WO2024163660A1

WO2024163660A1 - Devices and methods for guiding cells and monitoring electrical activities

Info

Publication number: WO2024163660A1
Application number: PCT/US2024/013841
Authority: WO
Inventors: Christos MICHAS; Jacob FLEMING; Kevin Gray; Shawn Mcguire
Original assignee: Curi Bio Inc
Current assignee: Curi Bio Inc
Priority date: 2023-01-31
Filing date: 2024-01-31
Publication date: 2024-08-08
Anticipated expiration: 2025-07-31

Abstract

Cell monitoring devices and systems for measuring electrical signals and their propagation within and between cells, cell assemblies and tissues are provided. Such cell monitoring devices include at least one guidance unit disposed on a substrate. The at least one guidance unit includes a plurality of seeding chambers, at least one guidance channel in communication with a first seeding chamber and a second seeding chamber, a co-culture chamber encompassing at least a portion of the at least one guidance channel, and a plurality of electrode arrays disposed along the at least one guidance channel.

Description

DEVICES AND METHODS FOR GUIDING CELLS AND MONITORING ELECTRICAL

ACTIVITIES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/442,293, filed on January 31, 2023, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates generally to the field of in vitro electrophysiology. In particular, the present disclosure provides devices, systems, and methods for measuring and assessing changes in electrical signals and their propagation within and between cells, cell assemblies and tissues, e.g., in response to a variety of user-controlled experimental conditions.

BACKGROUND

[0003] Cell-mediated electrical conduction is a process that enables the relay of signals and information across the human body. The impairment of such electrical conduction can lead to critical indications and diseases affecting the human health. Thus, an in vitro model of electrical conduction would be a useful tool that allows various investigations on the mechanism of diseases, discovery of therapeutics, and toxicity screening of compounds.

BRIEF SUMMARY

[0004] In an aspect, the present disclosure provides cell monitoring devices. Such cell monitoring devices include at least one guidance unit disposed on a substrate. The at least one guidance unit includes a plurality of seeding chambers, at least one guidance channel in communication with a first seeding chamber and a second seeding chamber, a co-culture chamber encompassing at least a portion of the at least one guidance channel, and a plurality of electrode arrays disposed along the at least one guidance channel.

[0005] In another aspect, the present disclosure provides systems that include the cell monitoring device and a non-transitory machine readable storage medium storing instructions, which when executed by a processor, performs operations, including: measuring electrical signals from the plurality of electrode arrays.

[0006] Non-limiting examples of the present disclosure will now be described in the following numbered clauses: [0007] Clause 1 : A cell monitoring device, comprising: at least one guidance unit disposed on a substrate, the at least one guidance unit comprising: a plurality of seeding chambers; at least one guidance channel having a first end and a second end, the first end of the at least one guidance channel being in communication with a first seeding chamber of the plurality of seeding chambers and the second end of the at least one guidance channel being in communication with a second seeding chamber of the plurality of seeding chambers; and a co-culture chamber encompassing at least a portion of the at least one guidance channel; a plurality of electrode arrays or electrode clusters disposed along the at least one guidance channel; and a cover; and an electrical interface in communication with the plurality of electrode arrays.

[0008] Clause 2: The device of clause 1 or any one or more clauses herein, wherein the at least one guidance channel comprises a plurality of guidance channels.

[0009] Clause 3 : The device of clause 2 or any one or more clauses herein, wherein any pair of seeding chambers of the plurality of seeding chambers is connected by at least one guidance channel of the plurality of guidance channels.

[0010] Clause 4: The device of clause 2, clause 3, or any one or more clauses herein, wherein at least one pair of seeding chambers of the plurality of seeding chambers is connected by at least two guidance channels of the plurality of guidance channels.

[0011] Clause 5: The device of any one of clauses 2-4 or any one or more clauses herein, wherein different pairs of seeding chambers of the plurality of seeding chambers are connected by at least one different guidance channel of the plurality of guidance channels.

[0012] Clause 6: The device of any one of clauses 2-5 or any one or more clauses herein, wherein at least one guidance channel of the plurality of guidance channels intersects with at least one other guidance channel of the plurality of guidance channels.

[0013] Clause 7: The device of any one of clauses 1-6 or any one or more clauses herein, wherein a cross-sectional area of at least one guidance channel of the plurality of guidance channels varies over one or more sections thereof.

[0014] Clause 8: The device of any one of clauses 1-7 or any one or more clauses herein, wherein the at least one guidance channel comprises a microchannel.

[0015] Clause 9: The device of clause 8 or any one or more clauses herein, wherein the at least one guidance channel has a linear trajectory. [0016] Clause 10: The device of any one of clauses 1-9 or any one or more clauses herein, wherein the guidance channel has a cross sectional area of 10 - 120 square pm.

[0017] Clause 11 : The device of any one of clauses 1-10 or any one or more clauses herein, wherein the guidance channel has a channel width of 20-40 gm and a height of 1-3 gm.

[0018] Clause 12: The device of clause 11 or any one or more clauses herein, wherein the guidance channel has a channel length less than 4000 gm.

[0019] Clause 13 : The device of any one of clauses 1-12 or any one or more clauses herein, wherein a cross-sectional width of the at least one guidance channel is 1 gm - 100 gm, a cross- sectional height of the at least one guidance channel is 1 gm - 100 gm, and a length of the at least one guidance channel is 0.1 mm - 10 mm.

[0020] Clause 14: The device of any one of clauses 1-13 or any one or more clauses herein, wherein at least some electrode arrays of the plurality of electrode arrays are disposed on opposing sides of the at least one co-culture chamber.

[0021] Clause 15: The device of any one of clauses 1-14 or any one or more clauses herein, wherein each electrode array of the plurality of electrode arrays comprises at least two electrodes disposed along the at least one guidance channel.

[0022] Clause 16: The device of clause 15 or any one or more clauses herein, wherein the at least two electrodes of at least one of the electrode arrays are exposed in the at least one guidance channel.

[0023] Clause 17: The device of clause 15 or clause 16 or any one or more clauses herein, wherein the at least two electrodes of at least one of the electrode arrays are disposed below at least one of an ion permeable material or a conductive material along the at least one guidance channel.

[0024] Clause 18: The device of any one of clauses 15-17 or any one or more clauses herein, wherein each electrode array of the plurality of electrode arrays comprises at least three electrodes disposed along the at least one guidance channel.

[0025] Clause 19: The device of any one of clauses 1-18 or any one or more clauses herein, wherein the at least one guidance channel comprises a plurality of guidance channels, and wherein at least one electrode of the plurality of electrode arrays is disposed along each guidance channel. [0026] Clause 20: The device of clause 19 or any one or more clauses herein, wherein at least two electrode arrays of the plurality of electrode arrays are disposed along each guidance channel. [0027] Clause 21 : The device of clause 19 or clause 20 or any one or more clauses herein, wherein at least some guidance channels of the plurality of guidance channels are parallel.

[0028] Clause 22: The device of any one of clauses 19-21 or any one or more clauses herein, wherein adjacent guidance channels of the plurality of guidance channels are electrically insulated from each other.

[0029] Clause 23 : The device of any one of clauses 19-22 or any one or more clauses herein, wherein a first electrode array is positioned at a first distance from a first seeding chamber along a first guidance channel of the plurality of guidance channels, and wherein a second electrode array is positioned at the first distance from the first seeding chamber along a second guidance channel of the plurality of guidance channels.

[0030] Clause 24: The device of any one of clauses 1-23 or any one or more clauses herein, wherein each electrode of the plurality of electrode arrays is configured to communicate electrical signals from the at least one guidance channel to the electrical interface.

[0031] Clause 25: The device of clause 24 or any one or more clauses herein, wherein at least some adjacent electrodes within at least one electrode array of the plurality of electrode arrays are spaced apart from each other by about: 50 pm - 500 pm, 75 pm - 325 pm, 75 pm - 250 pm, 75 pm - 200 pm, 75 pm - 150 pm, 75 pm - 125 pm, or 100 pm.

[0032] Clause 26: The device of clause 24 or clause 25 or any one or more clauses herein: wherein at least some electrodes within different electrode arrays of the plurality of electrode arrays are spaced apart by about: 100 pm - 10 mm, 100 pm - 5 mm, 500 pm - 5 mm, 1mm - 5 mm, or 1mm - 3 mm.

[0033] Clause 27: The device of any one of clauses 1-26 or any one or more clauses herein, wherein the plurality of electrode arrays comprises at least one stimulation electrode and at least two recording electrodes.

[0034] Clause 28: The device of clause 27 or any one or more clauses herein, wherein the at least one stimulation electrode is positioned in proximity to an end of the at least one guidance channel.

[0035] Clause 29: The device of any one of clauses 1 -28 or any one or more clauses herein, further comprising at least one additional electrode array at least partially disposed in at least one of the seeding chambers of the plurality of seeding chambers or the at least one co-culture chamber.

[0036] Clause 30: The device of clause 29 or any one or more clauses herein, wherein the at least one additional electrode array is configured to detect cells in one seeding chamber of the plurality of seeding chambers or in the at least one co-culture chamber.

[0037] Clause 31 : The device of any one of clauses 1-30 or any one or more clauses herein, wherein the cover does not cover at least an uncovered portion of the at least one guidance channel.

[0038] Clause 32: The device of any one of clauses 1-31 or any one or more clauses herein, wherein the cover covers at least a portion of the at least one guidance channel.

[0039] Clause 33 : A system comprising: a cell monitoring device, comprising: at least one guidance unit disposed on a substrate, the at least one guidance unit comprising: a plurality of seeding chambers; at least one guidance channel having a first end and a second end, the first end of the at least one guidance channel being in communication with a first seeding chamber of the plurality of seeding chambers and the second end of the at least one guidance channel being in communication with a second seeding chamber of the plurality of seeding chambers; a coculture chamber encompassing at least a portion of the at least one guidance channel; and a plurality of electrode arrays disposed along the at least one guidance channel; and a cover covering at least a portion of the at least one guidance channel; and an electrical interface in communication with the plurality of electrode arrays; and a non-transitory machine readable storage medium storing instructions, which when executed by a processor, performs operations, including: measuring electrical signals from the plurality of electrode arrays.

[0040] Clause 34: The system of clause 33 or any one or more clauses herein, further comprising an electronic instrument connectable to the electrical interface.

[0041] Clause 35: The system of clause 34 or any one or more clauses herein, wherein the electronic instrument comprises the non-transitory machine readable storage medium and the processor.

