some embodiments of any of the aspects, the axon outgrowth area has a width of at least 100 μm between the first cell growth area and the second cell growth area.

In some embodiments of any of the aspects, the axon outgrowth area is configured for axons to extend from the first cell growth area to the second cell growth area.

In some embodiments of any of the aspects, the axon outgrowth area comprises a nanopattern on the substrate.

In some embodiments of any of the aspects, the nanopattern comprises a series of parallel grooves and ridges with a proximal and distal end, wherein the proximal end of the grooves and ridges interfaces with the first cell growth area, and the distal end of the grooves and ridges interfaces with the second cell growth area.

In some embodiments of any of the aspects, the groove depth is between 100 nm and 600 nm, and the ridge width is between 400 nm and 1000 nm.

In some embodiments of any of the aspects, the axon outgrowth area comprises a series of parallel microchannels with a proximal and distal end, wherein the proximal end of the microchannels interfaces with the first cell growth area, and the distal end of the microchannels interfaces with the second cell growth area.

In some embodiments of any of the aspects, the microchannel width is between 1 μm and 50 μm, the interchannel width is between 1 μm and 150 μm, and the microchannel depth is between 50 nm and 5000 nm.

In some embodiments of any of the aspects, the device further comprises at least one barrier located between the first cell growth area and the second cell growth area, wherein the barrier is configured to separate a plurality of cell bodies of cells located on the first cell growth area from cells located on the second growth area.

In some embodiments of any of the aspects, the barrier is located at the interface between the first cell growth area and the axon outgrowth area.

In some embodiments of any of the aspects, the barrier is located within the axon outgrowth area, and wherein the barrier is configured to separate a plurality of cell bodies of cells located on the first cell growth area from cells located on the second growth area.

In some embodiments of any of the aspects, the barrier is configured to allow axons from cells located on the surface of the first cell growth area to extend into the axon outgrowth area.

In some embodiments of any of the aspects, the barrier is a non-removable or removable physical barrier.

In some embodiments of any of the aspects, the barrier is the same width as the axon outgrowth area.

In some embodiments of any of the aspects, the plurality of field potential electrodes (FPE) is arranged in an array.

In some embodiments of any of the aspects, the plurality of impedance electrodes (IE) is arranged in an array.

In some embodiments of any of the aspects, the field potential electrodes (FPE) are configured to receive an electrical signal via the electrical interface from a power source and configured to deliver an electrical stimulating signal to the surface of the first cell growth area.

In some embodiments of any of the aspects, the field potential electrodes (FPE) are configured to monitor any one of: spontaneous, electrically-paced, or optically-paced activity of cells in contact with the field potential electrode.

In some embodiments of any of the aspects, the field potential electrodes (FPE) are configured for one or both of: (a) monitor any one of: spontaneous, electrically-paced, or optically-paced activity of cells in contact with the field potential electrode; and (b) electrically stimulate cells present on the first cell growth area.

In some embodiments of any of the aspects, the field potential electrodes can electrically stimulate synaptic connections of cells present on the first cell growth area with the cells present on the second growth area.

In some embodiments of any of the aspects, the impedance electrodes (IE) are communicatively coupled via the electrical interface to at least one analyzing module in the form of an impedance analyzer, thereby permitting impedance monitoring from excitable cells attached to the surface of the second growth area.

In some embodiments of any of the aspects, the device further comprises a third cell surface area, wherein an edge of the third cell surface area interfaces with a proximal edge of the first cell growth area and the axon outgrowth area, or a distal edge of the second cell growth area and the axon outgrowth area.

In some embodiments of any of the aspects, the device further comprises a plurality of neuronal cells on the first cell growth area and a plurality of contractile cells or muscle cells on the second cell growth area.

In some embodiments of any of the aspects, the plurality of muscle cells on the second cell growth area is a 2D monolayer or an engineered 3D skeletal muscle construct.

In some embodiments of any of the aspects, the plurality of neuronal cells is derived from iPSCs obtained from a healthy subject or a subject with a neurodegenerative disease or disorder or a myopathy.

In some embodiments of any of the aspects, the plurality of muscle cells is derived from iPSCs obtained from a healthy subject or a subject with a neurodegenerative disease or disorder or a myopathy.

In some embodiments of any of the aspects, the plurality of neuronal cells comprise motor neurons, and the plurality of muscle cells comprise skeletal muscle cells.

In some embodiments of any of the aspects, the plurality of neuronal cells comprise sympathetic neurons, and the plurality of muscle cells comprise smooth muscle cells.

In some embodiments of any of the aspects, the plurality of neuronal cells comprise sympathetic neurons, and the plurality of muscle cells comprise cardiac muscle cells or cardiomyocytes.

