Non-destructive, long-term electrophysiological recording and stimulation of organoids

Abstract

The present disclosure is related to non-destructive, long-term electrophysiological recording and stimulation of organoids. For example, an example device includes biocompatible material and electrodes fabricated onto the biocompatible material. The example device, through controlled buckling and microfolding, forms a pocket for an organoid.

Description

NON-DESTRUCTIVE, LONG-TERM ELECTROPHYSIOLOGICAL RECORDINGAND STIMULATION OF ORGANOIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of United States Provisional Patent Application No. 63 / 730,036, filed December 10, 2024, and United States Provisional Patent Application No. 63 / 762,353, filed February 24, 2025, the entire contents of which are incorporated by reference in their entirety.BACKGROUND OF THE INVENTION

[0002] There are many technical challenges and difficulties associated with electrophysiological recording and stimulation of organoids.SUMMARY OF THE INVENTION

[0003] In accordance with various embodiments of the present disclosure, a device is provided. In some embodiments, the device comprises biocompatible material and electrodes fabricated onto the biocompatible material. In some embodiments, the device, through controlled buckling and microfolding, forms a pocket for an organoid.

[0004] In some embodiments, the device further comprises a platinum coating on the electrodes.

[0005] In some embodiments, the electrodes record electrical activity in the organoid.

[0006] In some embodiments, the electrodes stimulate electrical activity in the organoid.

[0007] In some embodiments, the electrodes capture spiking events associated with living neurons.

[0008] In some embodiments, the device further comprises petals.

[0009] In some embodiments, the device further comprises a silicone substrate with a hole.

[0010] In some embodiments, the petals are inserted through the hole of the silicone substrate to form the pocket.

[0011] In some embodiments, the biocompatible material comprises biocompatible polymer.

[0012] In some embodiments, the biocompatible polymer comprises parylene.

[0013] In some embodiments, the parylene is associated with a thickness of 8 pm.

[0014] In some embodiments, the electrodes comprise sensing electrodes.

[0015] In some embodiments, the sensing electrodes comprise gold nanomembranes.

[0016] In some embodiments, the gold nanomembranes are associated with a thickness of 200 nm.

[0017] In some embodiments, the gold nanomembranes functionalized with Platinum (Pt) Black or Pt are embedded in biocompatible polymer.

[0018] In some embodiments, the electrodes are associated with a density of approximately 40 electrodes / mm2.

[0019] In some embodiments, the electrodes are associated with a density of approximately 70 electrodes / mm2.

[0020] In some embodiments, the electrodes are associated with a density of more than 100 electrodes / mm2.

[0021] In some embodiments, the device further comprises an encapsulation layer, a Pt-Black layer, a gold layer, a chrome layer, and a substrate layer.

[0022] In some embodiments, the gold layer is between the encapsulation layer and the chrome layer.

[0023] In some embodiments, the chrome layer is between the gold layer and the substrate layer.

[0024] The foregoing illustrative summary, as well as other exemplary objectives and / or advantages of the disclosure, and the manner in which the same are accomplished, are further explained in the following detailed description and its accompanying drawings.BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1A to FIG. 1C provide schematic illustration highlighting the difference in interfacial coverage of electrical measurements for 2D MEA (as shown in FIG. 1A), microneedle electrodes (as shown in FIG. IB), and HotPocket (as shown in FIG. 1C). Compositional heterogeneity of organoids necessitates a conformal coverage of electrical measurement, which is offered by the HotPocket.

[0026] FIG. 2A and FIG. 2B provide representative platforms for organoid recording.

[0027] FIG. 2A shows surface recording mesh based on rose-petal electronics, including the half-sphere coverage that leads to a slight deformation of the organoid and the penetration of the electrode into the organoid that precludes the device reusability.

[0028] FIG. 2B shows the embedded recording mesh, illustrating that the embedded mesh can only record internal neurons that are generally less active than the surface counterparts, and that it does not allow reuse.

[0029] FIG. 3 illustrates immunohistochemical analysis of brain organoids including DAPI (for labeling all nuclei), PAX6 (for labeling radial glia / progenitor cells), and CTIP2 (for labeling deep-layer excitatory neurons).

[0030] FIG. 4 provides an overview of some features described in the present disclosure.

[0031] FIG. 5A to FIG. 5D provide design and preliminary tests of the HotPocket. Brightfield image (as shown in FIG. 5A) and fluorescent image (as shown in FIG. 5B) of the HotPocket (reference number 501 in FIG. 5B) holding an organoid (reference number 503 in FIG. 5B) transduced with a virus expressing GFP viewed from the bottom of the device. FIG. 5C and FIG. 5D illustrate the pocket opening viewed from the top of the device, where the organoid is inserted into the device using a standard wide-bore pipette tip (FIG. 5C illustrates a view prior to organoid insertion and FIG. 5D illustrates a view after organoid insertion).

[0032] FIG. 6A to FIG. 61 provide representative field potentials recorded from a HotPocket showing glutamate-sensitive activity in a cortical organoid. Temporal plot (as shown in FIG. 6A, FIG. 6D, and FIG. 6G), raster plot (as shown in FIG. 6B, FIG. 6E, and FIG. 6H), and representative waveforms (as shown in FIG. 6C, FIG. 6F, and FIG. 61) of the brain organoid at baseline (as shown in FIG. 6A to FIG. 6C), upon addition of 40 pM Glutamate (as shown in FIG. 6D to FIG. 6F), and upon addition of 40 pM TTX (as shown in FIG. 6G to FIG. 61), respectively. Data are gathered from an organoid treated with advanced maturation compounds. A raster plot illustrating 3 minutes of baseline activity (as shown in FIG. 6B and FIG. 6C), 40 uM glutamate (as shown in FIG. 6E and FIG. 6F), 2 uM TTX (as shown in FIG. 6H and FIG. 61). The waveform following each raster plot shows the average waveform (reference numbers 601, 603, 605, 607, 609, and 611), and individual spikes (reference numbers 602, 604, 606, 608, 610, and 612).

[0033] FIG. 6J illustrates raw data for a few channels under 20 uM glutamate simulation.

[0034] FIG. 7A to FIG. 7E illustrates fabricated compliant networks of HotPocket.

[0035] FIG. 7A provides a schematic illustration with exploded view of HotPocket pattern and cross-sectional view of the device’s layered structure.

[0036] FIG. 7B to FIG. 7E provide optical images of HotPocket showing its material flexibility (as shown in FIG. 7B and FIG. 7C) or planar representations to visualize the electrodes (as shown in FIG. 7D and FIG. 7E).

[0037] FIG. 7E and FIG. 7F further illustrates schematics of the device, which may feature 63 recording electrodes, 2 reference electrodes, and 9 petals designed to support and secure the organoid.

[0038] FIG. 7G illustrates that a small section of the electrode is exposed.

[0039] FIG. 7H illustrates impedance based on electrode coatings.

[0040] FIG. 8A to FIG. 8D illustrate mechanical simulation on strain distribution of HotPockets. FIG. 8A illustrates a 2D layout, while FIG. 8B to FIG. 8D illustrate pre-stretch at 75% (as shown in FIG. 8B), 100% (as shown in FIG. 8C), and 125% (as shown in FIG. 8D), respectively.

[0041] FIG. 9A provides conceptual illustration of the 3D rose-petal strategy to create microelectronic 3D HotPockets. FIG. 9A illustrates that the device is made of a stretchable elastomer which enhances electrode-organoid contact upon closure.

[0042] FIG. 9B illustrates a photograph of the device in a biosafety cabinet, with silicone padding beneath the plate to maintain the organoid’s temperature at approximately 37°C.

[0043] FIG. 9C is a microscopic image showing the folded 3D structure.

[0044] FIG. 10 provides representative images of side view and bottom view of HotPockets that can actively morph the pocket size to softly hold brain organoids with various sizes ranging from 1 mm to 2 mm in radius as labeled on top of the images. FIG. 10 further illustrates that the device can hold organoids between 2 to 3.4 mm in diameter, consistent with multiple differentiation protocols and differentiation times.

[0045] FIG. 11 A is an image showing that HotPocket can be fully immersed inside culturing environments with no disruption.

[0046] FIG. 1 IB provides a close-view image showing a HotPocket holding a brain organoid.