[0042] Clause 36: The system of any one of clauses 33-35 or any one or more clauses herein, wherein measuring the electrical signals includes measuring electrical signals at a plurality of electrode pairs, wherein each electrode pair comprises an electrode from a first electrode array and an electrode from a second electrode array of the plurality of electrode arrays. [0043] Clause 37: The system of clause 36 or any one or more clauses herein, wherein sampling the electrical signals at the plurality of electrode pairs comprises sampling the electrical signals at different times for different electrode pairs.

[0044] Clause 38: The system of any one of clauses 33-37 or any one or more clauses herein, wherein the instructions further include determining a conduction velocity of the electrical signals based upon the sampled electrical signals.

[0045] Clause 39: The device of any one of clauses 1-32 or any one or more clauses herein, wherein one or more electrode arrays of the plurality of electrode arrays is formed as an electrode cluster, optionally wherein the one or more electrode clusters are disposed on opposing sides of the co-culture chamber.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0046] Representative embodiments are described with reference to the following figures.

[0047] FIG. 1A is a fluorescent micrograph illustrating a seeded neuron according to an embodiment of the present disclosure.

[0048] FIG. IB is a fluorescent micrograph illustrating Schwann cell precursors according to an embodiment of the present disclosure.

[0049] FIG. 2A is a chart illustrating an exemplary operation of a system including a cell monitoring device embodying a plurality of guidance units, and an electronic instrument, according to an embodiment of the present disclosure.

[0050] FIG. 2B is an enlarged view of a cell monitoring device shown in FIG. 2A.

[0051] FIG. 2C is an enlarged view of the system including the cell monitoring device and the electronic instrument shown in FIG. 2A.

[0052] FIG. 3A illustrates a perspective view of aspects of a guidance unit according to an embodiment of the present disclosure.

[0053] FIG. 3B illustrates an enlarged perspective view of the guidance unit of FIG. 3 A taken from area “3B" shown in FIG. 3A.

[0054] FIG. 3C illustrates a perspective view of a guidance unit according to another embodiment of the present disclosure.

[0055] FIG. 3D illustrates an enlarged perspective view of the guidance unit of FIG. 3C taken from area “3D” shown in FIG. 3C. [0056] FIG. 4A is a schematic illustrating a cross-sectional view of the guidance unit of FIG. 3A taken along line “4A” shown in FIG. 3B.

[0057] FIG. 4B is a schematic illustrating a perspective cross-sectional view of the guidance unit of FIG. 3 A taken along line “4A” shown in FIG. 3B.

[0058] FIG. 5A is a schematic illustration of aspects of a co-culture chamber according to an embodiment of the present disclosure.

[0059] FIG. 5B is perspective view of aspects of a guidance unit incorporating a co-culture chamber of FIG. 5 A according to an embodiment of the present disclosure.

[0060] FIG. 6 is a chart illustrating a process for fabricating guidance units according to an embodiment of the present disclosure.

[0061] FIG. 7 is a fluorescence micrograph illustrating an axon cytoskeleton stained with Tuj 1

- green within a guidance channel according to an embodiment of the present disclosure.

[0062] FIG. 8A is a micrograph illustrating axonal extensions of neurons stained with DAPI

- blue according to an embodiment of the present disclosure.

[0063] FIG. 8B is a micrograph illustrating axonal extensions of neurons stained with Tuj 1

- green according to an embodiment of the present disclosure.

[0064] FIG. 8C is a micrograph illustrating axonal extensions of neurons stained with ChAT - red according to an embodiment of the present disclosure.

[0065] FIG. 8D illustrates an overlay of FIG. 8A - FIG. 8C with a phase contrast image in greyscale according to an embodiment of the present disclosure.

[0066] FIG. 9A shows aspects of a cell monitoring device comprising a guidance unit.

[0067] FIG. 9B is a schematic illustration of an alignment of the guidance channels with the electrodes of the device of FIG. 9A and the detected electrical signals propagating across each of the guidance channels.

[0068] FIG. 10A is a series of charts illustrating raw data traces of voltages against time as measured by multiple sequential electrodes within a single guidance channel of the device of FIG. 9A.

[0069] FIG. 10B is a schematic illustration of the transmitted peak groups shown in FIG. 10A. [0070] FIG. 10C is a chart illustrating a visualization of raw data from a single transmitted peak group shown in FIG. 10B. [0071] FIG. 11 A is a chart illustrating exemplary data acquisition and processing schemes using a device according to an embodiment of the present disclosure.

[0072] FIG. 1 IB is a series of charts illustrating simulated action potentials according to the exemplary data acquisition and processing schemes shown in FIG. 11 A.

[0073] FIG. 12 is a table illustrating a relationship between guidance channel length, conduction velocity, and sampling rates for tracking electrical signals propagating between electrodes according to an embodiment of the present disclosure.

[0074] FIG. 13 is a chart illustrating neuronal conduction velocity over time when using a guidance device according to an embodiment of the present disclosure.

[0075] FIG. 14A is a chart illustrating a dose response curve for Vincristine according to embodiment of the present disclosure.

[0076] FIG. 14B is a chart illustrating a dose response curve for Paclitaxel according to an embodiment of the present disclosure.

[0077] FIG. 14C is a chart illustrating a dose response curve for Oxaliplatin according to an embodiment of the present disclosure.

DETAILED DESCRIPTION

[0078] The present disclosure provides devices, systems, and methods for measuring and assessing changes in electrical signals and their propagation within and between cells, cell assemblies and tissues, e.g., in response to a variety of user-controlled experimental conditions.

[0079] Devices of the present disclosure provide structural or topographical cues embedded in or overlaid on electrode arrays or clusters to enable and control the extension of cells. For example, a neurosphere may be seeded in a seeding chamber of a cavity from which axons are directed to extend into a narrower section of the cavity where the electrical activity of an axonal growth process can be measured with the electrodes.

[0080] Embodiments further provide one or more guidance channels (e.g., microchannels) to guide the outgrowth of a smaller number of axons over the electrode arrays. With such structural or topographic control over cell extensions, it is possible to track an electrical signal as it serially propagates over the electrodes underlying the axonal growth processes and to calculate propagation metrics, such as the conduction velocity of the axonal process.

[0081] FIG. 1A is a fluorescent micrograph showing how neurons seeded in the form of a neurosphere tend to extend axons outside of the spheroid. Here, a neurosphere containing 5,000 cells extend neurites radially outward from the associated cell bodies. For the illustrative purpose, cells have been stained for the microtubule cytoskeleton (Tuj 1 - Red), the nuclear motor neuron marker islet- 1 (Isll - Green), and the mature motor neuron marker choline acetyltransferase (ChAT - Greyscale).

[0082] FIG. IB is a fluorescent micrograph of Schwann cell precursors stained for the neural crest lineage marker SlOOb. All scale bars represent 100pm. FIG. 1A - FIG. IB show that axonal outgrowth is rather irregular and accordingly does not permit controlled, reproducible tracking of an electrical signal (such as action potential) even if the substrate is furnished with sensors that can measure action potential on a cell level. The devices and systems of the present disclosure overcome such limitations, for example by providing microtopographic features on a culture substrate (e.g., guidance channels having micro-grooves/ridges and/or microchannels) that can direct and guide the migration/outgrowth of cells, preferably, in a unidirectional manner. [0083] Electrogenic cells tend to migrate or extend their conductive processes when seeded on a cell culture substrate. The present disclosure provides devices and systems that precisely guide such migrating or extending cell processes to come in proximity with electrode arrays arranged in a sequential manner vis-a-vis a seeding chambers connected by guidance channels guiding the cell processes and optional co-culture chambers for various support cells encompassing the guidance channels for interaction with the cell processes therein, thereby enabling the device to detect the propagation of the electrical signals at and between the electrodes. Accordingly, the devices disclosed herein provide an effective tool for evaluating the electrophysiological properties of electrogenic cells and their extensions in a reproducible, scalable manner.

[0084] FIG. 2 A - FIG. 2C show a system 200 of the present disclosure which measures and assesses changes in electrical signals within and between cells. The system 200 includes a cell monitoring device 202 (e.g., a consumable unit) and an electronic instrument 209 that physically and electrically interfaces with the cell monitoring device 202. Inventive aspects exist in the cell monitoring device 202, in guidance units 204 embodied therein, in the electronic instrument 209, and in the system 200 as a whole. As detailed beginning with FIG. 3 A, the cell monitoring device 202 includes one or more guidance units (e.g., in a multi-well format) that directionally guide propagation of cells seeded therein. See FIG. 2B.

[0085] FIG. 2A illustrates one method of using the system 200. First, selected cells are seeded into one or more seeding chambers of the guidance units 204 of the device 202. The seeding chambers are connected by one or more microtopographic features, e.g., guidance channels and optionally one or more co-culture chambers. Upon seeding, the microtopographic features formed within the guidance units 204 direct and guide the cells to develop the structures and electrophysiological connections reminiscent of the in vivo milieu. Once the cells/tissues seeded into the seeding chambers in the guidance units 204 start extending, migrating or physiologically developing in the desirable manner across the guidance channels, the device 202 can then be connected to the electronic instrument 209, which may be configured to stimulate the cells and/or record (i.e., measure) the electrical activity of the cell structures undergoing development within the guidance units 204.

[0086] As shown in FIG. 2B, the cell monitoring device 202 includes one or more guidance units 204 into which the cells are seeded and through which the cells extend. The guidance unit 204 is disposed on a substrate 201 (e.g., cell culture plate). The cell monitoring device 202 may further include a plurality of wells positioned such that each guidance unit 204 is aligned with one well. The substrate 201 can support a plurality of guidance units 204 (e.g., 6, 12, 24, 48, 96, 384 or 1536), each containing one or more guidance channels, where each guidance unit 204 is enclosed in a separate watertight well that can isolate the guidance unit 204 electrically, fluidically and, optionally, optically from each other.

[0087] The substrate 201 may comprise a base material attachable to a wall structure or lid 205, which in turn may be attachable to a device cover 219. In some embodiments, an adhesive layer serves as the medium that bonds the substrate 201 and the lid 205. The lid 205 may contain a plurality of wells, and once bonded to the substrate 201, the well-like structure isolates the guidance unit contained therein fluidically and electrically from outside the well. The substrate 201 may comprise a standalone layer or a plurality of stacked layers of materials, including elements of the guidance unit 204 including a base substrate, a layer of electrically conductive patterns (referred to as electrodes), a set of layers that selectively cover and insulate the electrodes (insulation layer, an ion permeable layer, and a conductive layer), and one or more layers that define microtopographic features over the electrodes and electrical insulation therebetween (such as seeding chambers, co-culture chambers, and guidance channels). The device cover 219 covers the assembled device to prevent the entrance of foreign bodies into the wells.

