Microphysiological analysis platform (MAP) for organoids / 3D tissue models

Abstract

Various examples include microfluidic devices or a microphysiological analysis platform (MAP) device. One example MAP device includes a set of arrays. An array of the set of arrays includes a set of chambers configured to contain a set of three-dimensional (3D) tissues. A chamber of the set of chambers is configured to contain a 3D tissue of the set of 3D tissues. The array also includes a perfusion system configured to exchange fluid with the set of chambers. The perfusion system facilitates uniform development of the set of 3D tissues. The array additionally includes a set of electrodes. A subset of the set of electrodes is configured to perform non-contact monitoring of electrophysiological signals in the organoid. The example MAP device also includes a flow control component configured to control the flow of fluid through the perfusion system.

Description

MICROPHYSIOLOGICAL ANALYSIS PLATFORM (MAP) FOR ORGANOIDS / 3D TISSUE MODELSRELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 662,239, filed June 20, 2024, the entirety of which is hereby incorporated by reference for all purposes.BACKGROUND

[0002] Three-dimensional (3D) tissue models, such as organoids, are crucial in biomedical research, as they provide more physiologically relevant environments than traditional two-dimensional (2D) cell cultures. They encompass organoids, spheroids, scaffold-based constructs, and bioprinted tissues, which replicate the complexity of native tissues. Organoids mimic organ development and function by self-organizing from stem cells. Spheroids are 3D aggregates used to study cell-cell interactions, metabolic gradients, and tumor behavior. Scaffold-based and bioprinted tissues enhance spatial organization and integrate extracellular matrix components, improving tissue fidelity. These 3D platforms support applications in developmental biology, disease modeling, drug discovery, toxicity assessment, and personalized medicine, offering greater biological accuracy and predictive capacity than conventional models.SUMMARY

[0003] A first example relates to a microfluidic device that includes a chamber configured to contain a three-dimensional (3D) tissue. The microfluidic device also includes a set of perfusion channels configured to transport fluid that facilitates precision control of molecular-level exchanges. The microfluidic device additionally includes a set of permeable barriers that fluidically couple the set of perfusion channels to the chamber. The chamber further includes a set of electrodes configured to perform non-contact monitoring of electrophysiological signals in the 3D tissue.

[0004] A second example relates to a microphysiological analysis platform (MAP) device that includes a set of arrays. An array of the set of arrays includes a set of chambers configured to contain a set of three-dimensional (3D) tissues. Achamber of the set of chambers is configured to contain a 3D tissue of the set of 3D tissues. The array also includes a perfusion system configured to exchange fluid with the set of chambers. The perfusion system facilitates uniform development of the set of 3D tissues. The array additionally includes a set of electrodes. A subset of the set of electrodes is configured to perform non-contact monitoring of electrophysiological signals in the organoid. The MAP device also includes a flow control component configured to control the flow of fluid through the perfusion system.

[0005] A third example relates to a method that includes introducing stem cells into a set of chambers of a microphysiological analysis platform (MAP). The method also includes differentiating the stem cells in the set of chambers into a set of three- dimensional (3D) tissues via fluid exchanged between the set of chambers and a set of perfusion channels. The method additionally includes applying a set of stimuli to the set of 3D tissues. The method further includes monitoring the response of the set of 3D tissues to the set of stimuli. The monitoring includes non-contact monitoring of electrophysiological signals in the set of 3D tissues.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1 illustrates a top view of an example microfluidic device, according to various examples discussed herein.

[0007] FIG. 2A illustrates a perspective view of another example microfluidic device, according to various examples discussed herein.

[0008] FIG. 2B illustrates three cross-sectional views of various stages in the development of an organoid within a microfluidic device.

[0009] FIG. 3 illustrates an example array of microfluidic devices connected by a single perfusion system that includes two perfusion channels.

[0010] FIG. 4 illustrates a cross-sectional view of a portion of an example array showing monitoring of multiple organoids.

[0011] FIG. 5 illustrates a block diagram showing an example microphysiological analysis platform (MAP) that includes two or more arrays of microfluidic devices.

[0012] FIG. 6 illustrates a block diagram of a second example MAP that includes two or more arrays of microfluidic devices.

[0013] FIG. 7 illustrates an exploded view of a specific example MAP similar to the MAP of FIG. 6.

[0014] FIG. 8 illustrates simulations of flow rate and shear stress on organoids in a hemispherical chamber that exchanges fluid with perfusion channels through endothelium-like barriers compared with fluid directly flowing through a chamber.

[0015] FIG. 9 illustrates a comparison between organoids grown according to various examples with fluid exchanged via endothelium-like barriers (top row) compared with organoids grown in a chamber with direct overflow fluid (bottom row).

[0016] FIG. 10 illustrates a comparison between organoid monitoring in a chamber according to various examples with organoid monitoring via a multielectrode array (MEA).

[0017] FIG. 11 illustrates a flow diagram of a method for employing a microfluidic device or MAP according to various examples.DETAILED DESCRIPTION

[0018] Various example systems and methods described herein provide or employ a microphysiological analysis platform (MAP) to culture three-dimensional (3D) tissue (e.g., organoids, spheroids, scaffold-based constructs, bioprinted tissues, etc.) and / or analyze the dynamics (e.g., biological / metabolic, chemical, electrophysiological, etc.) of the 3D tissue (e.g., organoids, etc.) in various scenarios and for various applications. The MAP employed by various examples includes at least one chamber and associated perfusion system(s) (e.g., a single chamber and perfusion system, an array of chambers and associated perfusion system, multiple arrays of chambers with associated perfusion systems, etc.) that facilitate development of 3D tissue (e.g, organoids, etc.) under conditions that maintain a balanced exchange between intrinsic cellular secretions and extrinsic factors delivered through perfusion dynamics that can model in vivo interstitial fluid flow, enabling more consistent and reproducible 3D tissue (e.g., organoid, etc.) generation than existing techniques. Non-contact electrodes of various examples allow for more sensitive monitoring of electrical activity in 3D tissue (e.g., organoid(s), etc.) while also avoiding the biological deviations caused by contact between the organoid and electrode. In various examples, 3D tissue (e.g., organoids, etc.) can be continuously monitored during development and / or in response to various stimuli (e.g., chemical, biological, electromagnetic, optical, thermal, mechanical, or ultrasonic stimuli).