[0047] FIG. 11C and FIG. 1 ID provide images showing the same organoid that has undergone insertion into and extraction from a HotPocket for 5 times (as shown in FIG. 11C) and 10 times (as shown in FIG. 1 ID), respectively.

[0048] FIG. 1 IE shows the live / dead staining for control organoids and those in the device for two hours. The live stain is calcein AM (reference number 1101), and the dead stain is propidium iodide (reference number 1103).

[0049] FIG. 12A provides images of a brain organoid before HotPocket and being enclosed inside HotPocket for 1 min, 1 day, and 12 days, respectively.

[0050] FIG. 12B provides a bright-field image of the same organoid, and the image is captured 6 days following a two-hour exposure in the HotPocket.

[0051] FIG. 12C illustrates changes in circularity over time.

[0052] FIG. 12D illustrates cytotoxicity test of brain organoids with and without being enclosed inside HotPocket for 1 day, respectively (N=6). FIG. 12D highlights the dead / live mean intensity ratio for five organoids in each group.

[0053] FIG. 13 provides a confocal microscope image of a brain organoid, showing optical slices of an 84-day differentiated organoid labeling all nuclei (To-PRO) showing several neuroepithelial buds and the formation of upper layer neurons (CUX1).

[0054] FIG. 14A to FIG. 14D illustrate successful generation of organoids from iPSCs harboring a mutation in the epilepsy-associated SLC35A2 gene. Organoids differentiated from iPSCs to day 18 from iPSCs harboring an indel (as shown in FIG. 14A), those with a S304P missense mutation (as shown in FIG. 14B), and isogenic controls (as shown in FIG. 14C). FIG. 14D provides representative example of electrical stimulation on organoids using HotPockets.

[0055] FIG. 15A to FIG. 15E illustrate neural networks from iPSC-derived neurons harboring pathogenic SLC35A2 variants (S304P and indel) display reduced firing (as shown in FIG. 15A) and asynchronous network connectivity (as shown in FIG. 15B) as assessed by spike train tiling coefficient (STTC) compared to WT control. Raster plots with each row showing the activity for each of the 16 electrodes present in each well for WT (as shown in FIG. 15C), S304P (as shown in FIG. 15D), and indel (as shown in FIG. 15E). A black tick represents a spike (action potential) detected at the electrode. When a spike is detected across electrodes, this is considered a network spiking event (indicated by the vertical lines).

[0056] FIG. 16 illustrates an example HotPocket for non-destructive, long-term electrophysiological recording of entire human brain organoids.

[0057] FIG. 17 highlights a need for personalized treatment (for example, epilepsy, autism, and Alzheimer’s affect over 11.7 million people while existing medications show inconsistentoutcomes) as well as a solution for drug screening via organoid-on-chip in accordance with some embodiments of the present disclosure.

[0058] FIG. 18 to FIG. 20 illustrate longitudinal, gentle communication with organoids provided by example HotPockets in accordance with some embodiments of the present disclosure. In particular, FIG. 18 highlights gentle, long-term engagement provided by example HotPockets, FIG. 19 highlights example HotPockets providing holistic recording over the whole 3D surface as the organoids grow, and FIG. 20 highlights example neural recording and spike events.

[0059] FIG. 21 A provides an example schematic illustration with exploded view of an example HotPocket pattern.

[0060] FIG. 2 IB compares an example HotPocket with a commercial counterpart (2D MEA), on the preparation steps for organoid recording.

[0061] FIG. 22A and FIG. 22B provide example optical images of HotPocket showing its material flexibility.

[0062] FIG. 23 A illustrates an example HotPocket with an organoid.

[0063] FIG. 23B illustrates an example HotPocket with organoids of various diameters.

[0064] FIG. 24 provides experimental images showing HotPockets mechanically wrapping around a brain organoids with high conformality.

[0065] FIG. 25A to FIG. 25D illustrate representative field potentials recorded from an example HotPocket showing glutamate-sensitive activity in a cortical organoid.

[0066] FIG. 25A shows example raw signals from a channel, including in the absence of an organoid (a noise profile), when the organoid is in base media showing limited activity, when the organoid is exposed to 20 uM glutamate leading to increased activity, and when TTX is added to the solution, which decreases activity as expected.

[0067] FIG. 25B provides a raster plot showing spikes called in each condition, demonstrating glutamate-sensitive activity, as expected. In particular, FIG. 25B shows that spikes were called using 5x local std from the mean threshold, that spikes were clustered using PCA, and that all spikes that clustered with the noise condition were excluded.

[0068] FIG. 25C shows spikes called across channels, illustrating activity recorded around the organoid in the glutamate condition with nearby channels showing similar activity.

[0069] FIG. 25D shows the representative spike pattern called in the organoid.BRIEF DESCRIPTION OF DRAWINGS

[0070] The description of the illustrative embodiments may be read in conjunction with the accompanying figures. It will be appreciated that, for simplicity and clarity of illustration, elements illustrated in the figures have not necessarily been drawn to scale, unless described otherwise. For example, the dimensions of some of the elements may be exaggerated relative to other elements, unless described otherwise. Embodiments incorporating teachings of the present disclosure are shown and described with respect to the figures presented herein.DETAILED DESCRIPTION OF THE INVENTION

[0071] Some embodiments of the present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the disclosure are shown. Indeed, these disclosures may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

[0072] As used herein, terms such as “front,” “rear,” “top,” etc. are used for explanatory purposes in the examples provided below to describe the relative position of certain components or portions of components. Furthermore, as would be evident to one of ordinary skill in the art in light of the present disclosure, the terms “substantially” and “approximately” indicate that the referenced element or associated description is accurate to within applicable engineering tolerances.

[0073] As used herein, the term “comprising” means including but not limited to and should be interpreted in the manner it is typically used in the patent context. Use of broader terms such as comprises, includes, and having should be understood to provide support for narrower terms such as consisting of, consisting essentially of, and comprised substantially of.

[0074] The phrases “in one embodiment,” “according to one embodiment,” “in some embodiments,” and the like generally mean that the particular feature, structure, or characteristic following the phrase may be included in at least one embodiment of the present disclosure, and may be included in more than one embodiment of the present disclosure (importantly, such phrases do not necessarily refer to the same embodiment).

[0075] The word “example” or “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any implementation described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other implementations.

[0076] If the specification states a component or feature “may,” “can,” “could,” “should,” “would,” “preferably,” “possibly,” “typically,” “optionally,” “for example,” “often,” or “might” (or other such language) be included or have a characteristic, that a specific component or feature is not required to be included or to have the characteristic. Such a component or feature may be optionally included in some embodiments, or it may be excluded.

[0077] The term “electronically coupled,” “electronically coupling,” “electronically couple,” “in communication with,” “in electronic communication with,” or “connected” in the present disclosure refers to two or more elements or components being connected through wired means and / or wireless means, such that signals, electrical voltage / current, data and / or information may be transmitted to and / or received from these elements or components.Overview of Example Embodiments of the Present Disclosure

[0078] Organoids are 3D models derived from induced pluripotent stem cells that mimic human brain development and function. Organoids contain electrophysiologically active neurons harboring disease-linked genetic variations, enabling genotype-phenotype modeling. Example embodiments of the present disclosure develop a non-invasive device to record electrophysiological activity from organoids, where individual organoids can be added or removed from the device without damage. Example embodiments of the present disclosure demonstrate a morphable and reusable multi-electrode array that does not require attachment factors and enables repeated use of the same organoid overtime. Future goals are to engineer synaptic plasticity within human organoids.

[0079] Testing of novel therapeutics for disorders of the brain has largely relied on animal models, which do not adequately capture human genetic background or cellular complexity. Organoids — 3D in vitro systems that recapitulate aspects of human development, cytoarchitecture, and function — are a promising model for studying therapeutics for epilepsy and other neurological disorders. Derived from induced pluripotent stem cells (iPSCs), organoids can generate neurons harboring disease-associated variants that are electrophysiologically active and can be used for in- depth investigations into how the genetic variants lead to brain phenotypes, like seizures. Organoidmodel systems can also be scaled for screening against a large number of pharmaceutical compounds targeting precise disease mechanisms. One key hurdle to using organoids for therapeutic screening in epilepsy is the development of devices for reproducible, non-destructive, and spatiotemporal electrical recording and stimulation of the entire organoid.