[0088] Various forms of the lid 205 and/or device cover 219 can be added to the device to provide additional functionalities. For example, in some embodiments, the lid 205 and/or device cover 219 comprises one or more optical stimulation apparatuses or light sources, e.g., that enable independent illumination of the wells for various imaging purposes or for optogenetic stimulation. In such embodiments, LEDs can serve as the light source, and the appropriate circuitry could be added to the cover to power the light source and provide control of the illumination properties, with use of appropriate external hardware and software. In other embodiments, the lid 205 and/or device cover 219 can be modified to encompass microfluidic features that can allow selective delivery of liquid solutions to the wells. Such embodiments may include ports in the lid 205 and/or device cover 219 where an external pumping mechanism can be connected to facilitate automated maintenance of cells and/or automated delivery of pharmacological agents, small molecules, biologic therapies, or toxins in the context of high- throughput screening. In still other embodiments, the lid 205 and/or device cover 219 may comprise electrical stimulation circuitry configured to electrically interface with one or more guidance units, e.g., to electrically stimulate cells therein.

[0089] In use, electrogenic cells are seeded in the guidance unit (e.g., in a microtopographic seeding chamber) and extend through one or more guidance channels, wherein electrodes of one or more electrode arrays or clusters record electrical signals from the cells (e.g., action potentials). Measuring the electrical signals across a plurality of electrodes enables analysis of the conduction velocity of the electrical signals and, by extension, electrophysiological properties of the cells.

[0090] An electrical interface 207 of the cell monitoring device 202 includes circuitry operably connected to a plurality of electrode arrays of each guidance unit 204. The electrical interface 207 includes one or more electrode contact pads, pins, spring-loaded probes, or other electrical interfacial structure configured to electrically communicate with an electrical interface of the electronic instrument 209.

[0091] As described below with respect to FIG. 6, embodiments of the devices of the present disclosure can be assembled by stacking the complete, patterned substrate 201 housing various microtopographic features defining the guidance units 204, an adhesive layer, and the lid 205. The guidance units 204 should be precisely situated within the wells of the lid 205; therefore, an alignment process may be implemented prior to the final bonding between the substrate and the lid. As described below, alignment features facilitating the precise alignment of the guidance units can be provided as part of the guidance unit 204 using various fabrication techniques mentioned above. [0092] As shown in FIG. 2C, the system 200 includes a data store 215 storing instructions 217 such as software logic (e.g., executable software code), firmware logic, hardware logic, or various combinations thereof, which when executed by a processor 213 (e.g., general processing units, graphical processing units, application specific integrated circuits), performs operations, e.g., processing electrical signals detected by one or more electrode arrays in the cell monitoring device 202, which are communicated to the electronic instrument 209 via the electrical interface 207. The instructions 217 may include logic embodying any whole or part of any method described herein. The processor 213, data store 215, and instructions 217 may be embodied in whole or in part in the electronic instrument 209 and/or other computing device 211.

[0093] The electronic instrument 209 may include one or more communications or electrical interfaces having circuits configured to enable communication with the cell monitoring device 202 and optionally with a remote server, base station, or other network element via the internet, cellular network, RF network, Personal Area Network (PAN), Local Area Network, Wide Area Network, or other network. Accordingly, the communications interface may be configured to communicate using wireless protocols (e.g., WIFI®, WIMAX®, BLUETOOTH®, ZIGBEE®, Cellular, Infrared, Nearfield, etc.) and/or wired protocols (Universal Serial Bus or other serial communications such as RS-234, RJ-45, etc., parallel communications bus, etc.). In some embodiments, the communications interface includes circuitry configured to initiate a discovery protocol that allows the device and other network element to identify each other and exchange control information. In an embodiment, the communications interface has circuitry configured to a discovery protocol and to negotiate one or more pre-shared keys. In an embodiment, the communications interface alternatively or additionally includes circuitry configured to initiate a discovery protocol that allows an enterprise server and the device to exchange information.

[0094] As used in this disclosure, a data store is a tangible machine-readable storage medium that includes any mechanism that provides (i.e., stores) information in a non-transitory form accessible by a machine (e.g., a computer, network device, personal digital assistant, manufacturing tool, any device with a set of one or more processors, etc.). For example, a machine-readable storage medium includes recordable/non-recordable media (e.g., read only memory (ROM), random access memory (RAM), magnetic disk storage media, optical storage media, flash memory devices, etc.). [0095] The instructions 217 may be embodied in a companion software that samples (i.e., measures or records) electrical signals from the electrode arrays of the cell monitoring device 202 and processes the electrical signals detected by the arrays of electrodes and deduces various propagation properties from the recorded signals which can provide valuable information on the robustness, maturity, and developmental forms and capabilities of the cellular models.

[0096] Various assays based on electrophysiological organ-on-chip models can be implemented in the instructions 217 with ease in a high throughput setting, in association with or independent of the electronic instrument 209. The organ-on-chip models of this disclosure can provide additional capabilities using co-culture platforms of the cell monitoring device 202 to develop and assay more complex models comprising many different types of cells. Additionally, this system 200 can be adapted (e.g., via one or more co-culture chambers) to allow localized applications of pharmacological agents, biologic therapies or toxins, and the safety and efficacy of various therapeutic strategies can be readily evaluated with the biological models developed with this device.

[0097] The systems 200 of the present disclosure can record the voltages that vary dynamically depending on the electrical activity of the cells overlying the electrodes, such as cross-membrane potentials or extracellular field potentials of the cells. Some embodiments of the system 200 also apply optical and/or electrical stimulation (in voltage or current) to the cells/tissues selected for testing. The electrical data recorded from the electrodes of the cell monitoring device 202 encompassing the guidance channels can be comparatively processed to deduce key electrophysiological metrics (such as conduction velocity of the cells) derived from the propagation of the electrical activity across the cells within the guidance channels. For example, prominent features of the observed signals (e g., the peak of an action potential) can be identified and then examined in relation to each other to identify a time shift or propagation time interval that can constitute an occurrence of a biologically meaningful event. Additionally, through the post processing of such voltage signals, additional metrics such as firing burst frequency, depolarization frequency, depolarization rate, repolarization rate, and rate of cell-cell communication can be easily derived using algorithms developed as part of the operating software. Electrical stimulation and/or optical stimulation can also be used to trigger electrical activity that propagates over the recording electrodes and to observe the evoked activity in response to different or varying stimulation patterns. [0098] System 200 can be used to quantify the effect of experimental conditions on the electrophysiological properties of the cell processes under test. Such conditions may include chemical compounds, inclusion or addition of other co-cultured cell types, genetic mutations, disease phenotypes, antigens, therapeutic agents, and external controls such as electrical and optical stimulation.

[0099] FIG. 3A - FIG. 3D depict representative guidance units 304 of the present disclosure which are similar to the guidance unit 204 shown in FIG. 2A - FIG. 2C, and thus may be embodied in any cell monitoring device of the present disclosure. In particular, FIG. 3A shows an example of a guidance unit 304 as seen within a well of a lid of a cell monitoring device, wherein one or more guidance channels are interposed between and connecting a plurality of seeding chambers. Each guidance channel features one or more electrode arrays or clusters, e.g., two or more electrode arrays. FIG. 3B is a magnified view of one of the electrode arrays or clusters situated near one end of the guidance channels. FIG. 3C shows an alternative guidance unit 304, and FIG. 3D shows a detail view thereof.

[0100] Referring to FIG. 3A and FIG. 3B, the guidance unit 304 is disposed on a substrate 301 upon which a precisely designed layout of electrode arrays and microtopographic features comprising various seeding chambers, one or more guidance channels, and one or more optional co-culture chambers is formed, e.g., as a plurality of layers disposed on the substrate 301. Each guidance unit 304 may be implemented on a cell-culture plate (i.e., the substrate 301) within a well-like structure in a configuration that can reproducibly and consistently direct the outgrowth or extension or migration of electrogenic cells along or over the electrode arrays. The composition of the layer(s) is described in more detail with respect to FIG. 4A and FIG. 4B.

[0101] Guidance unit 304 generally includes one or more seeding chambers, e.g., a plurality of seeding chambers, at least one guidance channel in communication with at least two guidance channels of the plurality of guidance channels, and one or more electrode arrays disposed along the at least one guidance channel.

[0102] In the illustrated example, the guidance unit 304 includes a plurality of seeding chambers (here, two seeding chambers 312 and 314) into which the cells/tissues can be seeded. For example, in some embodiments, the seeding chambers each comprise a microtopographic feature such as a recess formed in an insulating material. The shape and dimension of each seeding chamber may vary depending on the application. For example, in some embodiments, one or more seeding chambers is a circle (e.g., having a diameter of 2-5 mm). In some embodiments, one or more seeding chambers has a polygonal shape, (e.g., a rectangle having a side length between l-5mm).

[0103] The seeding chambers 312, 314 are connected by additional microtopographic features including one or more axon-guiding channels or micro guidance channels (here, three microchannels 306a, 306b, and 306c) that allow the cells in the seeding chambers 312, 314 to migrate or extend therethrough towards the other seeding chamber. FIG. 3B shows aspects of the guidance channels 306a - 306c in detail. In some embodiments, the one or more guidance channels include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a greater number of guidance channels.

[0104] Generally, each guidance channel is configured to direct axons outgrowing from neural cells seeded in the first seeding chamber therethrough towards the second seeding chamber. More specifically, each of the guidance channels (e.g., 306a) have a first end 308 and a second end 310, the first end 308 being in communication with a first seeding chamber of the plurality of seeding chambers (e.g., first seeding chamber 312) and the second end 310 being in communication with a second seeding chamber of the plurality of seeding chambers (e.g., second seeding chamber 314). In some embodiments, the at least one guidance channel is configured to guide axonal outgrowth therethrough while allowing myelination of the outgrown axons.

[0105] The microtopographic features defining the guidance channels are implemented using one or more layers of electrically insulating material such as SU8 or Polydimethylsiloxane (PDMS), and a cover (see FIG. 4A - FIG. 4B) is furnished, e.g., as part of the guidance unit 304, to seal at least a portion of the upper side of the guidance channels and optionally to increase the depth of the adjoining seeding chambers. In some embodiments, cover alignment features 322 such as grooves, ridges, keys, detents, or the like are furnished to facilitate precise positioning of the cover over the guidance unit. The guidance unit is surrounded by electrodes that may serve as a reference or ground electrode 318. The electrode traces 326 exit the guidance unit and connect to an electrical interface, which may comprise electrical contact pads, pins, and the like. [0106] The guidance channels 306a - 306c are characterized by their electrical insulation from each other (to prevent crosstalk) and smaller dimensions as compared to the seeding chambers, to direct or guide cell migration or extension on a fine scale. To enable electrical insulation, in some embodiments, adjacent guidance channels of the plurality of guidance channels are spaced apart by 1 pm - 100 pm.