[0019] Various examples can be employed in connection with any of a variety of human and / or non-human 3D tissue (e.g., organoids, spheroids, scaffold-basedconstructs, bioprinted tissues, etc.), including combinations of different 3D tissues (e.g., organoids, spheroids, scaffold-based constructs, bioprinted tissues, etc.) in separate chambers of an array of chambers (e.g., connected via a common perfusion system, etc.) of an example. However, for purposes of illustration, specific examples are discussed in the context of human brain organoids. In other examples, however, similar systems and techniques are employable in connection with other human or non-human 3D tissues (e.g., organoids, spheroids, scaffold-based constructs, bioprinted tissues, etc.).

[0020] Understanding cellular communication within brain organoids is pivotal for decoding brain development and neuropathogenesis. Existing techniques for analyzing organoids rely on non-physiological and invasive methods that disrupt naive cellular processes. Various examples culture organoids MAP for non-invasive electrophysiological readout within physiological fluidic dynamics, mirroring clinical electroencephalography (EEG, for brain organoids) / electrocardiography (EKG / ECG, for cardiac organoids) / electromyography (EMG, for muscle organoids) / electroretinography (ERG, for retinal organoids) / auditory brainstem responses (ABR, for cochlear, brainstem organoids) / etc. Through non-contact electrode integration, various examples enable long-term electrophysiological monitoring of organoids without compromising organogenesis within physiological dynamic environments. Additionally, various examples facilitate monitoring of organoids through multiple developmental stages. For example, in brain-regional modeling, examples capture the evolution of distinct brainwave patterns in midbrain organoids, from early brief bursts to broadband activities. The ability of various examples to administer controlled perturbations and simultaneously monitor responses (e.g., electrophysiological, chemical, optical, mechanical, acoustical, etc.) enables the tracking of pathological shifts in organoids (e.g., in brainwaves, etc.) with unprecedented temporal detail. Various examples facilitate analysis of physiology, pathogenesis, drug discovery, personalized medicine, etc.

[0021] Measuring electrical activity facilitates understanding how organoidbased models function, and for some organoids (e.g., brain organoids, etc.) physiological activities are fundamentally rooted in the electrophysiological signaling among cells. For models that lack visible functional expression, analyzing changes in electrical interactions among their cells provides various insights, such as how the intercellular network communicates, responds to stimuli, and evolves during diseaseprogression. Compared to existing techniques, various examples allow study of innate physiological dynamics through electrophysiological measurement by curtailing tissue disturbance and enabling long-term, longitudinal tracking, facilitating analysis of organoids in different scenarios, including capture of rare, progressive phenomena associated with organoid (e.g., brain, cardiac, pancreatic, muscle, retina, etc.) development and pathology across various time scales.

[0022] Various existing methodologies have notable limitations. Patch clamping provides insights into subcellular processes but can disrupt cell membranes and falls short of explaining cell-to-cell communication across broader networks. Multielectrode array (MEA) techniques, widely adopted to map single-cell activity distributions and infer network connectivity computationally, rely on direct contact between brain cells and rigid, flat electrodes, disrupting tissue environments and architectures. This constraint renders them less suitable for extended longitudinal studies. Moreover, conventional approaches decouple measurement from organoid generation, inflicting electrophysiological off-record periods that may miss transient developmental signals and potentially skew electrophysiological recordings due to organoid transfers and abrupt environmental changes.

[0023] Various examples include or employ a MAP as an integrated system that enables both culturing brain organoids and analyzing them (e.g., via electrophysiological dynamics, etc.) in situ without disturbing organogenesis, from stem cell stages to mature constructs, all within a physiological dynamic environment. The precise fluidic dynamics of various examples facilitate consistent organoid development while the integrated non-contact electrodes continuously capture intact electrophysiological activity (e.g., brainwaves, etc.) stemming from individual organoids without directly interfacing with cells. Prototype examples with midbrain organoids — a brain region implicated in numerous neuropathologies but not yet fully understood electrophysiologically — reveal the capability of these examples to capture distinct, region-specific brainwave patterns with unprecedented temporal detail in early development and pathological alterations, offering insights that are difficult to obtain by other means. Various examples are applicable in a range of scenarios, including developmental biology, drug discovery, and personalized treatment approaches for various conditions.

[0024] FIG. 1 illustrates a top view of an example microfluidic device 100 (e.g., of a MAP, etc.), according to various examples discussed herein. The examplemicrofluidic device 100 includes a chamber 110 configured to contain an organoid 120, a set of perfusion channels (the example device shows two perfusion channels 130 and 132, although other examples can employ one or more than two perfusion channels) that transport fluid (e.g., to cultivate, maintain, stimulate in connection with, etc. the organoid 120, etc.) and are fluidically coupled to (and exchange fluid) with the chamber 110 via permeable barrier(s) (e.g., a set of nanochannels such as example nanochannels 140 and 142 connecting the chamber 110 to the perfusion channels 130 and 132, respectively, etc.) that reduce fluid flow rate in the chamber and curtail stress on the organoid. The example device 100 also includes a set of non-contact electrodes (e.g., example electrodes 150, 152, and 154, etc.) that facilitate non-invasive monitoring of electrical activity within the organoid 120 (e.g., via the fluid, etc.).