In some embodiments of any of the aspects, the device further comprises an additional cell type on any one or more of: the first cell growth area, the second cell growth area or the axon outgrowth area.

In some embodiments of any of the aspects, the additional cell type is selected from any of: Schwann cells, microglia, astrocytes or satellite cells.

In some embodiments of any of the aspects, the additional cell type is Schwann cells, wherein the Schwann cells are located on the first cell growth area or axon outgrowth area, or both.

In some embodiments of any of the aspects, the neurodegenerative disease or a myopathy is selected from any of: CMT, ALS, SMA, myasthenia gravis, DMD, and a neuromuscular disease or wasting disorder.

In some embodiments of any of the aspects, the neuronal cells on the first cell growth area extend axons through the axon outgrowth area and into the second cell growth area comprising a plurality of muscle cells.

In some embodiments of any of the aspects, the neuronal cells that extend axons through the axon outgrowth area make synaptic connections with muscle cells present on the second growth area.

In some embodiments of any of the aspects, the first cell growth area comprises a nanopatterned surface, or the second cell growth comprises a nanopatterned surface, or both the first and the second cell growth surfaces comprise a nanopatterned surface.

In some embodiments of any of the aspects, the nanopattern on the first cell growth area is the same as the nanopattern on the second cell growth area.

In some embodiments of any of the aspects, the nanopattern on the first cell growth area is different to the nanopattern on the second cell growth area.

In some embodiments of any of the aspects, the nanopatterned surface provides anisotropic cues that promote improved levels of maturation of neuronal cell types, motor neurons and myocytes.

In some embodiments of any of the aspects, the device comprises an array of modules on the substrate.

In one aspect described herein is a system for measuring the electrical conductance from one cell type to a second cell type comprising: (a) a device as described herein, (b) an electronic interface for stimulation of field potential electrodes on the device, and (c) an electronic interface that permits recording of electrical activity from the impedance electrodes.

In one aspect described herein is a method for measuring the electrical conductance from one cell type to a second cell type comprising: (a) providing a device as described herein, wherein the device comprises a first cell type on the first cell growth area, and a second cell type on the second cell growth area, and wherein the first cell type extends axons across the axon outgrowth area to the second cell type in the second cell growth area; (b) providing electrical stimulation to the first cell type via the field potential electrodes, and (c) recording electrical activity of the second cell type via the impedance electrodes.

In some embodiments of any of the aspects, the first cell type is a plurality of neuronal cells.

In some embodiments of any of the aspects, the second cell type is a plurality of contractile or muscle cells.

In some embodiments of any of the aspects, the plurality of muscle cells is a monolayer or an engineered skeletal muscle construct.

In some embodiments of any of the aspects, the plurality of neuronal cells is derived from iPSCs obtained from a healthy subject or a subject with a neurodegenerative disease or disorder or a myopathy.

In some embodiments of any of the aspects, the plurality of muscle cells is derived from iPSCs obtained from a healthy subject or a subject with a neurodegenerative disease or disorder or a myopathy.

In some embodiments of any of the aspects, the plurality of neuronal cells comprise motor neurons, and the plurality of muscle cells comprise skeletal muscle cells.

In some embodiments of any of the aspects, the plurality of neuronal cells comprise sympathetic neurons, and the plurality of muscle cells comprise smooth muscle cells.

In some embodiments of any of the aspects, the plurality of neuronal cells comprise sympathetic neurons, and the plurality of muscle cells comprise cardiac muscle cells or cardiomyocytes.

In some embodiments of any of the aspects, the plurality of neuronal cells, or plurality of muscle cells, or both, are genetically modified cells to introduce one or more mutations for a neurodegenerative disease or myopathy.

In some embodiments of any of the aspects, the plurality of neuronal cells, or plurality of muscle cells, or both, are isogenic controls of a genetically modified cell that has one or more mutations introduced for a neurodegenerative disease or myopathy.

In some embodiments of any of the aspects, a method as described herein is used to assess electrical conductance across at least one neuromuscular junctions (NMJ) between an axon extended from the first cell type and the cell bodies of the second cell type.

In one aspect described herein is an assay for assessing an agent for modulation of electrical signaling from one cell type to another cell type, comprising: (a) providing a device as described herein, wherein the device comprises a first cell type located on surface of the first cell growth area, and a second cell type located on the second cell growth area, and wherein the first cell type extends axons across the axon outgrowth area from the first cell area to the second cell type in the second cell growth area; (b) contacting the first cell type, second cell type, or both, with an agent; (c) providing electrical stimulation to the first cell type via the field potential electrodes; (d) recording electrical activity of the second cell type via the impedance electrodes; and (e) detecting a change in electrical activity of the second cell type recorded via the impedance electrodes in the presence of the agent as compared to the absence of the agent.