[0107] In some embodiments, each guidance channel has a cross sectional area of 10 - 120 square pm, e.g., a rectangular cross section with a channel width of 20-40 pm (e.g., 30 pm), a height of 1-3 gm (e.g., 2 gm). The cross sectional shape of the guidance channel may have another shape, e.g., due to anisotropic formation processes. Each guidance channel may have a channel length of 250 - 10000 pm, e.g., less than 6000 pm or less than 4000 pm. Generally, the channel length, width, and height (depth) can be in the ranges of between 0.1 and 10 mm, 1 and 100 pm, and 1 and 100 pm, respectively.

[0108] In some embodiments, the at least one guidance channel comprises a plurality of guidance channels and any pair of the plurality of seeding chambers is connected by at least one guidance channel of the plurality of guidance channels. For example, each seeding chamber can be connected by one, two, three, or more guidance channels, depending on the type or nature of the cellular network to be studied. The illustrated embodiment includes three parallel guidance channels 306a - 306c, each having a linear trajectory. This configuration enables repeatable analysis of a cell culture migrating between the seeding chambers 312, 314. In some embodiments, one or more of the guidance channels has an arbitrary trajectory, e.g., a non-linear trajectory. Such a non-linear trajectory may enable a longer effective channel length on a substrate of a given size. In some embodiments, different pairs of seeding chambers of the plurality of seeding chambers are connected by at least one different guidance channel of the plurality of guidance channels. In some embodiments, at least one pair of seeding chambers of the plurality of seeding chambers is connected by at least two guidance channels of the plurality of guidance channels.

[0109] In some embodiments, one or more pairs of seeding chambers is connected by a guidance channel that is not the same guidance channel, and/or is otherwise not identical, to the guidance channel connecting another pair of seeding chambers. In some embodiments, one or more guidance channels may intersect at least one other of the guidance channels.

[0110] The guidance channels can also be designed to allow or exclude the entry of certain cells into the guidance channel. For example, in some embodiments, a cross-sectional area of at least one guidance channel of the plurality of guidance channels varies over one or more sections thereof, e.g., having a stepped down cross-sectional area, i.e., a constriction, between a co-culture chamber and one of the seeding chambers. Such an implementation prevents the co-localization of the supporting cells over the electrodes situated in the guidance channel, preventing the supporting cells from insulating the electrical communication between neurons and the electrodes. Such an implementation is particularly relevant if the supporting cells are myelinating cells (Schwann cells, oligodendrocytes) but could be generalized to any set of electrogenic and non-electrogenic cells. In some embodiments, the selective placement and distribution of the supporting cells may be accomplished by implementing another microtopography layer featuring one or more distinct co-culture chambers for the supporting cells and microtopographic features that can direct the supporting cells to migrate towards and into the designated areas of the guidance channels of the underlying layer. The supporting cells can be introduced as a suspension of individual cells, as cell clusters or as tissues.

[0111] Referring to FIG. 3 A and FIG. 3B, generally one or more electrode arrays underlie each of the guidance channels 306a - 306c. In some embodiments, the guidance unit includes a plurality of electrode arrays disposed along each guidance channel. For example, in the embodiment of FIG. 3A and FIG. 3B, two electrode arrays 316a, 316b formed as electrode clusters are disposed along guidance channel 306a. More particularly, electrode array 316a is disposed along guidance channel 306a in proximity to the first end 308 thereof, whereas electrode array 316b is disposed along guidance channel 306a in proximity to the second end 310 thereof. [0112] As shown in FIG. 3B, each electrode array comprises at least two electrodes spaced relatively close together and disposed along one of the guidance channels. In some embodiments, one or more of the electrode arrays may be further defined as an electrode cluster in consideration of relatively close spacing between adjacent electrodes, and/or in consideration of an observable grouping of electrodes. Generally, adjacent electrodes of an electrode array formed as an electrode cluster are spaced apart by a pitch of about: 50 pm - 500 pm, 75 pm - 325 pm, 75 pm - 250 pm, 75 pm - 200 pm, 75 pm - 150 pm, 75 pm - 125 pm, or about 100 pm. In some embodiments, each pair of adjacent electrodes of an electrode array is spaced apart by a common pitch. In other embodiments, one or more electrode arrays include pairs of electrodes that are spaced apart by different pitches. Either configuration may be appropriate for different signal processing techniques. In any embodiment, different electrode arrays may not overlap each other along the respective guidance channel. In any embodiment, a plurality of electrode arrays may be separated by a co-culture chamber or other structure along the guidance channel.

[0113] In comparison to electrode spacing within an electrode array, different electrode arrays within a single guidance channel may have a greater spacing between each other. For example, in some embodiments, at least some electrodes within different electrode arrays are spaced apart along a common guidance channel by about: 100 pm - 10 mm, 100 pm - 5 mm, 500 pm - 5 mm, 1mm - 5 mm, or 1mm - 3 mm. [0114] The electrode arrays are generally configured to detect or record action potentials propagating along axons or other cells outgrowing through the one or more guidance channels. Restated, each of the electrode arrays is positioned along one or more of the guidance channels 306a, 306b, 306c such that electrodes thereof are positioned to record electrical signals from said guidance channel, e.g., configured to detect action potentials propagating along axons outgrowing within said guidance channel 306a. In any embodiment, any one or more electrodes of the plurality of electrode arrays may be a recording electrode or a stimulation electrode.

[0115] In FIG. 3B, electrode array 316b is an electrode cluster that includes electrodes 324a, 324b, and 324c, each of which is disposed along guidance channel 306a in proximity to the second end 310 thereof near second seeding chamber 314. The electrodes 324a, 324b, and 324c have a consistent pitch or spacing. Electrode array 316a has an identical arrangement as electrode arrays 316b and is positioned in proximity to the first end 308 of the guidance channel 306a.

[0116] In some embodiments, each electrode array includes 2, 3, 4, at least 3, at least 4, or more electrodes disposed along a common guidance channel. Generally, utilizing a plurality of electrodes in each electrode array enables signal processing techniques that reduce computational errors (such as described with respect to FIG. 11 A - FIG. 1 IB). Accordingly, it is advantageous to utilize one or more electrode arrays over an individual electrode.

[0117] Like guidance channel 306a, each of guidance channels 306b and 306c also have two 3- electrode electrode arrays formed as electrode clusters: one each disposed along the respective guidance channel in proximity to each of the first end 308 and second end 310 thereof. For example, FIG. 3B shows that each of guidance channels 306a, 306b, and 306c have identical 3- electrode arrays disposed at a common distance away from the second seeding chamber 314, wherein the electrodes of the electrode arrays intersect the at least one guidance channel in a perpendicular orientation. Restated, in some embodiments, a first electrode array is positioned at a first distance from a first seeding chamber along a first guidance channel of the plurality of guidance channels, and a second electrode array is positioned at the first distance from the first seeding chamber along a second guidance channel of the plurality of guidance channels.

[0118] It is not necessary for the electrode array(s) to be respectively disposed in proximity to the first end or second end of the respective guidance channels. For example, in some embodiments such as that shown in FIG. 3C - FIG. 3D, each guidance channel includes electrode arrays disposed along the entire guidance channel. [0119] In some embodiments, one or more of the electrodes (e.g., 324a - 324c) is exposed to an interior of the respective guidance channel, e.g., to facilitate sampling of electrical signals from cells growing therein. The conductive properties of the electrodes can be further improved by coating exposed areas thereof with a second material, such as PEDOT. Accordingly, an electrode may have an “exposed” portion coated with a second material. This improvement can be achieved with various methods such as electroplating which can be implemented at any time in the fabrication protocol after the electrode deposition. The surface areas of various electrodes (i.e., the portions of the electrodes disposed along the guidance channel) can be optimally sized for their intended functionality (e.g., recording or stimulating) and can range, generally, from 10 pm² to 1mm². Thus, in any embodiment, at least a portion of one or more of the electrodes (e.g., a portion disposed along the guidance channel) is coated with a conductive material, e.g., PEDOT, to increase signal-to-noise ratio of the recorded signals.

[0120] Additionally or alternatively, in some embodiments, the electrodes (including the traces and portions extending along the guidance channels) may be overlaid with an ion-permeable layer of material (e.g., Nafion®) which allows the electrodes underlying the guidance channels to maintain electrical contact with the cells disposed thereon without being physical exposed to or in direct contact with the cells. Accordingly, in some embodiments, at least a portion of one or more of the electrodes (e.g., a portion disposed along the guidance channel) is covered by an ion-permeable material and optionally coated with a conductive material (e.g., PEDOT).

[0121] The cell processes traversing the guidance channels 306a are directed to extend or migrate across the electrode arrays 316a positioned along the guidance channels 306a in proximity therewith, thereby assuring the ability to measure electrophysiological changes undergoing the cell processes. The microtopographic features (e.g., seeding chambers, guidance channels, and/or co-culture chambers) can be designed to direct and control extensions of the cells over the electrode arrays 316a, 316b (e.g., unidirectional extension), thereby allowing the sequential detection of electrical signals (e.g., action potentials) by the electrode arrays 316a, which can be used to calculate various metrics such as conduction velocity of the cell processes. [0122] The traces of the electrode arrays 316a embedded in the guidance units 304 are routed to buses that terminate in an electrical interface, e.g., comprising contact pads, pins, or the like interfaceable with an external circuitry, such as an electronic instrument as described above with respect to FIG. 2A - FIG. 2C. [0123] Accordingly, the guidance unit 304 (and the devices and systems embodying such guidance units) allows for in situ measurement of conduction metrics, e.g., following a localized application of various conduction perturbations in the one or more co-culture chambers (described below). Furthermore, the one or more electrode arrays (e.g., 316a, 316b) enables more accurate measurements of conduction characteristics that traditional single-MEA electrode patterns cannot achieve.

[0124] The present disclosure thus provides a novel multi-MEA cell culture and analysis system that allows precise, compartmentalized, and multi-site in vitro monitoring of electrophysiological metrics and other biological interactions of various electrogenic cells. Further, the present disclosure presents an approach for spatially directing, stimulating, and quantitatively monitoring the propagation of electrophysiological signals within and between cell assemblies and tissues in vitro in a scalable and repeatable manner, in response to various compartmentalized or localized perturbations.

[0125] In some embodiments (see FIG. 5A - FIG. 5B), the guidance channels 306a traverse through one or more optional co-culture chambers, wherein other cells of various types and forms can be co-cultured biologically interacting with the traversing cell processes and, if desired, chemical compounds or therapeutic agents can be administered. The co-culture chambers enable independent perturbations with different conditions for the same underlying cell processes extending through the guidance channels. For example, supporting cells (e.g., Schwann cells, oligodendrocytes, microglia, astrocytes, etc.) can be selectively placed in the co-culture chamber(s) to enable biological interactions with electrogenic cells (e.g., neuronal axons) residing in the guidance channels. In some embodiments, the co-culture chamber(s) is configured to accommodate localized seeding of myelinating cells or glial cells in proximity to the outgrowing axons residing in the at least one guidance channel.