[0025] The chamber 110 shown in FIG. 1 has a circular cross-section, and several example chambers discussed herein have a hemispherical shape. In various examples, however, any of a variety of shapes and sizes are used for the chamber 110 (planar, cylindrical, paraboloid, toroidal, hexagonal, etc.), which in various examples is selected to accommodate uniform growth of the organoid 120. In various examples, at least the internal surfaces of the chamber 110 are formed of biocompatible material(s) (e.g., prototypes used a chamber formed from polydimethylsiloxane (PDMS) with a glass ceiling, etc.). In various examples, the chamber 110 accommodates stimulation of the organoid 120 through the walls of the chamber 110 via various techniques (e.g., optical, electrical, magnetic, mechanical, ultrasound, thermal, etc.) and / or measurement of the organoid 120 via various techniques. Measurement of the organoid 120 in various examples includes one or more of electrophysiological monitoring via non-contact electrodes (e.g., the electrodes 150, 152, and 154, etc.), various imaging techniques (e.g., optical techniques such as confocal imaging, calcium imaging, optical coherence tomography, etc.), etc.

[0026] The example device 100 is configured to accommodate a variety of different organoids as the organoid 120. In various examples, the organoid 120 is cultured in the chamber 110 (e.g., of an array of chambers, etc.) by introducing single-cell dissociated pluripotent stem cells into the chambers 110 (e.g., and in some examples, other chambers of an array of chambers, etc.) and employingselected differentiation techniques to guide the stem cells through progressive stages into the selected organoid 120.

[0027] The perfusion channels (e.g., the perfusion channels 130 and 132, etc.) are fluidically coupled with the chamber 1 10 via permeable endothelium-like barrier(s) (e.g., including nanochannels such as nanochannels 140 and 142, etc.) to provide fluid dynamics (e.g., flow rate, etc.) for the organoid 120 that models the in vivo interstitial fluid dynamics of the corresponding organ. Instead of directly providing fluid flow over the organoid 120, which increases stress on and affects the development of the organoid 120, the perfusion channels of various examples indirectly provide molecules to the organoid 120 via the barrier(s), which transport molecules in a manner similar to interstitial fluid. In various examples, the size, number, and other properties of the barrier(s) are selected to model the in vivo conditions of the organ correspond to the organoid 120. In FIG. 1 , the perfusion channels 130 and 132 are coupled to the chamber 110 at a substantially fixed distance by the barriers, around the majority of opposing halves of the circumference (e.g., greater than 90° arcs) of the chamber 110. In other examples, the relative position of the perfusion channels varies, such as to accommodate barrier(s) providing selected fluid dynamics for a given example.

[0028] In various examples, the perfusion channels 130 and 132 are coupled to an inlet reservoir that stores fluid to be provided to the organoid 120 via the barriers and an outlet reservoir that collects fluid after interaction with the organoid 120. Fluid collected in the outlet reservoir contains metabolites, secreted proteins, exosomes, extracellular vesicles, etc. from the organoid that can be analyzed in various examples, for example, to determine response to a potential treatment, drug toxicity, etc. Depending on the specific example, the flow of fluid through the perfusion channels is controlled in any of a variety of ways, such as via gravity by tilting the example device 100 (or a MAP comprising the example device 100, etc.), via pump(s), etc.

[0029] The electrodes (e.g., the electrodes 150, 152, and 154, etc.) of the example device 100 are separated from the organoid by a portion of the chamber 110. Although in various examples discussed herein electrodes are illustrated attached to a surface of the ceiling of the chamber 110 near the organoid 120, in other examples electrodes are at the same and / or other positions (e.g., on other portions of the surface of the chamber 110 not contacting the organoid 120, such aslocations not on the ceiling, etc.). In various examples, the electrodes are arranged to monitor electrical signals within the organoid 120 without physically contacting the organoid 120, to curtail morphological and other changes in the organoid 120 that result from contact.

[0030] In one specific example of the device 100, the chamber 110 is hemispherical with a diameter of around 2 mm, two perfusion channels 130 and 132 have a width of around 200 pm and a height of around 50 pm, and the barriers include nanochannels (e.g., nanochannels 140 and 142, etc.) with a width of around 4 pm and a height of around 2 pm. However, in various examples, the size and arrangement of elements can vary, for example, based on the type of organoid, selected fluid dynamics, etc.

[0031] Referring to FIG. 2A, illustrated is a perspective view of another example microfluidic device 200 (e.g., of a MAP, etc.), according to various examples discussed herein. The example microfluidic device 200 is similar to the example microfluidic device 100 and includes a hemispherical chamber 210 configured to contain an organoid 220, two perfusion channels 230 and 232 connected to the chamber 210 via a set of permeable barriers (e.g., respective sets of nanochannels such as nanochannels 240 and 242, etc.), and electrodes 250, 252, and 254 that facilitate non-contact monitoring of electrical activity within the organoid 120 and are attached to a side of a ceiling of the chamber 210 near the organoid 220 for non-contact monitoring.

[0032] FIG. 2B shows three cross-sectional views 260, 270, and 280 of various stages 222, 224, and 226 in the development of the organoid 220 within the microfluidic device 200. In various examples, organoid(s) (e.g., the organoid 120, the organoid 220, etc.) are cultured in the chamber(s) (e.g., the chamber 1 10, the chamber 210, etc.) of a microfluidic device (e.g., the device 100, the device 200, etc.) and / or a MAP that includes multiple microfluidic devices, allowing for continuous monitoring (e.g., via the electrodes 150, 152, 154, 250, 252, and / or 254, etc.) of the organoid during development of the organoid from stem cells (e.g., the stem cells 222, etc.) to an organoid (e.g., the partially developed organoid 224, the fully developed organoid 226, etc.).