[0080] The most common approaches to measuring electrophysiological activity in organoids are: i) attachment of a spherical organoid to two-dimensional multi-electrode arrays (MEA) using extracellular matrix proteins (as shown in FIG. 1 A), which can only record from neurons near the electrodes or ii) electrodes to be inserted into the organoid, which damage the cells around the insertion path (as shown in FIG. IB). These popular approaches have considerable limitations in that i) they may damage the organoids and limit their re-use, which is extremely costly because organoids require months of maturation to generate electrical activity; ii) they only record from a limited surface of the organoids, which likely miss recording from mature neurons as neuronal composition and maturity of organoids are heterogeneous along the surface; and iii) they are used for recording activity in the absence of stimulation, providing only unidirectional communication with the biological system. Stimulating neuronal activity using electrical methods will allow for investigations of activity-induced plasticity, such as long-term potentiation or depression.

[0081] Given the major limitations of these more commonly used approaches, 3D MEAs are created using buckled 3D scaffolds or grow organoids on mesh-like MEAs as reflect in FIG. 2A and FIG. 2B. While these techniques represent substantial improvements as compared to 2D or needle MEAs, these techniques still have several major limitations that preclude their practical utility as high-throughput organoid screening systems. As shown in FIG. 2A and FIG. 2B, the surface mesh electrodes provide uneven support that can deform a spherical organoid into an unintended disk shape (as shown in FIG. 2A). Furthermore, the slight penetration of the electrodes into the organoids (which are attached using extracellular matrix proteins (as shown in FIG. 2A)) effectively irreversibly bind the device to the organoid, preventing removal of the organoid from the device. Many organoid differentiation protocols require long-term culture in a spinning bioreactor to increase media diffusion and survival, which is not possible with this design. In addition, only one organoid can be measured within each device, which decreases throughput. These limitations also apply to the embedded mesh electrodes (as shown in FIG. 2B). The embedded mesh electrodes also only record internal neurons of an organoid, while example embodiments of the present disclosure measure activity at the surface of organoids because thecortical excitatory neurons (CTIP2) migrate out from the ventricular-like zone (PAX6) toward the surface (as shown in FIG. 3) and the center of the organoid often has a necrotic core due to poor media diffusion. As such, the neurons, which have electrophysiological activity, is often closest to the surface.

[0082] Finally, other devices such as the Buckling MEA and Shell MEA have relatively low electrode density (shell MEA has 3 electrodes, while buckling MEA has 20 electrodes) and are not able to conform to the size of organoids of different ages or differentiation protocols that generally vary between 1.5-4 mm in diameter.

[0083] In contrast, example embodiments of the present disclosure provide a reusable, high throughput microelectronic interface that allows for non-destructive, long-term, recording and stimulation of the entire surface of an organoid (as shown in FIG. 1C and TABLE 1 below). An example device according to example embodiments of the present disclosure (also referred to as a “HotPocket” in the present disclosure) provides a promising and practical approach to understanding neuronal response to external stimuli and identifying therapeutics that restore normal neuronal activity in organoids derived from individuals with neurological disorders.TABLE 1. Example Features of HotPocket

[0084] The HotPocket is a device that is morphable, biocompatible, reusable with 3D network that serves as a bidirectional interface for real-time tracking and manipulation of cellular electrophysiology using electrical stimulation (as shown in FIG. 5A to FIG. 5D). This tool fdls a unique gap amongst current commercially available MEAs that measure activity in only a small part of an organoid or damage the organoid, preventing repeated measurements (as shown in FIG. 1A and FIG. IB). The present disclosure demonstrates the practical impacts of example HotPockets in detecting electrophysiological signals, with long temporal range (> 9 months) and high spatial resolution (> 70 el ectrode s / mm2) of the entire surface of multiple organoids, sequentially, repetitively and in high throughput. Thus, the HotPocket is a valuable tool for many academic labs studying neuronal activity in human organoids, especially in large quantity.

[0085] With 65 million people affected worldwide, epilepsy is one of the most common chronic neurological diseases and creates a major burden in seizure-related disability, mortality,comorbidities, stigma, and costs. Medication has limited efficacy for approximately 30% of individuals with epilepsy and elicits adverse effects that dramatically decrease quality of life. While gene discovery has identified hundreds of genetic variants associated with epilepsy risk, translating these gene discoveries into therapeutics has been hindered by minimal functional understanding of disease pathophysiology. Without high-fidelity model systems with physiologically relevant phenotypes to provide a mechanistic understanding of these disorders as well as devices to reliably and efficiently measure those phenotypes, discovery of effective therapeutics will be severely limited.

[0086] The present disclosure demonstrates the utility of this device for long-term recording and stimulation of neurons to understand how network activity (e.g. the spike time tiling coefficient (STTC)) is coordinated in biological systems and disrupted in epilepsy. Here, examples of the present disclosure uses iPSC-derived organoids harboring mutations strongly associated with epilepsy as a “positive control”: to ensure the ability to detect robust, biologically driven electrophysiological differences across two genetically defined groups using the device. The HotPocket technology provides richer data than planar MEAs because HotPocket measures the entire spatial (across the surface) and temporal (across development of an organoid) extent of an organoid, and likely measure more features, as compared to measurements using a planar MEA. While this application is focused mainly on technological innovation, reproducibility, and validation with application to epilepsy mutations as a positive control, a greater amount of data may in the future enable more utilities such as drug screening to reverse electrophy si ologi cal ly associated deficits in epilepsy and other disorders.

[0087] The interdisciplinary approach and technological advancements make the HotPocket technology uniquely well aligned with the mission of the National Institute of Neurological Disorders and Stroke in seeking fundamental knowledge about the brain and nervous system and in using that knowledge to reduce the burden of neurological diseases. The HotPocket can increase the utility of organoids for future therapeutic discovery including screening compounds or using electrical stimulation to determine if they ameliorate altered network activity observed in epilepsy.

[0088] The present disclosure resolves key hurdles in recording and stimulating electrophysiological activity in organoid models and validation in a genetic model of epilepsy, and enhances understanding of neuronal communication within a human model system.

[0089] Some examples of the present disclosure are both technical and biological, and include:

[0090] 1 a reusable, rose-petal inspired electronic, biocompatible device for electrical recording and stimulation of organoids in large quantities, including (a) construction with biomaterials to ensure a long-term, benign interface with living organoids, (b) a morphable, soft scaffold that folds to enable structural adaptation to fit many sizes of organoids that are generated with different protocols or change size as they differentiate, (c) a device that does not require attachment factors to make close contacts so enables insertion and removal of an organoid from the device, (d) microelectronic sensors integrated in the folded structure offer optimized sensing continuously and spatiotemporally, (e) increased electrode density of individual sensing and stimulation sites to enable near cellular-level resolution, (f) localized neuronal stimulation using electrical methods, and

[0091] 2. rigorous, repetitive, and long-term testing with clinically relevant organoids harboring epilepsy-associated mutations, including (a) rigorous reproducibility and biocompatibility testing of the HotPocket device with human cortical organoids and comparability against currently used 2D MEA approaches, (b) organoids derived from iPSCs harboring SLC35A2 mutation and isogenic controls provide an ideal validation platform with clear expected electrophysiological phenotypes, and (c) device generation and validation sets the stage for high- throughput therapeutic screening to reverse epilepsy phenotypes.

[0092] In some embodiments, the HotPocket contains 64 recording electrodes and 2 reference electrodes (as shown in FIG. 7A). The electrodes use gold nanomembranes (thickness 200 nm) prepared by a sputter deposition system. Exposing 64 contact pads located on the inner surface of the nest-like pocket allows recording of changes in voltage potential proximal to neurons in the organoid, thus forming a 3D spatiotemporal recording profde of the entire organoid. Other regions of the device are fully encapsulated with a medical-grade parylene to enable electrical insulation and a biocompatible interface. The device buckles, forming an elastic pocket where a spherical organoid can be easily inserted. Each electrode can be used as both a channel for recording electrical activity in an organoid, or reversed to stimulate electrical activity in the organoid. For example, multiple iterations of HotPockets have been developed that now fit snugly around an organoid such that it remains in the pocket without damaging it, and can be both inserted and removed from the pocket using common wide bore pipette tips. In some embodiments, neuronal activity are successfully recorded from the HotPockets enclosing organoids in the absence of stimulation, upon addition of Glutamate (with dose-dependent increasing in neuronal activity from10-40 uM) , and with very limited activity after application of the sodium channel antagonist (TTX), demonstrating consistent neural behaviors upon chemical stimulation and suppression, respectively (as shown in FIG. 6 A to FIG. 61). Organoids recorded in the HotPocket show viability for at least 3 months following the recording, highlighting that this approach is non-destructive. This represents a clear demonstration of the feasibility of the HotPocket for recording activity in human organoids.