[0126] In some embodiments having at least one co-culture chamber, at least some electrode arrays of the plurality of electrode arrays are disposed on opposing sides of the at least one coculture chamber.

[0127] Furthermore, other types of cells, chemical compounds or therapeutic agents can be administered in the optional co-culture chamber(s) to induce a localized effect on the conduction or other electrophysiological changes of the cell processes within the guidance channels 306a. The electrode arrays 316a underlying the conducting cell processes are situated along the guidance channels 306a, e.g., either side or both sides (and optionally on or underneath) of the co-culture chambers, and thus are configured to monitor the electrical activity of the cell processes across the guidance channels 306a and measure the effects of co-culture of other cells or other localized perturbations on the propagation of electrophysiological signals.

[0128] In embodiments comprising one or more co-culture chambers (as shown in FIG. 5A - FIG. 5B), the electrogenic cells can be co-cultured with supporting cells (e.g., neurons and myelinating cells) in the co-culture chamber where the topographic features can be further designed to control the spatial distribution of the electrogenic and supporting cells (e.g., myelinating cells can be directed to be only in contact with neuronal axons in the co-culture chambers). In such embodiments, the system incorporating the device of the present disclosure can be used to quantify the effects on conduction of the experimental conditions on the supporting cells as well. In other embodiments, the electrogenic cells can be co-cultured with downstream cells in a separate seeding chamber, to observe the effect of the electrical signal on the downstream cells (e.g., motor neurons forming junctions with myotubes/fibers) in healthy or pathological conditions, or in response to experimental conditions imposed in the co-culture chamber. In other embodiments, the guidance units of the present disclosure can facilitate the interconnection of multiple electrogenic cell populations and enable the observation of electrical communications between such populations on an individual or network level (e.g., multiple interconnected seeding chambers, each featuring neuron populations that collectively form a neuronal network).

[0129] FIG. 3C shows an alternative guidance unit 304 having an identical construction to the guidance unit of FIG. 3A - FIG. 3B, but featuring more numerous electrode arrays disposed along each of the guidance channels (see FIG. 3D), as well as one or more stimulation electrodes 330 respectively disposed in one or more of the seeding chambers 312, 314 in proximity to the first end 308 and/or second end 310 of the guidance channels.

[0130] Such stimulation electrodes 330 are configured interface with the cells/tissues seeded in the seeding chambers and thus can be used as recording electrodes and/or stimulation electrodes to apply an electrical signal to propagate along the guidance channels. Because the electrodes disposed along the guidance channels are generally used to record electrical signals, the plurality of electrode arrays may therefore comprise at least one stimulation electrode and at least two recording electrodes. Restated, the stimulation electrode 330 may be considered part of an additional electrode array. Thus, the at least one additional electrode array may be at least partially disposed in at least one of the seeding chambers. In some embodiments, one or more of the seeding chambers includes a plurality of electrodes disposed thereunder, any number of which may be stimulation electrodes or recording electrodes.

[0131] As shown in FIG. 3D, the construction of guidance unit 304 is very similar to that shown in FIG. 3A. Namely, three linear, parallel, and electrically insulated guidance channels 306a, 306b, and 306c are in communication with seeding chambers 312, 314. Likewise, a plurality of electrode arrays is disposed along each guidance channel, each electrode array having the cluster construction shown in FIG. 3B. However, in comparison to the embodiment of FIG. 3A, the guidance unit 304 of FIG. 3C and FIG. 3D includes numerous electrode arrays evenly spaced apart along each guidance channel. In the illustrated embodiment, each guidance channel is provided with eight electrode arrays. For example, guidance channel 306c is provided with electrode arrays 328a, 328b, 328c, 328d, and so on. Such additional electrode arrays facilitate the sampling of electrical signals propagating along each guidance channel, and computation of electrophysiological properties (e.g., conduction velocity) with finer resolution and lower error.

[0132] FIG. 4A depicts a schematic cross section of the guidance unit 304 taken along the dashed line “4A” in FIG. 3B, showing various layers and topographical features (not to scale). FIG. 4B depicts a three-dimensional cross-sectional perspective view taken along the same section.

[0133] Substrate 301 may include one or more layers of materials, including a mechanically robust layer of material(s) used in cell culture plates, such as polystyrene, polycarbonate, polyethylene terephthalate, or glass. One purpose of the substrate 301 is to host the subsequent, more elaborate layers and topographical features of other materials. Unless otherwise defined, the term “substrate” shall mean a single base layer, e g., a single base layer comprising any of the materials defined above. Optionally, the term “substrate” may be defined as a plurality of substrate layers, each substrate layer comprising one or more of the materials above. The substrate 301 may have a thickness of 1-10000 pm, 1000-10000 pm, 100-1000 pm, 1-100 pm, 1- 50 pm, 1-10 pm, or 1- 5 pm.

[0134] The substrate 301 and all layers thereon are formed in an aligned fashion so that the microtopographic features and the electrodes follow a precise, predetermined alignment pattern. This aligned pattern maintains proper proximity of electrogenic cells to the electrodes situated in a precisely defined manner. In some embodiments, the material(s) of the substrate 301 can be made transparent, e g., for imaging purposes. [0135] The electrodes (e.g., electrode 324a) are disposed on the substrate 301 as an electrode layer of electrically conductive patterns and may constitute any one or more conductive materials, including gold, platinum, titanium oxide, PEDOT, or iridium tin oxide. The traces 326 of the electrodes are routed to the outer side of the substrate 301 and terminate in an electrical interface (e.g., contact pads) adapted for interfacing with external circuitry. The electrode layer includes a layer of the electrode material (e.g., a single layer, such as of gold) and optionally one or more additional layers of electrode enhancing material (e.g., PEDOT). The electrode layer may be defined by its location between, on one side, the substrate 301, and on a second side, at least one of an electrode insulation layer 332, a microtopographic feature layer 334, or a cover 320.

[0136] The electrode insulation layer 332 includes one or more layers is disposed on top of the electrode-bearing substrate (e.g., on top of electrode material 662). Portions of the electrode insulation layer 332 may be selectively removed (as with photolithography) to expose portions of the electrodes for recording or stimulation. Electrode insulation layer 332 may comprise SU8, PDMS, or other suitable electrically insulating layer. In some embodiments, the electrode insulation layer 332 does not include at least one of the seeding chambers, guidance channels, or co-culture chambers formed therein. In some embodiments, the electrode insulation layer 332 includes a single layer of insulating material. In some embodiments, the electrode insulation layer 332 includes a plurality of layers of insulating material (e.g., a plurality of layers of common insulating material which are applied sequentially). The electrode insulation layer 332 may have a thickness of 1-1000 pm, 100-1000 pm, 1-100 pm, 1-50 pm, 1-10 pm, or 1- 5 pm. The electrode insulation layer 332 may be defined by its location between, on one side, at least one of the substrate 301 or the electrode layer, and on the other side, at least one of the microtopographic feature layer 334 or the cover 320.

[0137] The microtopographic feature layer 334 comprising one or more layers of a microtopographic material is disposed on top of the electrode insulation layer 332 and patterned to create the microtopographic features such as at least one of the seeding chambers, guidance channels 306a-c, or co-culture chambers. As shown, the microtopographic features (namely guidance channels 306a-c align with the electrodes 324a. Microtopographic feature layer 334 may comprise a photoresist such as SU8 or other suitable material such as PDMS. In some embodiments, the microtopographic feature layer 334 includes a single layer having one or more recesses or voids defining one or more microtopographic features such as a seeding chamber, guidance channel, and/or co-culture chamber. In some embodiments, the microtopographic feature layer 334 includes a plurality of layers (e.g., having a common material) having one or more recesses or voids defining one or more microtopographic features such as a seeding chamber, guidance channel, and/or co-culture chamber. The microtopographic feature layer 334 may have a thickness of 1-100 pm, 1-50 pm, 1-10 pm, or 1- 5 pm (e.g., 2 pm). The microtopographic feature layer 334 may be defined by its location between, on one side, at least one of the substrate 301, the electrode layer, or the electrode insulation layer 332, and on the other side, the cover 320.

[0138] The cover 320 (optionally removable) is configured to cover at least a portion of the microtopographic features, e.g., at least a portion of at least one of the seeding chambers, guidance channels 306a-c, or co-culture chambers. As with the electrode insulation layer 332 and microtopographic feature layer 334, the cover 320 may comprise PDMS or a photoresist such as SU8, or other material. In some embodiments, the cover 320 does not cover at least an uncovered portion of the at least one guidance channel, such as a portion encompassed by a co-culture chamber.

[0139] Any of the foregoing materials or features of the layers of substrate can be selectively chosen to be hydrophilic, hydrophobic, cell-adhering or cell-repellent. Preferably, any of the electrode insulation layer 332, microtopographic feature layer 334, and cover 320 are biocompatible and inherently suitable for direct cell culture or be modifiable with standard cell culture techniques to allow adherent cell culture. Furthermore, any insulating, microtopography or cover materials can be oxygen-permeable to improve oxygen transfer to the cultured cells/tissues.

[0140] FIG. 5A illustrates a conceptualization of a co-culture chamber model of a guidance unit 502. Neurosphere 521 is seeded in a seeding chamber 512, from which axons 523 extend into guidance channel 506, wherein an electrode array 516a (formed as an electrode cluster that includes electrodes 524a, 524b, and 524c) records action potentials therefrom. A co-culture chamber 525 is provided as an enlargement of the guidance channel 506 which may have an open top, removable cover, or other access means. In other words, co-culture chamber 525 is formed as a recess in the microtopographic layer(s), and having an enlarged channel width as compared to the guidance channel along which it lies. One or more such co-culture chambers may be provided in any guidance unit of the present disclosure along one or more guidance channels. [0141] Myelinated axons 527 extend through the co-culture chamber 525 toward the exclusion channel 529 (here, a Schwann cell exclusion chamber), which allows the axons 523 to continue into the guidance channel 506, where electrode array 516b (namely, electrodes 524d, 524e, and 524f) record action potentials thereof. As shown, electrode arrays 516a, 516b are disposed along the guidance channel 506 on opposing sides of co-culture chamber 525. Of note, guidance channel 506 is also an exclusion chamber.