[0033] FIG. 3 illustrates an example array 300 of microfluidic devices (e.g., the device 100, the device 200, etc.) connected by a single perfusion system that includes two perfusion channels (e.g., the perfusion channel 330). The examplearray 300 includes a set of chambers such as chamber 310 (e.g., each of which can be similar to the chamber 110, the chamber 210, etc.) that are each configured to house an organoid during development and / or analysis in connection with one or more use cases (e.g., drug discovery, personalized diagnostics / treatment(s), etc.). Fluid for the organoids in the chambers is provided via perfusion channel(s) (e.g., the perfusion channel 330, etc.) of a perfusion system fl uidically coupled to the chambers via a set of permeable barriers (e.g., sets of nanochannels, etc.) that simulate biological interstitial flow for the organoid, facilitating uniform organoid development. In addition to monitoring visually and / or via imaging of the organoid(s), the array 300 includes a set of electrodes 350 near each organoid for electrophysiological monitoring. The array 300 additionally includes a reference electrode 352. Each of the electrodes 350 and the reference electrode 352 are connected to contact pad(s) for output (e.g., to an amplifier; filter(s) that remove potential interference such as at frequencies of EM stimulation, 60Hz; etc.).

[0034] In various examples, the perfusion channels (e.g., the perfusion channel 330, etc.) facilitate uniformity and reproducibility of the organoids formed within the set of chambers (e.g., the chamber 310, etc.). The perfusion channels in the array 300 are fluid ical ly coupled with (e.g., via barriers, etc.) each of the chambers in a subset of the array sequentially based on a direction of fluid flow (e.g., with the chambers on the left side of FIG. 3 in a first subset and the chambers on the right side of FIG. 3 in a second subset, etc.), for example, with fluid exchanged with the chamber 310 upstream of the fluid exchanged with the chamber below chamber 310, which is upstream of the next chamber for further exchange, etc. Based on various examples and analysis of prototypes, the use of perfusion channels that exchange fluid with a set (or subset) of chambers in sequence was found to result in more uniform and reproducible organoid development than conventional systems or techniques.

[0035] FIG. 4 illustrates a cross-sectional view of a portion of an example array 400 showing monitoring of multiple organoids. In the array 400 organoids are in chambers such as chamber 410 (e.g., similar to the chamber 110, the chamber 210, the chamber 310, etc.), with perfusion channels such as perfusion channel 430 (e.g., similar to the perfusion channels 130, 132, 230, 232, 330, etc.) providing fluid to the chambers. Electrodes such as electrodes 450 are connected to contact pad(s) such as contact pad 452, both of which are exposed at defined openings in acommon passivation layer 460 in array 400, allowing for external interfacing. In the array 400, layer 460 is connected to layer 470 (e.g., via deposition, etc.). In a prototype similar to the example array 400 of FIG. 4, the chambers are formed from PDMS based on a mold created via photolithography, the electrodes were deposited on a glass layer 470 via electron-beam applicator as a 5nm titanium layer adjacent to the layer 470 followed by a 50nm platinum layer, and the layer 460 is a 100 nm SiC>2 layer also deposited on the layer 470. The materials, thicknesses, and topologies of layers can vary between examples (e.g., among biocompatible materials for materials and surfaces that interact with the organoid, via contact or fluid, etc.). Signal(s) from the contact pad(s) are output to an amplifier 480 (e.g., a multi-channel head stage amplifier, etc.) and compared to a reference voltage (e.g., ground, such as from a reference electrode such as reference electrode 352, etc.) and output (e.g., to a display, etc.) to simultaneously monitor electrophysiological activity of a set of organoids.

[0036] Referring to FIG. 5, illustrated is a block diagram showing an example MAP that includes two or more arrays (e.g., array 510 and array 520, etc.) of microfluidic devices (e.g., the example device 100, the example device 200, etc.). The first array 510 (and similarly, the Nth array 520) includes one or more chambers 512 (or similarly, chamber(s) 522, which can be similar to, for example, the chamber 1 10, the chamber 210, etc.) for culturing and / or monitoring organoids, which receive fluid via a perfusion system 514 (or perfusion system 524), which can include an inlet reservoir, perfusion channel(s) (e.g., the perfusion channel(s) 130, 132, 230, 232, etc.) that provide fluid to the chamber(s) 512 (or chamber(s) 522) via permeable barriers (e.g., that include nanochannels such as the nanochannels 140, 142, 240, 242, etc.), and an outlet reservoir. In a given array (e.g., the array 510, the array 520, etc.), different chamber(s) can store the same or different chemical or biological material can be provided to the organoids via the perfusion system (e.g., candidate drugs for drug discovery, etc.). Additionally or alternatively, in various examples the organoids within the chamber(s) can be stimulated via one or more additional stimulation component(s) (e.g., optical, electrical, magnetic, thermal, ultrasound, etc.).

[0037] The MAP 500 facilitates non-contact monitoring of organoids via multiple techniques. For example, the organoids can be monitored via fluid collected from the outlet reservoir(s) of perfusion system(s) and / or via measurementcomponent(s) 518 (or measurement component(s) 528, etc.), which in various examples includes one or more of electrodes (e.g., the electrodes 150, 152, 154, 250, 252, and / or 254, etc.), imaging system(s) (e.g., optical imaging, calcium imaging, optical coherence tomography, etc.), etc.

[0038] In various examples, the flow of fluid through the perfusion system(s) is controlled via one or more flow control component(s) 530, such as tilt system(s) that control flow of one, some, or all arrays via gravity (e.g., by controlling the angle of the array(s) 510 and / or 520 relative to a direction of a gravitational force), pump(s), etc.