[0093] Examples of the present disclosure establish a technological foundation that enables long-term, bidirectional, high-throughput stimulation and recording at the biotic-abiotic interface sequentially and repetitively with a series of organoids, allowing both fundamental study of neuronal communication as well as therapeutic development.Example Communication Networks For Hosting Microscale Electrical Sensors And Stimulators

[0094] Example embodiments of the present disclosure provide soft, deformable communication networks that host microscale electrical sensors and stimulators. For example, functional nanomembranes (such as monocrystalline silicon, gold, copper, poly(3,4- ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS), and others) supported on a polymeric film (such as poly dimethyl siloxane, parylene, and polyimide) can enable high stretchability while maintaining excellent voltage sensing performance. Microelectrodes recording may have high electrode density (20 - 100 / mm2).

[0095] However, many two-dimensional layouts cannot support formation of geometrically complex three-dimensional (3D) constructs in a reconfigurable and deterministic fashion to record from and stimulate the entire surface of an organoid. The present disclosure develops classes of compliant 3D frameworks that incorporate microscale sensors for high-sensitivity measurements and stimulation of action potentials generated on the entire surface of organoids.

[0096] For example, examples of the present disclosure provide microelectrode design. In some embodiments, microscale and biocompatible electrical sensors are designed and fabricated for recording neuron activity and electrical stimulators for triggering firing events. In some embodiments, soft microelectrodes with a coating scheme are developed to optimize for minimal impedance to measure the membrane potentials of neurons with minimal noise. Microelectrodes are the gold-standard technology for recording action potentials. Example embodiments of the present disclosure use gold nanomembranes as sensing electrodes for the electrical microsensor(as shown in FIG. 7A to FIG. 7E). For example, the electrical microstimulators uses gold nanomembranes (for example, with a thickness of 200 nm) embedded inside a biocompatible polymer (for example, parylene with a total thickness of 8 pm) to provide the needed biocompatibility, safety, and longevity. Electron beam evaporation will form thin fdms of chrome (for example, Cr with a thickness of 10 nm) and gold (for example, Au with a thickness of 200 nm) on the parylene. Photolithography and wet etching will yield patterns of interconnections and microelectrodes. Spin coating and curing another layer of parylene (for example, with a thickness of 4 pm) will create an insulating fdm on these conductive features to ensure protection of the sensing electrodes from unintended contact with biofluids. Selectively exposing certain areas at designated microscale sites of the microelectrodes via reactive ion etching will allow platinum coating on the electrodes and form intimate contacts with neighboring cells that can enable effective transfer of ionic charges across the biotic-abiotic interface. Sputter deposition of a thin layer of platinum effectively lowers the electrode impedance, thus improving the signal-to-noise ratio. Example microelectrodes in accordance with some embodiments of the present disclosure not only provide sensing functions to capture spiking events of living neurons, but can also be used as a microscale stimulator that triggers voltage-gated ion channels located at the cellular membrane to modulate neural activity. Example embodiments of the present disclosure have successfully achieved ~40 electrodes / mm2density in some examples (as shown in FIG. 7A to FIG. 7E). In some embodiments, ~70 electrodes / mm2density of electrode recording for the HotPocket is achieved, with 128 electrodes in total. The number of recording sites can be easily scaled up to more than 100 electrodes / mm2using photolithography and cleanroom processes to enable high spatial resolution of neural recording. The performance of electrical stimulation can be tested by immersing the soft microelectrode into phosphate-buffered saline and analyzing the signal quality measured from one electrode in response to a series of stimulation (for example, with amplitude ranges from 1 mV to 1000 mV and frequency ranges from 10 Hz to 10 kHz) generated from the neighboring electrode.

[0097] Various embodiments of the present disclosure leverage microelectrode robustness and mechanical softness to ensure mechanical stability of the compliant 3D framework that embeds the sensing electrodes. In some embodiments, the layer of sensing electrodes are designed at the mechanical neutral plane so that mechanical deformation of the whole device generates negligible strain on the sensing electrodes. For example, example embodiments of the present disclosure usefinite element analysis to calculate the strain distribution of the 3D framework and understand the range of mechanical deformation and optimize the structural design (as shown in FIG. 8A to FIG. 8D). In some embodiments, the mechanical stability of the device are tested with a cyclic bender to examine whether its impedance maintains below 50 KQ after 100, 1000, and 10000 cycles of repeated bending. As shown in FIG. 8A to FIG. 8D, the simulation of strain distribution on the soft microelectrodes confirms that the maximum strain is below 0.2%, well below the fracture threshold of platinum-coated gold (5%). Additionally, the resultant 3D-constructed frameworks preserve the high softness that ensures biocompatibility for long-term organoid recording and stimulation. TABLE 2 below illustrates some features of electrodes.ABLE 2 Some Features of Electrodes

[0098] Although there is a remote possibility that certain microsensors will be challenging to integrate together in high density due to the fabrication precision and intrinsic property of soft materials that limit the resolution, example embodiments of the present disclosure provide alternative lithography methods should this unlikely event occur. For example, example embodiments of the present application may utilize advanced transfer printing technology that would allow heterogeneous integration of multi-materials after each individual component is fabricated using traditional photolithography with optimum resolution.

[0099] Another potential challenge is to ensure structural robustness when undergoing elastic deformation. If breakage occurs in current HotPocekt design at an unacceptable frequency, example embodiments of the present disclosure may use biocompatible elastomer (e g. polyurethane) to serve as the substrate materials for the HotPocket design.

[0100] As the electrode density is increased, the reduced size of the sensing pads will inevitably show increased impedance. If such an increase interferes with the sensing quality, an example coating of MXene (a biocompatible metallic 2D materials with relatively low impedance) or Pt Black may be applied on the sensing pad of HotPockets to further lower the interfacial impedance at the recording site for improved signal fidelity.Example Transformation of Planar Electronic Network into a 3D Morphable Nest Structure

[0101] Various embodiments of the present disclosure develop a rose-petal strategy that transforms a planar electronic network into a 3D morphable nest structure with microfluidics for repetitive insertion of organoids.

[0102] Many routes to 3D microstructures and nanostructures (including ion-beam lithography, layer-by-layer growth, multiphoton lithography, printing-based fabrication, and holographic lithography) are often limited to enable structural morphability only for certain high- performance materials such as metallic membranes and silicon. Recently developed methods exploit concepts in self-assembly and mechanically guided assembly to address this limitation with remarkable capability of compatible integration into modern planar technologies established in the semiconductor industry. However, strong reliance on a planar base or template for anchoring precludes the implementation of those approaches in a broader horizon of applications, especially in making morphable devices interfacing with biological tissues. Origami and kirigami, utilized in some schemes, have significant potential in generating morphable mesostructures in 3D that contain sensors and effectors.

[0103] In some embodiments, the sensors and stimulators are integrated with a folded rosepetal strategy to wrap around the entirety of the 3D organoid. For example, example embodiments of the present disclosure develop a rose-petal strategy that will allow transformation of the 2D microelectronics network into 3D HotPocket with dynamic shape morphability and fluidic interconnections. To enable 3D transformation of the microelectronic network, some embodiments of the present disclosure utilize programmable rose-petal concepts that transform a 2D microelectronic and microfluidics network into complex 3D geometry (as shown in FIG. 9A). Stemmed from this, the HotPocket utilizes microfolding as the assembly strategy.

[0104] The process starts with fabrication of 2D soft microelectronic and microfluidics systems through an integrated approach that includes photolithography, laser patterning, transfer printing, and materials bonding. Then, integrating a 2D precursor with a folding host through a transfer-printing process prepares the compliant sensing and stimulation network embedded with fluidics channels for 3D transformation. Strategically bending the host at various degrees of angle translates the origami effect to the guest 2D precursor into a specially engineered 3D mesostructure. FIG. 10 shows preliminary results on compliant 3D frameworks of microelectronicsystems, fabricated by the microfolding and buckling approach, to demonstrate the structural versatility and morphability that can enable structural formation of the proposed HotPocket.