[0142] FIG. 5B shows an embodiment of a guidance unit 502 identical to that shown in FIG. 3A and FIG. 3B except that it incorporates a co-culture chamber 525, e.g., to drive myelination of the extended axons. Accordingly, all of the terminology and structures previously described with respect to FIGS. 3A-3B are expressly incorporated again here. The co-culture chamber 525 is disposed between electrode arrays 516a, 516b (each of which is formed as an electrode cluster as shown in FIG. 3B); restated, electrode arrays 516a, 516b are disposed on opposing sides of the co-culture chamber 525. Accordingly, the guidance unit 502 depicted in FIG. 5B enables myelination of the central section of the extended axons while excluding Schwann cells from regions of the guidance channels containing electrodes, thereby facilitating accurate detection of propagating action potentials along the guidance channels. Thus, the co-culture chamber in this embodiment serves as the myelination chamber. In some embodiments, at least one additional electrode array may be at least partially disposed in the co-culture chamber 525, e.g., to stimulate and/or record cell populations therein.

[0143] The bilateral electrode arrays shown in FIG. 5A - FIG. 5B (i.e., electrode arrays disposed on opposing sides of the co-culture chamber) characterize the electrical signals that traverse the axonal guidance channels and their propagation. Each electrode array may be used to measure action potential metrics and conduction metrics locally at the location of the arrays of electrodes, and to calculate the metrics of conductions across the co-culture chamber. The segregated nature of the co-culture chamber enables the selective and localized perturbation of the system and the localized observation of the perturbation’s effect on the conduction metrics. Such perturbations include the co-culturing of supporting cells, the co-culturing of different types of supporting cells, the addition of compounds that are potentially cytotoxic to the electrogenic cells or to the supporting cells, or the addition therapeutic agents that target any of the cell types present in the guidance unit of the device. The segregated nature of the co-culture chamber also enables experimenting with the timing of the perturbation relative to the time of seeding in the seeding chambers, or relative to the age of any of the cells in the seeding chambers or that will be seeded in the co-culture chamber.

[0144] In some embodiments, the guidance unit comprises a plurality of co-culture chambers disposed along the one or more guidance channels, wherein each co-culture chamber is isolated from another and is in proximity to a designated set of electrodes. Such an embodiment enables application of different conditions (as described above) to the same guidance channel and the underlying extended/migrating cell processes in a spatially and temporally independent manner and to monitor, independently and locally, the effects of each experimental condition. In some embodiments, the guidance unit 502 comprises a plurality of electrode arrays disposed on each opposing side of the one or more co-culture chambers.

[0145] FIG. 6 describes a process for fabricating representative guidance units of the present disclosure. Structural terms have the meanings previously defined herein. Structural terms recited below have the meanings heretofore assigned unless otherwise stated.

[0146] At step 676, an electrode material 662 is deposited on a clean substrate 660.

[0147] At step 678, an electrically insulating material 664 (i.e., an electrical insulation layer) is deposited on top of the electrode-bearing substrate (e.g., on top of electrode material 662). In some embodiments, the insulating material 664 is deposited in a spatially selective manner to maintain partial exposure of the electrode material 662 on the surface of the substrate 660, e.g., to reveal electrodes 666 at locations corresponding to one or more guidance channels.

[0148] Alternatively, one or more layers of insulating material 664 can be uniformly deposited over the electrode material 662 and then selectively removed to expose the electrode material 662 at predetermined locations, e.g., corresponding to the one or more guidance channels and or seeding chambers. The exposed areas can vary in size depending on the designated function of the corresponding electrode (e.g., stimulation or recording). The locations of the exposed area are also predetermined based on the intended signal propagation path (the guidance channels). The selective exposure of the electrodes can be achieved using conventional photolithography, or by adhering a pre-patterned layer of insulator on the electrode-bearing substrate.

[0149] At step 680, a topographic material 668 (i.e., a microtopography layer) is deposited on top of the insulated substrate and is patterned to create the microtopographic features 670. In this step, the topographic material 668 can be selectively removed to resurface the electrode areas and to form topographic material 668. The microtopographic feature 670 may vary in size and generally comprise three categories: (i) features that form the seeding chambers (ii) features that form the microchannels (guidance channels) between the seeding chambers, and (iii) one or more co-culture chambers disposed along the one or more guidance channels, as described above.

[0150] The microtopographic feature 670 should align with the electrodes, as the guidance channels should cross over the underlying exposed electrodes and direct the cells to come to proximity with the electrodes. As described above, the number of seeding chambers, guidance channels connecting the chambers, the trajectory of these channels, and the number and position of electrodes within or along each channel and in any chamber depend upon, and can be customized or optimized for, a specific application. Exemplary guidance units are shown in FIG. 3 A - FIG. 3B, FIG. 4A - FIG. 4B, and FIG. 5 A - FIG. 5B. However, the processes described herein applies to all configurations.

[0151] In some methods, additional layers and materials are added to the micropatterned substrate to add or enhance functionalities. For example, in some embodiments, it is desirable to increase the height of the walls defining the seeding chambers to minimize the probability of cells escaping the chamber during seeding. Additionally or alternatively, in some embodiments, it is desirable to add a ceiling to the guidance channels to prevent migrating/extending cells from exiting the guidance channels before reaching the other seeding chambers, and/or to electrically shield the guidance channels for higher signal-to-noise ratio of data detected by the electrodes underlying the guidance channels. Thus, the process heretofore described may further include optional step 682, in which a layer of material implemented to form cover alignment features 672. In optional step 684, the cover 674 is then placed on top of and in alignment with the microtopographic features, to complete assembly of the guidance unit.

[0152] The exemplary embodiments of the guidance units shown in FIG. 3A - FIG. 4B show such alignment features on the micropatterned substrate that can be used to accomplish the aligned positioning of the top layer that covers the guidance channels and increases the height of the seeding chambers. In one implementation of the process, some of the layers can be made of a photosensitive material such as SU8 and the selective exposure can be accomplished using photolithography. In another implementation of the process, steps 682, 684 can be omitted, and the cover can be fabricated as part of the microtopography layers in step 680. Additionally, multiple replicas of a guidance unit can be fabricated in parallel on the same substrate to produce a device featuring multiple units (e.g., multi -well plate). The microtopographically patterned substrate can then be attached to the well-structure or lid (as can be seen in FIG. 2A) to form a multi-well device containing multiple guidance units that are fluidically, electrically or, optionally, optically insulated from each other.

[0153] In other embodiments of the present disclosure, the micropatterning processes can be repeated multiple times to create a stack of microtopography maps, each map directing the final location of different cell seedings. For example, a specific area or areas along a portion of guidance channels may be patterned to provide one or more additional seeding chambers or coculture chambers where supporting cells (e.g., Schwann cells, oligodendrocytes, microglia, astrocytes, etc.) can be selectively placed for biological interactions with electrogenic cells (e.g., neuronal axons) residing in the guidance channels.

[0154] As discussed above, the guidance channels can also be designed to exclude the entry of certain supporting cells into the guidance channel. The selective placement and distribution of the supporting cells may be enabled by implementing one or more additional microtopography layers featuring one or more distinct co-culture chambers for the supporting cells and microtopographic features that can direct the supporting cells to migrate towards and into the designated areas of the guidance channels of the underlying layer.

[0155] FIG. 7 shows neurons in a partially open-topped micro guidance channel according to another guidance unit of the present disclosure, as imaged using immunofluorescence, which displays partial neuronal guidance over the open guidance channel. The upper portion of FIG. 7 is a top fluorescence micrograph with axon cytoskeleton stained for Tuj 1 - Green. The vertical short-dashed line shows the edge of the closed portion of the micro guidance channel. The parallel long-dashed lines denote the edges of the covered portion of the micro guidance channel. The lower portion of FIG. 7 is a cross-sectional elevation schematic of the micro guidance channel of the guidance unit, showing each region of the device (not to scale) aligned with the image above. As shown, the covered and uncovered portions of the guidance channel effectively guided directional neuronal extension.

[0156] FIG. 8A - FIG. 8D show the axonal extensions of neurons exiting from a guidance channel of an embodiment of a guidance unit of the present disclosure, as imaged by phase contrast microscopy and immunofluorescence, demonstrating clear axonal guidance and cell body exclusion through the guidance channel. FIG. 8A is a 20x micrograph of sample stained for nuclei with DAPI - blue. FIG. 8B is a 20x micrograph stained for microtubule cytoskeleton, Tuj l - green. FIG. 8C is a 20x micrograph stained for motor neuron specific protein ChAT - red. FIG. 8D depicts an overlay of the 20x micrographs of Tuj 1 - green and ChAT - red with a phase contrast image in grey scale. All scale bars in FIG. 8A - FIG. 8D represent 100pm.

[0157] FIG. 9A shows one embodiment of a device comprising a guidance unit 904, which comprises a microtopography layer 936 formed of PDMS disposed on a multielectrode array 916 (MEA). The guidance unit 904 is disposed at the center of a well 938 of a 6-well plate or lid produced by Axion Biosystems, part no. M384-tMEA-6W.

[0158] The multielectrode array 916 comprises a plurality of electrodes 924, in this example, organized in a 2.1 mm x 2.1mm lattice of 8x8 electrodes. The vertical and horizontal pitch between the electrodes 924 is 300pm.

[0159] The PDMS piece has a plurality of parallel guidance channels 906 formed therein.

Each of the guidance channels 906 has a height (i.e., depth) of 2pm, a channel width of 30pm, and a channel length ranging between 2.5mm and 3mm.

[0160] FIG. 9B illustrates how electrodes 924 disposed along a guidance channel 906 of the guidance unit 904 record electrical signals from cells therein. As shown in the plot on the right side of FIG. 9B, the electrical signals propagating across electrogenic cells in the guidance channels are detected by each electrode underlying that guidance channel 906, allowing sequential detection of the propagating signals.

[0161] FIG. 10A - FIG. 10C show methods of using the guidance unit 904 of FIG. 9A to identify the peak and time interval of electrical signals, here action potentials, transmitted across the multiple electrodes disposed along a guidance channel. Such methods are repeatable and equally advantageous with guidance units of the present disclosure.

[0162] FIG. 10A shows raw data traces of voltages against time derived from multiple sequential electrodes within a single guidance channel. The top plot is for a first electrode, the middle plot is for a second electrode, and the bottom plot is for a third electrode. The first, second, and third electrodes are spaced apart along a guidance channel of a guidance unit of the present disclosure, e.g., that shown in FIG. 9A - FIG. 9B. As shown, action potentials are recorded by the respective electrodes and plotted against time. Action potential peaks are identified (e.g., using a peak finding algorithm; black filled circles denote the peaks). For example, the first-in-time action potential peaks of electrodes are respectively identified as peaks 1040a, 1040b, 1040c.