[0039] Additionally, in various examples the MAP 500 includes an output component 540 that generates outputs to a user. In various examples, the output component 540 includes one or more of an amplifier, a display, a local or remote memory for storing data, etc.

[0040] FIG. 6 illustrates a block diagram of a second example MAP 600 that includes two or more arrays (e.g., array 610 and array 620, etc.) of microfluidic devices (e.g., the example device 100, the example device 200, etc.). The example MAP 600 is one example of a MAP 500. The MAP 600 includes two or more arrays, of which arrays 610 and 630 are shown, which are similar to the arrays 510 and 520 of FIG. 5. The arrays 610 and 630 include chambers 612 and 632 (e.g., which are similar to the chambers 512 and 522, etc.), the perfusion channel(s) 614 and 634, inlet reservoirs 616 and 636, outlet reservoirs 618 and 638, stimulation component(s) 620 and 640, and electrodes 622 and 642, respectively. The perfusion channel(s) 614 and 634 receive fluid from the inlet reservoirs 616 and 636, respectively, which includes fluid to facilitate growth, maintenance and / or stimulation of organoids in chamber(s) 612 and 632 via permeable barriers between the channel(s) 614 / 634 and the chamber(s) 612 / 632, respectively, and in various examples includes additional biological and / or chemical material to be provided to the organoid(s) (e.g., for drug discovery, toxicity analysis, etc.). The outlet reservoirs 618 and 638 collect fluid (e.g., for analysis, etc.) that has passed through the perfusion channels 614 and 634 after exchanging fluid with the chamber(s) 612 and 632, respectively. Optionally, one or more stimulation (e.g., optical, electrical, magnetic, ultrasound, thermal, etc.) component(s) 620 or 640 are included for individual arrays 610 or 630 or for the example MAP 600 to apply additional stimuli to organoids of the arrays 610 and / or 630. Electrophysiological signals of the organoids in the chamber(s) 612 and 632 are detected via the electrodes 622 and 642, respectively, and provided to one or moreoutput component(s) 660 (e.g., amplifier(s), display(s), memory, etc.). A tilt system 650 is configured to change the angle of the MAP 600 relative to gravity, thereby controlling the flow of fluid from inlet reservoir 616 through perfusion channel(s) 614 (which exchange fluid with chamber(s) 612) to outlet reservoir 618 and from inlet reservoir 636 through perfusion channel(s) 634 (which exchange fluid with chamber(s) 632) to outlet reservoir 638.

[0041] FIG. 7 illustrates an exploded view of a specific example MAP 700 similar to the MAP 600 of FIG. 6. The MAP 700 is assembled from a printed circuit board 710 with a set of pins 712 (e.g., spring loaded pins in various examples, etc.), a fluidic layer 720 (e.g., formed of PDMS, etc.) that includes one or more (e.g., eight are shown in FIG. 7) arrays of organoid chambers 722 and associated perfusion channels 724, an electrode layer 730 that includes electrodes 732 for non-contact monitoring of organoids and contact pads 734 that electrically align with the pins 712, and a reservoir layer that includes inlet reservoirs 742 and outlet reservoirs 744 that fluidically align with the perfusion channels 724 to form perfusion systems that provide fluid to the organoids in the chambers 722.

[0042] Many existing electrophysiological methods (e.g., multielectrode array (MEA) techniques, etc.) disrupt cellular conditions and have prolonged off-record periods during which data is not obtainable. In contrast, various examples employ a microfluidic device and / or a MAP comprising a set of microfluidic devices, providing an integrated system that facilitates the simultaneous culturing and electrophysiological monitoring of organoids under physiologically advanced conditions. Various examples include an integrated chamber (e.g., the chamber 100, the chamber 200, etc.) with non-contact electrodes (e.g., the electrodes 150, 152, and 154, the electrodes 250, 252, 254, etc.), configured to support long-term electrophysiological assessment of individually cultured organoids (e.g., the organoid 120, the organoids 220, 224, and / or 226, stem cells 222, etc.). The geometry of the chamber of various examples stabilizes the organoid, enabling continuous monitoring (e.g., visual and electrophysiological tracking, etc.) from micrometer-scale spheroids to millimeter-sized brain organoids over extended periods. Similarly to clinical EEG / EKG / EMG, the non-contact electrodes of various examples capture tissue-wide electrophysiological signatures generated by the organoid while avoiding foreign material interference and preserving natural biological progression. The non- disruptive design of various examples enables prolonged monitoring of individualorganoids, allowing for capture of data related to the emergence, evolution, and pathological transformation of organoid (e.g., brain network, etc.) dynamics.

[0043] The fluidic design of various examples provides a physiologically relevant dynamic environment that facilitates robust organoid growth, electrophysiological activity (e.g., neural activity, cardiac activity, pancreatic activity, or activity of any organoids, etc.), and continuous monitoring. Integrated perfusion channels (e.g., perfusion channel(s) 130, 132, 230, 232, etc.) and endothelium-like barriers (e.g., that include nanochannel(s) such as nanochannel(s) 140, 142, 240, 242, etc.) deliver media (e.g., fluid(s)) in a controlled, dynamic manner, preserving cellular mechanics and communication. FIG. 8 shows simulations of flow rate and shear stress on organoids in a hemispherical chamber that exchanges fluid with perfusion channels through endothelium-like barriers (the left and center images, respectively) compared with fluid directly flowing through a chamber (right images). In the left image of FIG. 8, the flow rate throughout the chamber remains below 0.1 mm / s. As can be seen in the range of stresses in the scales accompanying the center and right images, the maximum stress seen in various examples (where all stresses shown are at or below 1 .0x10-5dyne / cm2) is substantially lower than the stresses on almost the entirety of the organoid in the chamber with direct fluid flow (where the majority of the stresses shown exceed 9x10-5dyne / cm2).