[0105] In some embodiments, the HotPocket design utilizes microfolding assembly strategies as described in FIG. 9A. Specifically, example embodiments use controlled buckling to enable the HotPocket to form an overall shape emulating a mesoscale pocket. Then, the deterministic microfolding strategy provides an approach to mechanically finetune the size of the pocket in a reversible manner to be able to fit multiple sizes of organoids. FIG. 10 shows 3D printed organoids of varying radii from 1 mm to 2 mm and the same HotPocket device that is capable of making close contact with all of these organoids.

[0106] In some embodiments, a mechanical interlocking device is developed for enhanced biocompatibility and shape morphability. The integration between each of the individual components (sensors, stimulators, compliant frameworks, substrates, and others) requires sophisticated bonding at the interface between the sensors and the substrate. Many methods of bonding rely on adhesive materials including UV-crosslinkable polymers and adhesive composites, which may introduce potentially toxic elements or complicate the fabrication process. Here, example embodiments of the present disclosure provide a mechanical interlocking strategy that integrates individual components using mechanical force instead of introducing additional bonding materials, thus ensuring high biocompatibility of the integrated system. Furthermore, the proposed mechanical interlocking allows reversible bonding of the components, thus enabling structural reconfiguration to adapt the growing size of organoids over a long term (> 9 months) or different organoid protocols.

[0107] The proposed mechanical interlocking strategy in accordance with some embodiments of the present disclosure involves components such as a flexible microelectronic framework and a silicone substrate with an opening hold in the center, as shown in FIG. 9A. In some embodiments, the flexible microelectronic framework is designed to have nine interlocking petals, which can be inserted through the hole of the silicone substrate, and buckled together to form a flexible pocket that can be easily adjusted by stretching the silicone substrate in order to ensure holding of an organoid even as its size is changing over time. In some embodiments, the interlocking structure based on silicone templates is also designed to provide additional support for organoids and reinforce the structural integrity of the device, which is slightly larger, allowing tunability for different sizes of organoids. In some embodiments, the entire scaffold can maintain its structuralintegrity and flexibility without any adhesive material, thus making the device easy to assemble, customize, and sterilize using washes of 70% ethanol for long-term safety and biocompatibility (as shown in FIG. 11A to FIG. HD), even after repeatedly insertion and extraction of brain organoids with HotPocket (as shown in FIG. 11 A to FIG. 1 ID).

[0108] In some embodiments, the HotPocket successfully recorded neuronal activity (as shown in FIG. 5A to FIG. HD). In some embodiments, 15 HotPockets with 128 recording / stimulation sites for each are made. The compliant mechanical construct and shape morphability of those HotPockets allow conformal holding of organoids with diameters ranging from 1 mm to 4 mm in diameter, consistent with all known cortical organoid differentiation protocols. In some embodiments, electrical measurements and electrical stimulation (0.1 ms pulses at 0.8-2.0 mA and 60-100 Hz) from the 15 HotPockets individually (as shown in FIG. 14D) are collected and compared, 80 each of which will be holding a living organoid that shares the same genetic background and growth environment (as described below). These devices may be used with various types of organoids for studying autism spectrum disorders, neurogenesis, neuronal development, fragile X syndrome, and others.

[0109] Although there is a small possibility that the parylene-based encapsulation layer precludes formation of HotPocket with targeted pocket size, example embodiments of the present disclosure provide alternative methods should this unlikely event occur. Specifically, example embodiments of the present disclosure use alginate-based hydrogel with mechanical elasticity and surface chemistry close to embryonic tissue to form a soft HotPocket. The hydrogel matrix would also provide an easy venue for incorporating growth factors in close contact with living organoids for sustaining growth.Example Evaluation of Devices According to Embodiments of the Present Disclosure

[0110] Various embodiments of the present disclosure evaluate the functionality and chronic stability of the HotPocket using organoids modeling a genetic form of epilepsy.

[0111] Organoids are an in vitro model of the human brain and enable the measurement of electrophysiological activity of human neurons after extended differentiation times. Organoids can be derived from individuals harboring a genetic mutation associated with a disease to allow mechanistic study of mutational effects. In addition, environmental stimuli or therapeutic compounds can be applied to organoids making them suitable for drug screening approaches.

[0112] In some embodiments, device evaluation and characterization rely on the connection established between the MPIs, which are located in close proximity to each other and enable frequent interaction and iterative device improvement. The operation of the 3D microelectronic scaffold will be tested in the following ways:

[0113] (1) testing immunohistochemistry to evaluate changes in organoid structure and cell type composition due to exposure to the HotPocket,

[0114] (2) testing real-time detection of neuronal activity from a brain organoid in response to channel activators and blockers,

[0115] (3) evaluating the efficacy of electrical stimulation using the HotPocket for brain organoids, and

[0116] (4) evaluating the ability of HotPocket to detect electrophysiological signals in organoids modeling a form of genetic epilepsy.

[0117] In testing immunohistochemistry to evaluate changes in organoid structure and cell type composition due to exposure to the HotPocket, and in order to ensure biocompatibility of the compliant 3D framework, example embodiments of the present disclosure use medical -grade parylene as the encapsulation material that provides direct physical contact with organoids. In some embodiments, the exceptional thermal stability, chemical resistance, and tensile strength enable parylene-based encapsulation as an ideal strategy for long-term operation in biological environments. Example embodiments of the present disclosure perform immunohistochemical analysis to determine if cell type proportions change after exposure to the HotPocket device (as shown in FIG. 5A to FIG. 5D). The immunohistochemical analysis will be performed on tissue- cleared organoids (using the iDISCO+ protocol), and analyzed using a pipeline. The differences in cell type proportions for organoids recorded in the HotPocket and complementary organoids may be determined from the same differentiation that were never exposed to the HotPocket. In some examples, all nuclei (TOPRO3) and major cell types present in the organoid are imaged (as shown in FIG. 13) including radial glia (PAX6), intermediate progenitors (TBR2), lower layer neurons (CTIP2), and upper layer neurons (BRN2). In some embodiments, 10 organoids recorded in the HotPocket for 3 separate 1-hr recording sessions at 6 months differentiation and 10 which will not be exposed to the HotPocket are tissue-cleared, imaged, and quantified in order to quantify differences. Because of the biocompatibility of the materials and the short duration within the pocket, there is likely no differences in cell type proportions caused by exposure to the HotPocket.

[0118] In testing real-time detection of neuronal activity from a brain organoid in response to channel activators and blockers, example embodiments of the present disclosure establish a robust culturing protocol to enable prolonged growth of self-organizing 3D human cortical organoids for over 12 months based on a mini-spinning bioreactor protocol and reproducible protocols. Such prolonged culture allows maturation of organoids that acquire a high degree of cellular diversity and neuronal maturation, including spontaneously active neuronal activity. The first question being addressed is if the HotPocket can identify reproducible, longitudinal spontaneous, and evoked activity. Next, differentiations of one iPSC line (PGP1, male) are performed and differentiated 6 separate times. Each differentiation will produce ~96 organoids. For each differentiation, spontaneous activity for at least 10 organoids at 0, 2, 4, 6, and 9 months post differentiation within the HotPocket are recorded. To determine which ion channels and receptors contribute to spontaneous electrophysiological activity, activity following application of a sodium channel antagonist (TTX), NMDA receptor antagonist (APV), AMPA receptor antagonist (CNQX), or a GABAA receptor antagonist (bicuculline) are recorded. Furthermore, the ability to pharmacologically evoke (using KC1, AMPA, glutamate) or modulate activity via application of GABA, dopamine, and serotonin are tested. Parameters that are measured by the HotPocket include mean firing rate, burst rate, burst duration, density of spikes in bursts, and local field potentials (LFPs) to globally assess presumptive synaptic activity. Assessing changes in these parameters over time will allow identification of physiological parameters of maturation and how these correlate with morphological and genetic expression patterns. Additionally, the ability to induce plasticity pharmacologically pairing bath application of glycine to induce long-term potentiation (chemical LTP) and serotonin or dopamine for long-term depression (LTD) with 0.1 Hz sampling of local field potentials are assessed. The reproducibility and variance of each of the measurements across time using phenotypic correlations are determined, where it is expected to have correlations > 0.8 across all electrophysiological measures for the 6 differentiations to indicate high reproducibility, and via repeated measures ANOVA, where it is not expected to have mean differences in each measure across the 6 differentiations.