[0163] FIG. 10B is a schematic representation of the transmitted peak groups. Peaks identified on the first electrode of the electrode array (top plot of FIG. 10A) are examined in relation to the peaks detected from the sequential electrodes (middle and bottom plots of FIG. 10 A) to identify groups of action potential peaks occurring within a given time frame (At). For example, peaks 1040a, b, c are plotted against time. Peaks detected by every electrode in a sequence that are in the correct order and within the determined time frame (At) are classified as a transmitted peak group (identified in the schematic as three sequential peaks with color matched peak marks). For example, peak group 1042 includes the first-in-time peaks 1040a, b, c.

[0164] FIG. 10C depicts the visualization of raw data from a single transmitted peak group, e.g., peak group 1042. One or more peak groups can then be examined to determine the time delay between the first and subsequent peaks. This gives a time for propagation of the action potential traversing a known distance between the electrodes within a guidance channel. Subsequently, the mean time delays, associated conduction velocities, and/or other parameters can be calculated from this derivation.

[0165] The systems of the present disclosure thus include methods and data acquisition hardware accompanied with a software program comprising logic stored as instructions, including the application of various stimulation protocols and the acquisition and processing of data. In particular, the advantages of multiple electrodes arrays, as applied to the signal processing, can be utilized to improve the quality or accuracy of data acquired from the conduction measurements obtained using the devices and guidance units of the present disclosure. The method of acquiring and processing data can be optimized by evaluating various processing patterns and sample rates. This is particularly relevant to improve the accuracy of propagation velocity estimates because different processing patterns can improve or degrade the range of errors in the calculated estimates.

[0166] FIG. 11 A - FIG. 1 IB depicts various data acquisition and processing methods using the devices and guidance units of the present disclosure, i.e., those with a plurality of electrode arrays. Any method described herein which includes sampling electrical signals from the plurality of electrode arrays may be specifically implemented according to any of the following processing methods.

[0167] The following sampling methods are implemented where the propagation of an action potential across two arrays of electrodes is simulated and recorded by the electrodes using different sampling strategies. The sampling across the various paired electrodes in the two electrode arrays, e.g., with staggered sampling between the different pairs, improves the accuracy of conduction velocity measurements (compared to synchronous sampling). Furthermore, the size and relative positions of the electrodes (e.g., distance between the electrodes within an electrode array) and the relative positions of, or the distance between, different electrode arrays in the guidance channels can be varied to improve the performance of the data acquisition and processing methods described herein.

[0168] In FIG. 11A - FIG. 11B, action potentials travelling over the electrode arrays are simulated and their propagations are recorded by the electrodes and processed according to the six processing conditions referred to as Conditions 1 to 6 in FIG. 11 A, which comprise both synchronous techniques (Conditions 1-3) and asynchronous or staggered sampling strategies (Conditions 4-6). The recording is repeated “Number of samples” times and the mean conduction velocity is calculated. In FIG. 1 IB, this experiment is repeated 1000 times and standard deviation of the 1000 means is extracted as the error.

[0169] Returning to FIG. 1 1 A, in this simulation, the electrodes are split into two arrays 1 144, 1146 of three electrodes each disposed along a common guidance channel, as shown in the device of FIG. 3A - FIG. 3B. The electrode pitch within each array 1144, 1146 is 100pm and the pitch between the two arrays 1144, 1146 is 2mm. The sampling frequency is 30kHz, and the sampling period, Ts, is set at 33ms.

[0170] In each of the processing Conditions 1-6, the conduction velocity is independently calculated based on the recordings of each electrode pair (each electrode pair including one electrode from each of arrays 1144, 1146) and then an average conduction velocity is calculated across all pairs (or combinations of pairs). Condition 5 and Condition 6, which combine the staggered sampling and averaging, yields the lowest error at high velocities.

[0171] By building on such evaluation of the processing conditions, the present disclosure provides methods for improving data acquisition and analytics that allows for accurate tracking of fast conduction velocities that certain plain or synchronous techniques (e.g., Condition 1 and 4 in FIG. 11 A - FIG. 1 IB) cannot achieve or track at all, as further shown in FIG. 12.

[0172] Referring to FIG. 12, a key question to be answered in tracking an electrical signal traveling between two electrodes of an electrode pair in embodiments of the present disclosure is: if an electrical signal is propagating at a velocity V (m/s), what is the minimum sample rate S (in kHz) to track the signal between two electrodes of the electrode pair spaced at D (pm)? This question is particularly relevant to the application of the present disclosure because in vivo velocities in myelinated nerves are in the range of 10-120 m/s and the sample rate S in relation to the electrode distance D should be set to enable the accurate measurement of the conduction velocities in the range of at least, and preferably over and beyond, those of the myelinated nerves. It can be assumed that a signal can be tracked if the time it needs to propagate between the electrodes exceeds one sampling period.

[0173] According to FIG. 12, with a sampling rate of less than 12.5 kHz, a signal travelling at a conduction velocity V can be detected serially by two electrodes spaced at D (pm), up to the velocities shown in white. With a sample rate between 12.5 kHz and 30 kHz, the signal can be detected at the conduction velocities up to (or beyond, in certain conditions) those shown in gray. With a sampling rate exceeding 30 kHz (as used in the simulation in FIG. 11 A), the signal can be detected at the conduction velocities up to (or beyond, in certain conditions) those shown in black.

[0174] In view of the foregoing disclosures, embodiments of the present disclosure may be advantageously used to characterize the electrical activity of various electrogenic cells and tissues or to characterize their responses in the electrical activity to external factors, as applied to basic life science research, disease modeling, and therapeutics development, among others. Potential diseases that can be modeled with this device include various forms of neuropathies such as but not limited to multiple sclerosis, spinal muscular atrophy, Charcot-Marie-Tooth syndrome, Guillain-Barre syndrome, diabetic neuropathy, chemotherapy induced neuropathy, amyotrophic lateral sclerosis, Parkinson’s, and Alzheimer’s disease. External factors that can be applied with various embodiments of the present disclosure include the addition of chemical compounds, the inclusion of additional co-cultured cells, the induction of genetic mutations in any of the present cell types, the use of cells/tissues derived from patients, the addition of microbial antigens, the addition of therapeutic agents, the application of electrical or optical stimulation, among others. For example, various embodiments of the present disclosure can be used to investigate the effect of an external factor on the conduction velocity of neurons when the external factor impairs the conduction within neurons or the functionality of surrounding myelinating cells or the integrity of the surrounding myelin layers. Similarly, the present disclosure can be adapted to investigate the effect of conduction changes on downstream cells/tissues, such as skeletal muscle cells, by providing each guidance unit with the chambers featuring neurons interactively connected to the chambers featuring the downstream cells (like skeletal muscles). The present disclosure can also be used to investigate the network properties of multiple clusters of electrogenic cells or model their network functions. For example, each chamber in the device can feature a different type of neurons present in the central or peripheral nervous system, modeling a communication network of such intercellular nervous system.

[0175] As described above, one advantageous application of the devices and guidance units of the present disclosure is for tracking action potential propagations along neuronal axons. Various embodiments implemented according to the present disclosure demonstrate the capability of this device to culture and direct axonal outgrowth through the guidance channels formed in this device, as validated by imaging the axons (FIG. 7 - FIG. 8D). The guidance channels aligned to an underlying electrode array allow the sampling of extracellular field potentials propagating along the electrode arrays. The recordings were processed to extract the peaks of the extracellular field potentials and to calculate the axonal conduction velocity (FIG. 10A - FIG. 10C).

[0176] FIG. 13 shows neuronal conduction velocity over time in FIG. 13, which are represented in line graphs showing means (points) and 95% confidence intervals (error bars) for at least n=50 events for 3 separate guidance channels of the device of FIG. 9A across fifteen days in vitro, demonstrates the utility of the present disclosure in the modeling and measurement of various axonal activities in a highly reproducible and high throughput manner. As shown, the conduction velocities stabilize across time. Such results would be repeatable on devices and guidance units of the present disclosure.

[0177] In application, the devices and guidance units of the present disclosure can be advantageously used for in vitro modeling of neuropathic diseases and in vitro therapeutics screening. For example, FIG. 14A - FIG. 14C show dose-dependent effects of chemotherapeutic agents on the axonal conduction velocities, as measured by the guidance unit of FIG. 9A and which would be repeatable using devices and guidance units of the present disclosure. Here, the device was used to generate dose response curves of the normalized conduction velocities for Vincristine (FIG. 14A), Paclitaxel (FIG. 14B), and Oxaliplatin (FIG. 14C), expressed as 100 equivalent to the conduction velocity for the lowest drug dose and 0 equivalent to the slowest conduction detected across all doses. Points represent mean values (n=4) and error bars represent upper and lower 95% confidence intervals of the mean. Fitted dose response curves were generated with a nonlinear least square fit of a 4-parameter logistic model. These response curves demonstrate the reduction in the axonal conduction velocity in a dose dependent manner to chemotherapeutic agents known to cause peripheral neuropathy, replicating the in vivo effects of these agents on the conduction velocity of axons. [0178] In view of all the advantages of the present disclosure heretofore described, the present disclosure presents guidance units, devices, and systems for spatially directing, stimulating, and quantitatively monitoring the propagation of electrical signals within and between cells, organoids, and tissues in vitro in a scalable and repeatable manner in response to compartmentalized, localized perturbations that can be easily customized for cell biology, drug screening, and therapeutics applications, particularly in the field of various neuronal diseases involving peripheral and central nervous systems. The present disclosure provides a microphysiological system that allows precise, compartmentalized, and multi-site in vitro monitoring of electrophysiological properties detectable in biological interactions of electrogenic cells. In particular, due to its versatility and scalability as a high throughput device, the present disclosure can become an essential tool for researchers engaged in drug discovery and development as well as basic life science research.

[0179] Advantageously, devices of the present disclosure overcome shortcomings of conventional two-dimensional (2D) MEAs which do not effectively and reproducibly model electrical conduction because they are not generally equipped to control directional migration and/or directional extensions of cells. Moreover, monitoring and identifying cell extensions with MEAs is not trivial because cell extensions are random, requiring substantial effort to identify the trajectory of an individual cell extension.

[0180] As another advantage, the structural cues of the seeding chambers, guidance channels, and co-culture chambers may be used to provide spatial separation of cell bodies and cell extension processes interacting therewith to model biological mechanisms involving, and electrophysiological communications among, distinct cell populations and tissues. For example, it could be advantageous to create a plurality of seeding chambers featuring embedded neurospheres and narrow cell-guiding (or communication) channels that can biologically connect to other chambers. The neurospheres may then extend the processes through such narrow guidance channels and form synapses with the neighboring neurospheres, effectively modeling a neuronal network. Similarly, one may use a similar system to model heterotypic junctions and the communications therethrough, such as neuronal cells forming a neuromuscular junction with separate myotubes.