[0044] Unlike conventional dish cultures or direct-flow systems that disrupt secretory signaling, various examples maintain a balanced exchange between intrinsic cellular secretions and extrinsic factors delivered through perfusion dynamics, enabling more consistent and reproducible organoid generation. FIG. 9 shows a comparison between organoids grown according to various examples with fluid exchanged via endothelium-like barriers (top row) compared with organoids grown in a chamber with direct overflow fluid (bottom row). As can be seen in FIG. 9, various examples provide uniform organoid growth, in contrast to the irregular growth of direct flow systems. FIG. 10 shows a comparison between organoid monitoring in a chamber according to various examples (at 1000) with organoid monitoring via a multielectrode array (MEA, at 1010). As can be seen, the contact with electrodes in MEA systems causes developmental deviations observed in the MEA-plated organoids, in terms of both morphology and cell composition. For example, analysis of midbrain organoids tested on MEA systems showed accumulation of electrophysiologically less active astrocytes on the electrode surface, disrupting thespatial propagation of electrical signals and hindering transmission to the recording sensor from more electrophysiologically active neurons situated farther from the electrode. The direct formation of stem cell aggregation and on-chip differentiation via the MAP of various examples facilitates in-situ electrophysiology of network activities of an organoid (e.g., a brain construct, etc.) without disturbing tissue architecture. In contrast, the MEA approach misses early-stage electrophysiological development because organoids are initially cultivated on a non-electrophysiological platform until they reach early stages. Additionally, the MEA approach can have less sensitive readouts of population activity due to the influence of the electrodes on cell composition, such as abnormal astrocyte accumulation due to contact between the organoid and the rigid surface. The time-lapse signals shown below 1000 and 1010 represent example measurements from comparisons between an MEA approach and example prototypes.

[0045] The scalability of various examples supports high-throughput analysis within a compact platform (e.g., one example prototype is 12.5 cm x 6.5 cm, accommodating 80 organoids across eight arrays, allowing for eight independent perfusion dynamics, etc.). Various examples demonstrate statistically powered, physiologically advanced organoid electrophysiology achieved through simultaneous longitudinal recordings of the complete organoid activities via a large-scale, embedded, non-contact electrode array. In various examples, seamless fluidic dynamics are ensured through a pump-based system or a custom tilting system that eliminates complications associated with conventional tubing and pumps.

[0046] Various examples facilitate the cultivation and / or analysis of organoids in connection with a range of scenarios, including modeling developmental biology, drug screening and discovery, precision medicine based on organoids cultured from a specific patient, and drug toxicity testing. As specific examples, prototypes were employed for culturing and studying brain organoids in connection with organogenesis and neurotoxin-induced pathology. While specific examples involved human brain organoids, these examples are applicable to any human and nonhuman organoids or three-dimensional tissues.

[0047] In a first example use case, a prototype example MAP was employed to track (e.g., electrophysiologically, chemically / metabolically, etc.) brainwave development during brain organogenesis. Neural network activity is a fundamental hallmark of brain development, yet its temporal progression is difficult to monitor viaconventional techniques. In the first example use case, brain organoids were differentiated directly on-chip from pluripotent stem cell aggregates within a MAP to monitor the progression of neural activity. The perfusion system allowed for differentiation into selected organoids (e.g., midbrain organoids in the first example use case) using fluidic controls for molecular exchanges between the perfusion channels (e.g., perfusion channel(s) 130, 132, 230, 232, etc.) and chambers (e.g., chamber(s) 110, 210, etc.). The outlet reservoir(s) (e.g., outlet reservoir(s) 618, 638, etc.) and non-contact electrodes (e.g., electrode(s) 150, 152, 154, 250, 252, 254, etc.) - as well as various imaging techniques such as confocal imaging, calcium imaging, optical coherence tomography, etc. - allowed for the ability to monitor the organoids throughout development and in response to various stimuli without the organoid changes induced by contact-based techniques such as MEA.

[0048] In the second example use case, a prototype MAP was employed to monitor mature midbrain organoids throughout exposure to a neurotoxin, modeling Parkinson's disease. Mature midbrain organoids in a prototype MAP example were exposed to 1 -methyl-4-phenylpyridinium (MPP+), a well-established neurotoxin widely used to model Parkinsonism in vivo and in vitro, to monitor pathological features of Parkinsonism, including dopaminergic neuronal loss and complex disruptions in neural network dynamics. Low-dose (10 pM) MPP+was chronically administered to the mature organoids via perfusion, and activity changes were continuously monitored.

[0049] The non-invasive, non-contact readout, enabled by electrodes of various examples, facilitates the continuous capture of faithful biological signals, including brainwaves, over extended periods. Prototype examples were examined in a direct, head-to-head comparison between MAP and a conventional contact-based electrophysiological system (MEA) to validate the advantage of non-contact readout, evaluating a range of developmental and functional parameters. Two parallel cohorts of midbrain organoids were examined: one continuously cultured and monitored by a prototype MAP example from pluripotency through maturation (e.g., as shown in FIG. 10 at 1000), and the other transferred to MEA at the neural progenitor stage to capture early electrophysiological activity using the contact-based readout (e.g., as shown in FIG. 10 at 1010). MAP reliably tracked the evolving patterns of neural activity, revealing a continuous trajectory toward complex brainwave dynamics as shown in FIG. 10 at 1000. In contrast, MEA recordings failed to capture the fullelectrophysiological progression as shown in FIG. 10 at 1010. While higher- amplitude signals were initially observed — likely due to closer proximity between the electrodes and the tissue — the signals diminished during further development, resulting in reduced signal quality and loss of midbrain-specific electrophysiological features expected at later stages.