[0119] Next, a similar experiment is performed to measure electrophysiological activity in the HotPocket across 3 additional genotypically different iPSC lines (two females and one male) that are previously generated. These will allow assessment of whether different lines have within donor reproducibility and cross-donor differences using intraclass correlation coefficients and ANOVA.Genetic effects have been previously found to be major drivers of gene expression differences in iPSCs, though this has not yet been measured for electrophysiological phenotypes. It is expected to be higher within donor, than across donor correlations of electrophysiological phenotypes. Finally, a device is generated that will be usable regardless of organoid differentiation protocol. The field has as yet not settled on a gold standard differentiation protocol, so it is important to demonstrate the effectiveness of the HotPocket using multiple protocols. Another commonly used approach is called cortical spheroids, which is performed the same both within- and cross-donor experiments using the same cell lines for this alternative and commonly used protocol as well. While it is not necessarily expected to have high correspondence between electrophysiological activity across protocols, it is expected to have consistent phenotypes within donor and protocol.

[0120] In evaluating efficacy of electrical stimulation using the HotPocket for brain organoids, having experimental access to not only recording electrical activity, but also evoking electrical activity allows for the study of stimulus-specific features such as long-term potentiation and longterm depression, as well as circuit formation. In some embodiments, HotPockets can evoke electrical activity directly using the same recording electrodes, following an established procedure. Connecting a selected section of recording electrodes to the output portal of the Control System (for example, RHS stim / recording system, Intan Technologies, Inc.) allow those electrodes to serve as stimulation electrodes. Example embodiments of the present disclosure perform systematic electrical stimulation, separately on organoids, over a period of time (e.g., 3 months) with continual recording of neural activities to capture the effects of electrical stimulation on organoid growth and epileptic pathology (as shown in FIG. 14D). The stimulation procedure (e.g., duration, intensity, frequency, etc.) will use parameters from existing literature designed to evoke LTD or LTP (LTD electrical: 0.1ms pulse 1 Hz for 15 min, LTP electrical: 0.1 ms pulse, 100Hz for 1 sec).

[0121] When evaluating the ability of HotPocket to detect electrophysiological signals in organoids modeling a form of genetic epilepsy, in order to assess the ability of the HotPocket technology to detect biologically relevant electrophysiology in an organoid model, some embodiments of the present disclosure use an iPSC-derived organoid model of SLC35A2 epilepsy. SLC35A2 is located on the X chromosome and encodes a transporter that moves UDP -galactose into the Golgi apparatus to act as a substrate for the formation of galactose-containing glycans. Early post-zygoitcally acquired de novo variants are associated with a congenital disorder ofglycosylation that is associated with severe, early-onset form of epilepsy and developmental delays. For example, somatic variants that arise during embryonic brain development to lead to focal neocortical epilepsy and a mild malformation of cortical development have been identified. The association of loss-of-function variants in SLC35A2 has since been further corroborated by multiple different laboratories.

[0122] To further explore the mechanisms by which loss-of-function variants in SLC35A2 give rise to seizures, CRISPR-Cas9 is used to edit a missense variant (S304P) that was identified in an individual with epilepsy into an induced pluripotent stem cell (iPSC) derived from a healthy male (wildtype, WT). They also created a knockout line by introducing frameshift insertiondeletion (indel) variants in the same iPSC line to mimic nonsense variants that have also been reported in individuals with SLC35A2 epilepsy. Those same iPSC lines are used to differentiate cortical organoids. Dual SMAD inhibition protocol is then used to differentiate each of the three iPSC lines (WT, S304P, indel) into cortical excitatory neurons in a 2D model system and measured electrical activity using a planar multi el ectrode array (Axion Biosystems). It is shown that the disease-causing SLC35A2 variants generate robust and highly reproducible network electrophysiological changes in iPSC-derived neurons harboring disease-causing SLC35A2 variants (as shown in FIG. 14A to FIG. 14D). Further investigations show that the addition of bicuculline, a GABAergic antagonist, partially rescued the asynchronous firing in SLC35A2- variant harboring networks suggesting that an increased proportion of GABAergic neurons are being produced as a result of the genetic variants in SLC35A2. This is further supported by an increase in GAB Aergic-specific markers as assessed with quantitative real-time PCR in SLC35 A2- variant harboring neurons compared to the WT. Collectively, these data suggest that loss-of- function variants in SLC35A2 alter the differentiation trajectory towards the GABAergic fate, which consequently generates asynchronous, hypoactive networks. Despite these clear electrophysiological effects of the mutation in neurons in this 2D model system, there is no evidence that individuals with SLC35A2-associated epilepsy have more GABAergic neurons in their brain tissue. This suggests that the changes being observed are either early-stage changes that are not captured in the limited time scale that is possible in the 2D model system (~60 days) or that the effects present differently in the 2D model system where there is more limited cellular diversity. A critical next step is to study the network electrophysiological effects of the variants inorganoids where the effects of the mutations can be studied in much longer periods of development (6 months to a year) and in the presence of more diverse cell types.

[0123] Given the robust effects of the disease-causing SLC35A2 variants in this 2D neuronal model, some embodiments of the present disclosure use these same lines to generate cortical organoids to demonstrate the utility of HotPockets to detect electrophysiological changes associated with SLC35A2 variants. Example embodiments of the present disclosure differentiate these same isogenic iPSC lines using the previously described differentiation protocol, to the same time points previously described, and assess those measures that were previously found to be reproducible. Example embodiments of the present disclosure measure at least 10 organoids per genotype within the HotPocket. While the effects of pathogenic SLC35A2 variants in an organoid model have not been studied, given the robust changes in the 2D model system (as shown in FIG. 15A to FIG. 15E), it is expected that the electrophysiological changes will be readily detectable in the organoid model using the HotPocket technology. Example embodiments of the present disclosure also explore the effects of bicuculline on electrophysiological signals in the organoid to evaluate the ability of HotPockets to detect the effects of pharmacologic treatment.

[0124] To provide a benchmark for the HotPocket performance, example embodiments of the present disclosure measure neuron activity from it. These signals will be compared with those generated by both a commercial 2D MEA system (Maestro Pro, Axion Biosystems), which is a well-established in vitro monitoring platform for neurons, and a patch clamp setup, which a gold standard for ion channel screening of neuron cells. As both systems induce irreversible fatal damage to organoids after measurements while HotPocket will preserve organoid viability, example embodiments of the present disclosure conduct multiple phases of benchmark testing of the HotPocket in different organoids, and assess consistency of discovered phenotypes between organoids harboring an epilepsy mutation and isogenic control across different electrophysiological measurement techniques.

[0125] Phase 1 : HotPocket compared with patch clamp.

[0126] In order to determine whether these electrophysiological phenotypes are consistent regardless of electrophysiological measurement technique, example embodiments of the present disclosure perform patch clamp electrophysiology. For example, ~6-month organoids from 10 organoids per genotype are embedded into low melting point agarose and sectioned into 250 um slices. Continuing this example, 3-5 cells per organoid are recorded assaying the same parametersand pharmacological manipulations assayed with the HotPocket. Furthermore, these experiments enable direct assessing the synaptic properties and ionic conductance of single cells, validating the electrophysiological properties of organoids in a manner inaccessible to the HotPocket.

[0127] Phase 2: HotPocket compared with planar MEA.

[0128] One advantage of HotPocket is the nondestructive insertion and extraction of organoids. Thus, example embodiments of the present disclosure first perform mapping measurement of spike events using a HotPocket with 128 recording sites on a cortical organoid. Then, extraction of the cortical organoid and placing it on a commercial MEA device (Axion BioSystems, Inc.) allow mapping measurement of a partial area of the same organoids. Example embodiments of the present disclosure analyze the correlations between the two mapping measurements (described above) from HotPocket and the commercial MEA device, respectively, and quantify the similarities. Example embodiments of the present disclosure repeat the same experiment with 10 organoids per genotype to examine the consistency in fidelity performance. For example, correlations > 0.8 between electrophysiological techniques will provide strong confidence that recordings are similar.