[0181] It is also advantageous to provide an in vitro electrical conduction model that can also simulate and accurately monitor the events occurring in and around the extended processes of the cells. Conduction impairment of the nervous system is often caused by localized malfunctions in or around axons. Such malfunctions may be associated with functional impairments of cells surrounding axons, leading to various diseases involving the central and peripheral nervous system (such as multiple sclerosis, Charcot-Marie-Tooth, Parkinson’s, Alzheimer’s, diabetic neuropathies and cancer-neuropathies). Thus, it is advantageous to provide an in vitro conduction measurement system that can model the extra-axonal milieu which allows one to, locally and selectively, perturb this milieu and locally measure the conduction properties of the extended axons in response thereto. Such extra-axonal milieu may be modeled in the form of the co-culture chambers described herein, which may be in contact with or encompass one or more axon guidance channels of the model and provides the localized inclusion of supporting cells (i.e. Schwann cells, immune cells, oligodendrocytes, astrocytes, microglia, etc.) as part of the extra- axonal milieu.

[0182] In some embodiments, migration of certain cell types through an axon-guiding channel is prevented by appropriately sizing the guidance channel connecting the seeding chambers. Thus, with the use of the segregated chambers and the proper sizing of the guidance channels connecting the chambers, the cell population in a seeding chamber can be advantageously dissociated from the cell population in other seeding chambers as well as from the extra-axonal cell population and the other conditions in a co-culture chamber. In some embodiments, at least one seeding chamber of the plurality of seeding chambers or the at least one co-culture chamber is configured to accommodate dissociated cells, cell organoids, or extracts of tissues.

[0183] It should be noted that various changes can be made to the embodiments of the present disclosure as could be reasonably contemplated in view of the above-described description by any person skilled in the art. The following claims are presented as examples of embodiments of the present disclosure, but these claims should not be construed to limit other claims or other embodiments disclosed herein.

[0184] The detailed description set forth above in connection with the appended drawings, where like numerals reference like elements, are intended as a description of representative embodiments of the present disclosure and are not intended to represent the only embodiments. Each embodiment described m this disclosure is provided as an example or illustration and should not be construed as preferred or advantageous over other embodiments. The illustrative embodiments provided herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Similarly, any steps described herein may be interchangeable with other steps, or combinations of steps, in order to achieve the same or substantially similar result. Further still, one or more features of any embodiment may be combined with one or more features of one or more embodiments to form additional embodiments, which are within the scope of the present disclosure.

[0185] Generally, the embodiments disclosed herein are non-limiting, and the inventors contemplate that other embodiments within the scope of this disclosure may include structures and functionalities from more than one specific embodiment shown in the FIGURES and described in the specification. It will be appreciated that variations and changes may be made by others, and equivalents employed, without departing from the spirit of the present disclosure. Accordingly, it is expressly intended that all such variations, changes, and equivalents fall within the spirit and scope of the present disclosure as claimed. For example, the present disclosure includes additional embodiments having combinations of any one or more features described above with respect to the representative embodiments.

[0186] In the foregoing description, specific details are set forth to provide a thorough understanding of representative embodiments of the present disclosure. It will be apparent to one skilled in the art, however, that the embodiments disclosed herein may be practiced without embodying all the specific details. In some instances, well-known process steps have not been described in detail in order not to unnecessarily obscure various aspects of the present disclosure.

[0187] The present application may include references to directions, such as “first,” "second," "vertical," "horizontal," "front," "rear," "left," "right," "top," and "bottom," “below,” “around,” etc. These references, and other similar references in the present application, are intended to assist in helping describe and understand the particular embodiment (such as when the embodiment is positioned for use) and are not intended to limit the present disclosure to these directions or locations.

[0188] The present application may also reference quantities and numbers. Unless specifically stated, such quantities and numbers are not to be considered restrictive, but exemplary of the possible quantities or numbers associated with the present application. Also in this regard, the present application may use the term "plurality" to reference a quantity or number. In this regard, the term "plurality" means any number that is more than one, for example, two, three, four, five, etc. The term "about," "approximately," etc., means plus or minus 5% of the stated value. The term "based upon" means "based at least partially upon." The term "between" includes the values recited in connection therewith. The expressions “at least one of A, B, or C"; “at least one of A, B, and C"; and “at least one of A, B, and/or C" have the same meaning, i.e., any one of the following conditions satisfy all of the foregoing expressions: A; B; C; AB; AC; BC; ABC.

Claims

CLAIMS What is claimed is:

1. A cell monitoring device, comprising: at least one guidance unit disposed on a substrate, the at least one guidance unit comprising: a plurality of seeding chambers; at least one guidance channel having a first end and a second end, the first end of the at least one guidance channel being in communication with a first seeding chamber of the plurality of seeding chambers and the second end of the at least one guidance channel being in communication with a second seeding chamber of the plurality of seeding chambers; and a co-culture chamber encompassing at least a portion of the at least one guidance channel; a plurality of electrode arrays disposed along the at least one guidance channel; and a cover covering at least a portion of the at least one guidance channel; and an electrical interface in communication with the plurality of electrode arrays.

2. The device of claim 1, wherein the at least one guidance channel comprises a plurality of guidance channels.

3. The device of claim 2, wherein any pair of seeding chambers of the plurality of seeding chambers is connected by at least one guidance channel of the plurality of guidance channels.

4. The device of claim 2, wherein at least one pair of seeding chambers of the plurality of seeding chambers is connected by at least two guidance channels of the plurality of guidance channels.

5. The device of claim 2, wherein different pairs of seeding chambers of the plurality of seeding chambers are connected by at least one different guidance channel of the plurality of guidance channels.

6. The device of claim 2, wherein at least one guidance channel of the plurality of guidance channels intersects with at least one other guidance channel of the plurality of guidance channels.

7. The device of claim 1, wherein a cross-sectional area of at least one guidance channel of the plurality of guidance channels varies over one or more sections thereof.

8. The device of claim 1, wherein the at least one guidance channel comprises a microchannel.

9. The device of claim 8, wherein the at least one guidance channel has a linear trajectory.

10. The device of claim 1, wherein the guidance channel has a cross sectional area of 10 - 120 square pm.

11. The device of claim 1, wherein the guidance channel has a channel width of 20-40 pm and a height of 1-3 pm.

12. The device of claim 11, wherein the guidance channel has a channel length less than 4000 pm.

13. The device of claim 1, wherein a cross-sectional width of the at least one guidance channel is 1 pm - 100 pm, a cross-sectional height of the at least one guidance channel is 1 pm - 100 pm, and a length of the at least one guidance channel is 0. 1 mm - 10 mm.

14. The device of claim 1, wherein at least some electrode arrays of the plurality of electrode arrays are disposed on opposing sides of the at least one co-culture chamber.

15. The device of claim 1, wherein each electrode array of the plurality of electrode arrays comprises at least two electrodes disposed along the at least one guidance channel.

16. The device of claim 15, wherein the at least two electrodes of at least one of the electrode arrays are exposed in the at least one guidance channel.

17. The device of claim 15, wherein the at least two electrodes of at least one of the electrode arrays are disposed below at least one of an ion permeable material or a conductive material along the at least one guidance channel.

18. The device of claim 15, wherein each electrode array of the plurality of electrode arrays comprises at least three electrodes disposed along the at least one guidance channel.

19. The device of claim 1, wherein the at least one guidance channel comprises a plurality of guidance channels, and wherein at least one electrode of the plurality of electrode arrays is disposed along each guidance channel.

20. The device of claim 19, wherein at least two electrode arrays of the plurality of electrode arrays are disposed along each guidance channel.

21. The device of claim 19, wherein at least some guidance channels of the plurality of guidance channels are parallel.

22. The device of claim 19, wherein adjacent guidance channels of the plurality of guidance channels are electrically insulated from each other.

23. The device of claim 19, wherein a first electrode array is positioned at a first distance from a first seeding chamber along a first guidance channel of the plurality of guidance channels, and wherein a second electrode array is positioned at the first distance from the first seeding chamber along a second guidance channel of the plurality of guidance channels.

24. The device of claim 1, wherein each electrode of the plurality of electrode arrays is configured to communicate electrical signals from the at least one guidance channel to the electrical interface.

25. The device of claim 24, wherein adjacent electrodes of at least one electrode array of the plurality of electrode arrays are spaced apart by a pitch of 75-150 pm.

26. The device of claim 25, wherein at least some electrodes within different electrode arrays of the plurality of electrode arrays are spaced apart by 100 pm- 10 mm.

27. The device of claim 1, wherein the plurality of electrode arrays comprises at least one stimulation electrode and at least two recording electrodes.

28. The device of claim 27, wherein the at least one stimulation electrode is positioned in proximity to an end of the at least one guidance channel.

29. The device of claim 1, further comprising at least one additional electrode array at least partially disposed in at least one of the seeding chambers of the plurality of seeding chambers or the at least one co-culture chamber.

30. The device of claim 29, wherein the at least one additional electrode array is configured to detect cells in one seeding chamber of the plurality of seeding chambers or in the at least one coculture chamber.

31. The device of claim 1, wherein the cover does not cover at least an uncovered portion of the at least one guidance channel.

32. A system comprising: a cell monitoring device, comprising: at least one guidance unit disposed on a substrate, the at least one guidance unit comprising: a plurality of seeding chambers; at least one guidance channel having a first end and a second end, the first end of the at least one guidance channel being in communication with a first seeding chamber of the plurality of seeding chambers and the second end of the at least one guidance channel being in communication with a second seeding chamber of the plurality of seeding chambers; a co-culture chamber encompassing at least a portion of the at least one guidance channel; and a plurality of electrode arrays disposed along the at least one guidance channel; and a cover covering at least a portion of the at least one guidance channel; and an electrical interface in communication with the plurality of electrode arrays; and a non-transitory machine readable storage medium storing instructions, which when executed by a processor, performs operations, including: measuring electrical signals from the plurality of electrode arrays.

33. The system of claim 32, further comprising an electronic instrument connectable to the electrical interface.

34. The system of claim 33, wherein the electronic instrument comprises the non-transitory machine readable storage medium and the processor.

35. The system of claim 32, wherein measuring the electrical signals includes measuring electrical signals at a plurality of electrode pairs, wherein each electrode pair comprises an electrode from a first electrode array and an electrode from a second electrode array of the plurality of electrode arrays.

36. The system of claim 35, wherein sampling the electrical signals at the plurality of electrode pairs comprises sampling the electrical signals at different times for different electrode pairs.

37. The system of claim 32, wherein the instructions further include determining a conduction velocity of the electrical signals based upon the sampled electrical signals.