[0050] MAP’s superior monitoring fidelity was reinforced by preserved tissue morphology and developmental consistency. Organoids cultured in MAP maintained their 3D architecture, exhibited consistent developmental trajectories with high interorganoid reproducibility (e.g., as seen in the top row of FIG. 9). Continuous perfusion and electrical operation did not perturb biological development, as evidenced by similarity to control groups lacking perfusion or electrical monitoring, affirming the non-disruptive nature of various examples. In contrast, organoids cultured on MEAs developed structural abnormalities, including partial flattening and aberrant cellular outgrowths, which likely compromised the native network organization. These morphological distortions were accompanied by altered expression of midbrainspecific genes and increased variability across organoids. As a result, electrophysiological signals captured by MEA, though superficially midbrain-like, may lack developmental authenticity and statistical robustness. In contrast, various examples employing microfluidic devices (e.g., MAP examples, etc.) accurately capture signals from unaltered organoids.

[0051] Various examples also provide the ability to detect functional responses to dopaminergic stimuli with high fidelity, in contrast to conventional systems. The non-contact electrodes of various examples maintain stable electrical baselines and sustain sensitivity to non-contact signals over extended periods without fouling or degradation. This enabled MAP to consistently capture robust and reproducible responses to both L-DOPA and MPP+in midbrain organoids. In contrast, contact-based MEA recording failed to reliably detect such responses, reflecting their intrinsic insensitivity to non-contact signals. While dopaminergic neurons — the primary targets of these agents — were present in MEA-grown organoids, poor signal acquisition was likely due to their limited proximity to electrodes and the accumulation of astrocytes along the recording interface.

[0052] Thus, various examples provide multiple advantages over conventional electrophysiological platforms, enabling more accurate modeling and monitoring of organoid development and disease-related dynamics.

[0053] Various examples include and / or employ an advanced microphysiological platform that enables comprehensive, real-time, non-invasive electrophysiological monitoring of organoids, including developmental and pathological brainwave genesis on-chip. Various examples support the entire organogenesis from stem cells to mature organoids within a physiological environment, maintaining architectural integrity while enabling long-term longitudinal tracking (e.g., optical, electrophysiological, etc.). The non-contact electrodes of some examples mimic an EEG configuration, providing uncompromised readouts of tissuewide electrophysiological dynamics, revealing network communication within organoid circuitry. Various examples enhance electrophysiological readout by avoiding electrode-organoid contact, which can otherwise lead to biological deviations and unstable readouts. Additionally, various examples curtail off-record periods inherent to conventional methods, such as differentiation phases in non- electrophysiological setups or delays in electrode contact acclimation, allowing various examples to detect rare but vital electrophysiological events.

[0054] Prototype MAP examples detected changes in brainwaves that are not detectable via conventional systems. Monitoring of organoid electrophysiology via various examples can provide insights into early, disease-relevant neural alterations, highlighting its potential to uncover biomarkers of early-stage dysfunction for therapeutic intervention.

[0055] Various examples provide multiple capabilities lacking in conventional systems, for example, enabling (1 ) intact brain organogenesis on-chip for highly consistent organoid modeling — from stem cells to uniform organoid arrays — through precise control of biophysical fluid dynamics; (2) long-term longitudinal monitoring of electrophysiological activity from early embryonic stages, offering insights into electrophysiological development (e.g., neurophysiological brainwave genesis, etc.); and (3) real-time exploration of pathogenesis in associated organs (e.g., neuropathogenesis, etc.), capturing electrophysiological (e.g., brainwave, etc.) dynamics with unprecedented temporal details. Additionally, various examples offer further advantages over conventional systems, including the ability to address common challenges associated with organoid models, such as standardization, lack of vascularization, and scalability. Various examples have diverse applications, including facilitating organoid research by enabling comprehensive studies of organoid genesis, pathogenesis, and pharmacology, among others.

[0056] In view of the foregoing structural and functional features described above, example methods will be better appreciated with reference to FIG. 11 . While, for purposes of simplicity of explanation, the example methods of FIG. 11 are shown and described as executing serially, it is to be understood and appreciated that the illustrated order does not limit the present examples, as some actions could in other examples occur in different orders, multiple times and / or concurrently from that shown and described herein. Moreover, it is not necessary that all described actions be performed to implement a method.

[0057] Referring to FIG. 11 , illustrated is a flow diagram of a method 1100 for employing a microfluidic device or MAP according to various examples. In other examples, the blocks of the example method 1100 are a set of machine-readable instructions on a non-transitory machine-readable medium or are a set of operations performed by a processor executing machine-readable instructions as the operations. Method 1100 provides one specific example involving organoids, but other examples employ similar steps in connection with other 3D tissues (e.g. spheroids, scaffold-based constructs, bioprinted tissues, etc.).

[0058] At block 1 110, method 1100 includes introducing stem cells, primary cells, cell lines, etc, into one or more chambers (e.g., a single chamber of a microfluidic device, one or more chambers of a MAP, etc.). In various examples, the selected stem cells vary, including based on a selected application. For example, in personalized medicine applications, the stem cells can be those of a selected individual or primary cells.

[0059] At block 1 120, method 1100 includes differentiating the stem cells into one or more selected organoids via fluid exchange between the chambers and associated perfusion channels. The specific differentiation protocol(s) employed (e.g., sequence(s) of chemicals provided to the organoid via the fluid, etc.) varies based on the selected organoid(s). In various examples, the selected organoid(s) are of one type (e.g., corresponding to the same organ, etc.) or include organoids of one or more types.

[0060] At block 1 130, method 1100 includes applying one or more selected stimuli to the organoid(s), which, in various examples, depends on the scenario, such as developmental biology with genetic defects, drug discovery, testing potential treatments, or assessing drug toxicity, etc.. In various examples, the one or more stimuli are one or more of materials (e.g., chemical, biological, etc.) added to thefluid exchanged with the chamber(s), electrical, magnetic, mechanical / ultrasound, thermal stimuli, etc.