[0129] A potential challenge is that successful execution of evaluating the functionality and chronic stability of the HotPocket using organoids modeling a genetic form of epilepsy is dependent on the success of developing soft, deformable communication networks that host microscale electrical sensors and stimulators and developing a rose-petal strategy that transforms a planar electronic network into a 3D morphable nest structure with microfluidics for repetitive insertion of organoids. In accordance with some embodiments of the present disclosure, more than 20 prototype devices are generated with demonstrated capability in neural recording and stimulation, which pave the way for further application-specific improvement and show clear feasibility of developing soft, deformable communication networks that host microscale electrical sensors and stimulators and developing a rose-petal strategy that transforms a planar electronic network into a 3D morphable nest structure with microfluidics for repetitive insertion of organoids (as shown in FIG. 4 to FIG. 14D). In accordance with some embodiments of the present disclosure, iterative improvements in multiple aspects of device generation (e.g. conductive coating on sensing sites, material softening, and structural morphing) will lead to reproducible measurements of biologically driven neuronal activities.

[0130] Example embodiments of the present disclosure describes a highly useful product, the HotPocket, that is designed as a recording and stimulation device to interface with human brain organoids. Example embodiments of the present disclosure provide: i) a complete set of materials (semiconductors, conductors, interlayer / gate dielectrics, etc.) and design strategies that enable electrical stimulation and recording with the entire organoid at high spatiotemporal resolution, ii) active adaptation of pocket size and improved biocompatibility of materials construction that avoids adhesives to allow easy insertion and removal of organoids, and iii) demonstration of the HotPocket to utilize brain organoids as an effective and efficient platform for studying neurological development and disorders, and for screening new drugs.

[0131] The opportunity to enable thorough, long-term, bidirectional communications with entire brain organoids at high spatiotemporal resolution opens the door to new ways of studying neurodevelopment and neurological disorders. Example embodiments of the present disclosure enable correlating molecular, cellular, and physiological scale experiments with cognitive and clinical studies, which could lead to personalized treatments, and reduce costs associated with traditional clinical trials. In addition, the generation of brain organoids from patient material enables a range of therapeutic agents to be tested in the proposed HotPocket.

[0132] Organoids, derived from induced pluripotent stem cells (iPSCs), are 3D in vitro systems that recapitulate aspects of human brain development, cytoarchitecture, and function. Organoids can be used to generate relevant cell types on the genetic background of individuals with a neuropsychiatric disorder, can be scaled for therapeutic screening, and can produce stimulus-responsive electrophysiological activity after months of differentiation. Current popular approaches to measuring electrophysiological activity in organoids require: i) electrodes in the form of mesh or needles to be inserted into the organoid; or ii) attachment of a 3D organoid to two-dimensional multi-electrode arrays (MEA) using extracellular matrix proteins; or iii) mesh electrodes that forms irreversible attachment with organoid and prevents repetitive usage. Such methods damage the organoids, limiting their re-use at a later point, and only record electrical activity of the inner core or a partial area of an organoid, thus missing the vast majority of signal around the entire organoid surface where most neurons are found. Organoids require months of differentiation to generate electrical activity, so longitudinal measurements from the same organoid would greatly decrease experimental costs but are difficult with current systems. Also, differentiation protocols produce organoids of different sizes, so devices to measureelectrophysiological activity need to adjust to the size of the organoid. Example embodiments of the present disclosure solve these challenges by developing a 3D microelectronic-organoid recording platform used to longitudinally measure neuronal activity around the entire organoid surface without damage. The present disclosure also validates the device using an organoid model of epilepsy where abnormal electrophysiological activity is expected.

[0133] Thus, the present disclosure develops a soft, biocompatible microelectronic 3D network, which is referred to as HotPocket, for long-term, bidirectional communication with organoids. The present disclosure provides materials, manufacturing strategies, and modeling tools for seamless interfacing with clinically relevant organoids, a step toward enabling personalized medicine for neurological and psychiatric disorders.

[0134] For example, example embodiments of the present disclosure enable fabricating high- density electrodes onto biocompatible materials. Example embodiments of the present disclosure develop a novel integration technology to assemble microscale electrical sensors and stimulators into a single biocompatible, soft circuit. The fabrication scheme utilizes the microfabrication and soft lithography techniques. Example embodiments of the present disclosure ensure microelectrodes have sufficient signal quality for both recording and stimulation and are able to withstand the strain generated by the 3D morphable device.

[0135] Example embodiments of the present disclosure fold electrode-containing devices to closely fit around 3D organoids. For example, example embodiments of the present disclosure develop a rose-petal approach to fold the soft microelectrodes into a hollow droplet structure surrounding the organoid. Example embodiments of the present disclosure develop a morphing mechanism so the structural size of the pocket can change, allowing compatibility with differentsized organoids from different differentiation protocols and as they grow through time. In accordance with some embodiments of the present disclosure, 25 HotPockets with 128 recording / stimulation sites for each are generated.

[0136] Example embodiments of the present disclosure evaluate the functionality and biocompatibility of the HotPocket using organoids modeling a genetic form of epilepsy. For example, some embodiments of the present disclosure differentiate organoids from established iPSC lines to determine if placement of organoids within HotPockets affects their cellular composition or organization, determine the level of reproducibility of neuronal recordings within and across organoids and donors, and demonstrate electrical stimulation of organoids. Exampleembodiments of the present disclosure utilize established models of clinically relevant organoids from individuals with epilepsy to determine if genetically driven changes in neuronal activity can be observed using our new device. Example embodiments of the present disclosure compare the results to commercially available 2D MEA platforms to determine consistency of electrophysiological phenotypes across measurement techniques.

[0137] As such, example embodiments of the present disclosure develop and validate an advanced system for recording and stimulating brain organoids has the potential to greatly increase the phenotypes able to be studied in neuronal 3D cultures, opening up possibilities of understanding network behavior through development, how brain networks are altered in neuropsychiatric disorders like epilepsy, and could be used to screen drugs to reverse disorder associated effects. Example embodiments of the present disclosure allow future possibilities of modeling behavior in organoids, using a closed-loop system where neuronal stimulation is driven by activity, which enables emerging opportunities to study synthetic artificial intelligence.Example Implementation of Example Embodiments of the Present Disclosure

[0138] Three-dimensional (3D) human brain organoids derived from induced pluripotent stem cells (iPSCs) provide human-specific cellular composition, cytoarchitecture, and spontaneous network activity, offering a powerful platform to investigate mechanisms of neurological disease and to screen candidate therapeutics. Yet, widespread translation of organoid models into discovery and screening pipelines has been limited by the lack of devices that can reproducibly record and stimulate the entire organoid non-destructively over long time scales. Existing solutions typically compromise between coverage, invasiveness, reuse, and throughput: planar multielectrode arrays (MEAs) require immobilizing a spherical organoid onto a 2D surface with attachment factors and thus sample only a small fraction of its surface; penetrating microneedle / laminar electrodes damage tissue along the insertion path; and recent 3D meshes or buckled / shell MEAs improve geometric access but still deform organoids, bind irreversibly through partial penetration or protein adhesives, offer relatively low electrode counts, or cannot accommodate the wide range of organoid diameters produced by different differentiation protocols and developmental stages. These constraints reduce signal fidelity, preclude repeated measures on the same specimen after bioreactor culture, and limit bidirectional interrogation of developing neural circuits.

[0139] To address these gaps, example embodiments of the present disclosure develop HotPockets that provide morphable, biocompatible, reusable 3D microelectronic interfaces that gently envelop an entire organoid to enable high-density, long-term extracellular recordings and on-device stimulation. In some embodiments, the morphability of HotPockets allows the HotPockets to accommodate organoids of various sizes.

[0140] In some embodiments, HotPockets integrate 63-64 recording sites plus reference electrodes within a soft, rose-petal-like architecture that buckles into an elastic “pocket,” bringing electrodes into conformal contact over the full organoid surface. In some embodiments, the conductive network (for example, Cr / Au with Pt coatings) is encapsulated in medical-grade parylene for electrical insulation and biocompatibility. In some embodiments, organoids can be introduced and removed using a standard wide-bore pipette tip, enabling repeated measurements without damaging the specimen. During testing, HotPockets capture robust activity that increased with glutamate and was suppressed by TTX, and organoids remained viable months after recording, providing evidence of a non-destructive interface suitable for longitudinal studies.