[0061] At block 1 140, the method 1100 includes monitoring the response of the organoid(s) to one or more stimuli. In various examples, the monitoring includes one or more of electrophysiological monitoring via non-contact electrodes, chemical / biological monitoring based on analysis of fluid collected after exchange with the chamber(s), imaging (e.g., optical imaging, calcium imaging, optical coherence tomography, etc.).

[0062] What has been described above are examples. It is, of course, not possible to describe every conceivable combination of components or methodologies, but one of ordinary skill in the art will recognize that many further combinations and permutations are possible. Accordingly, the disclosure is intended to embrace all such alterations, modifications, and variations that fall within the scope of this application, including the appended claims. As used herein, the term "includes" means includes but not limited to, the term "including" means including but not limited to. The term "based on" means based at least in part on. Also as used herein, the term "set" means one or more elements (e.g., where the elements can be anything, such as datasets, nodes, relationships, etc.), and a “subset” of a set A refers to any set B where every element of set B is an element of set A (note that every set A is a subset of itself, as every element of set A is an element of set A). Additionally, where the disclosure or claims recite "a," "an," "a first," or "another" element, or the equivalent thereof, it should be interpreted to include one or more such elements, neither requiring nor excluding two or more such elements.

[0063] In this description, unless otherwise stated, "about," "approximately" or "substantially" preceding a parameter means being within + / - 10 percent of that parameter. Modifications are possible in the described embodiments, and other embodiments are possible, within the scope of the claims.

Claims

CLAIMSWhat is claimed is:1 . A microfluidic device, comprising: a chamber configured to contain a three-dimensional (3D) tissue; a set of perfusion channels configured to transport fluid that facilitates precision control of molecular-level exchanges; a set of permeable barriers that f luid ically couple the set of perfusion channels to the chamber; and a set of electrodes configured to perform non-contact monitoring of electrophysiological signals in the 3D tissue.

2. The microfluidic device of claim 1 , wherein a permeable barrier of the set of permeable barriers comprises a set of nanochannels that fluid ically couple a perfusion channel of the set of perfusion channels to the chamber.

3. The microfluidic device of claim 1 , wherein the set of perfusion channels is coupled to an inlet reservoir configured to contain the fluid prior to exchange with the chamber.

4. The microfluidic device of claim 1 , wherein the set of perfusion channels is coupled to an outlet reservoir configured to contain the fluid after exchange with the chamber.

5. The microfluidic device of claim 1 , wherein a flow control component controls the flow of fluid through the perfusion channels to facilitate the precision control of the molecular-level exchanges.

6. The microfluidic device of claim 5, wherein the flow control component comprises a tilt system configured to control an angle of the device relative to a gravitational force.

7. The microfluidic device of claim 5, wherein the flow control component comprises at least one pump.

8. The microfluidic device of claim 1 , wherein the chamber has a circular crosssection.

9. A microphysiological analysis platform (MAP) device, comprising: a set of arrays, wherein an array of the set of arrays comprises: a set of chambers configured to contain a set of three-dimensional (3D) tissues, wherein a chamber of the set of chambers is configured to contain a 3D tissue of the set of 3D tissues; a perfusion system configured to exchange fluid with the set of chambers, wherein the perfusion system facilitates uniform development of the set of 3D tissues; and a set of electrodes, wherein a subset of the set of electrodes is configured to perform non-contact monitoring of electrophysiological signals in the 3D tissue; and a flow control component configured to control the flow of fluid through the perfusion system.

10. The MAP device of claim 9, wherein the perfusion system comprises: a set of perfusion channels configured to transport the fluid; and a set of permeable barriers that f luid ically couple the set of perfusion channels to the set of chambers, wherein a subset of the set of permeable barriers fluidically couples the set of perfusion channels to the chamber.11 . The MAP device of claim 10, wherein a permeable barrier of the subset of the set of permeable barriers comprises a set of nanochannels that fluidically couple a perfusion channel of the set of perfusion channels to the chamber.

12. The MAP device of claim 10, wherein the perfusion system comprises an inlet reservoir coupled to the set of perfusion channels, wherein the inlet reservoir is configured to contain the fluid prior to exchange with the set of chambers.

13. The MAP device of claim 10, wherein the perfusion system comprises an outlet reservoir coupled to the set of perfusion channels, wherein the outlet reservoir is configured to contain the fluid after exchange with the set of chambers.

14. The MAP device of claim 9, further comprising a flow control component configured to control the flow of fluid through the perfusion system.

15. The MAP device of claim 14, wherein the flow control component comprises a tilt system configured to control an angle of the device relative to a gravitational force.

16. The MAP device of claim 14, wherein the flow control component comprises at least one pump.

17. The MAP device of claim 9, wherein the chamber has a circular cross-section.

18. The MAP device of claim 9, further comprising a stimulation component configured to apply at least one of an electromagnetic stimulus, an optical stimulus, a thermal stimulus, a mechanical stimulus, or an ultrasonic stimulus to the 3D tissue.

19. The MAP device of claim 9, further comprising an amplifier configured to amplify signals from the set of electrodes.

20. A method, comprising: introducing stem cells into a set of chambers of a microphysiological analysis platform (MAP); differentiating the stem cells in the set of chambers into a set of three- dimensional (3D) tissues via fluid exchange between the set of chambers and a set of perfusion channels that facilitate uniform development of the set of 3D tissues; applying a set of stimuli to the set of 3D tissues; monitoring the response of the set of 3D tissues to the set of stimuli, wherein the monitoring comprises non-contact monitoring of electrophysiological signals in the set of 3D tissues.

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