[0141] In some embodiments, HotPockets are designed for mechanical conformity without adhesives. In some embodiments, finite-element analyses (FEA) and benchtop studies show that opening the device on a pre-stretched elastomer substrate, placing the organoid centrally, and releasing the substrate yields a uniform wrap that preserves organoid circularity and minimizes strain on the microelectrodes, supporting whole-surface coverage while maintaining tissue integrity. In some embodiments, HotPockets also address a practical challenge in organoid research: size variability. Devices conform to organoids spanning standard mature diameters (~2- 3.4 mm) across protocols, and FEA demonstrates stable electrode-tissue contact across radii tested, enabling one device family to serve different culture timelines and lines.

[0142] Relative to incumbent and emerging systems summarized in comparative analyses, HotPockets uniquely combine reusability, full-surface access, adaptability to different sizes, and bidirectional interfacing, capabilities that are simultaneously absent in planar MEAs, penetrating arrays, shell / buckling MEAs, and mesh-based constructs. This positioning directly targets the bottleneck that has limited organoids as scalable, physiologically relevant testbeds for disorders such as epilepsy, where longitudinal, whole-surface network phenotyping and controlled stimulation quantify development, plasticity, and pharmacologic response.

[0143] Various embodiments of the present disclosure provide the HotPocket platform with practical advantages that enable non-destructive, long-term electrophysiology of entire human brain organoids, including, but not limited to, the following:

[0144] i) Short preparation time. Because HotPockets do not require attachment factors or tissue penetration, organoids can be loaded, positioned, and recorded within minutes; rapid swap- in / swap-out workflows allow sequential measurements across specimens during a single session.

[0145] ii) Reusability. The same device supports repeated insertions and removals of the same or different organoids without compromising viability or geometry, as evidenced by live / dead assays and repeated handling (5-10 cycles) in accordance with some embodiments of the present disclosure.

[0146] iii) Flexible, conformal attachment. The morphable, rose-petal architecture wraps the organoid uniformly to maximize electrode-tissue contact over the full surface while minimizing mechanical strain and deformation.

[0147] iv) Adaptivity to various sizes. A single HotPocket design family accommodates organoids of multiple diameters produced by different protocols or maturation states (-2-3.4 mm demonstrated), ensuring broad applicability across laboratories and timelines.

[0148] Together, these features define a practical, scalable interface for whole-organoid, bidirectional electrophysiology that preserves specimen integrity while enabling longitudinal and comparative measurements across lines, ages, and conditions. The present disclosure details the device design and fabrication, quantitative mechanical and electrical characterization, and demonstrations of pharmacologic modulation and stimulation paradigms enabled by HotPockets.

[0149] In accordance with some embodiments of the present disclosure, HotPocket concept enables whole-surface, non-destructive interfacing with human brain organoids.

[0150] For example, FIG. 5 A to FIG. 5D provide example concepts and loading workflow in accordance with some embodiments of the present disclosure. Brightfield and fluorescence views shown in FIG. 5A to FIG. 5D demonstrate the behavior of the device: a petal-like microelectronic “pocket” gently envelops an organoid and establishes broad-area contact without adhesives or penetration. Bottom-view fluorescence shows the GFP-positive organoid fully enclosed within the blue-autofluorescent pocket (as shown in FIG. 5A and FIG. 5B), while top-view images before and after insertion illustrate a simple pipette-load, release, and record workflow (as shown in FIG.5C and FIG. 5D). This rapid, alignment-tolerant loading is foundational for short preparation times and repeat measurements on the same specimen.

[0151] FIG. 5 A illustrates an example device 501 in accordance with some embodiments of the present disclosure comprises biocompatible material 503 and electrodes 505 fabricated onto the biocompatible material 503. In some embodiments, the biocompatible material 503 comprises biocompatible polymer. In some embodiments, the biocompatible polymer comprises parylene. In some embodiments, the parylene is associated with a thickness of 8 pm.

[0152] FIG. 9A illustrates 3D rose-petal strategy and design rationale in accordance with some embodiments of the present disclosure. The schematic clarifies how the petal geometry is tuned to (i) open widely for loading, (ii) collapse elastically to conform to the spherical surface, and (iii) distribute bending / strain away from metal traces. This strategy achieves whole-surface access while minimizing shear and compressive forces on soft tissues — a precondition for non-destructive long-term use.Fabrication, layout, and comparison to planar MEAs

[0153] FIG. 7A illustrates an example device 700 comprising an encapsulation layer 701, a gold layer 703, a chrome layer 705, and a substrate layer 707. In some embodiments, the gold layer 703 is between the encapsulation layer 701 and the chrome layer 705. In some embodiments, the chrome layer 705 is between the gold layer 703 and the substrate layer 707.

[0154] In some embodiments, sensing electrodes 709 of the example device 700 comprise gold nanomembranes on the gold layer 703. FIG. 7D and FIG. 7E further illustrate portions of example gold nanomembranes. In some embodiments, the gold nanomembranes are associated with a thickness of 200 nm as shown in FIG. 7A. In some embodiments, the gold nanomembranes are embedded in biocompatible polymer (for example, the encapsulation layer 701 shown in FIG. 7A).

[0155] FIG. 7F, FIG. 21 A and FIG. 2 IB illustrate example layouts and user-facing workflows. For example, FIG. 7F illustrates the 2D electrode layout and FIG. 21 A illustrates exploded schematic that show a high-density network integrated into flexible petals and insulated except at the recording / stimulation sites. A side-by-side comparison with a commercial 2D MEA as shown in FIG. 2 IB highlights the reduction of preparation steps, enabling high throughput screening of organoids. Specifically, planar MEAs typically require attaching a spherical organoid onto a plane (adhesives / coatings, immobilization time, delicate positioning), which restricts coverage andcomplicates recovery. In contrast, HotPockets avoid immobilization chemistry and leverage the device’s intrinsic mechanics to establish contact, shortening setup and enabling rapid swap- in / swap-out experimentation.

[0156] In some embodiments, an example comprises a Pt-Black layer. In some embodiments, the gold nanomembranes are functionalized with Platinum (Pt) Black or Pt. In the example shown in FIG. 21a, the example device 2100 comprises a parylene C layer 2101 (for example, an encapsulation layer), a Pt-Black layer 2103, a gold / chrome layer 2105, and another parylene C layer 2107 (for example, a substrate layer).

[0157] In some embodiments, the electrodes of the example device comprise sensing electrodes (also referred to as “recording electrodes”). For example, FIG. 7F illustrates sensing electrodes 711, reference electrodes 713, and ACF cable connection region 715. In some embodiments, the electrodes are associated with a density of approximately 40 electrodes / mm2. In some embodiments, the electrodes are associated with a density of approximately 70 electrodes / mm2. In some embodiments, the electrodes are associated with a density of more than 100 electrodes / mm2.

[0158] FIG. 7B, FIG. 7C, FIG. 22A, and FIG. 22B illustrate fabricated compliant networks. Optical images shown in FIG. 7C, FIG. 22A, and FIG. 22B verify that the released devices retain mechanical compliance and integrity across petals, interconnects, and pads. Together with the schematic (as shown in FIG. 7B), these data establish a robust microfabrication route compatible with repeated handling. In practice, the combination of thin-film metals and polymer encapsulation supports the mechanical cycles associated with opening, loading, and releasing the petals, consistent with the intended reusability of the platform.Mechanics of conformal wrapping and contact stability

[0159] FIG. 8A to FIG. 8D and FIG. 23A to FIG. 23B illustrate example finite-element analysis. Simulations on the 2D layout and organoid-wrapped states (as shown in FIG. 8A and FIG. 23A) show that petal bending localizes strain in the polymer substrate rather than the conductor lines. In some embodiments, the pre-stretch level modulates the final wrap: moving from 100% to 125% pre-stretch (as shown in FIG. 8C and FIG. 8D) increases the contact pressure uniformly and reduces gaps at petal boundaries, providing a tunable parameter to balance conformality and ease of loading. Simulations across organoid diameters (as shown in FIG. 23B)predict stable electrode-tissue proximity over a wide size range, a prerequisite for cross-line, and cross-age studies.

[0160] As shown in FIG. 7F, in some embodiments, the device comprise soft, foldable petals. In some embodiments, the petals are interlocked and connected at the center of the petal pattern. As further shown in FIG. 9A, in some embodiments, the device, through controlled buckling and microfolding, forms a pocket 901 for an organoid 903. Similar to various examples described herein, the device comprises interlocking petals 905 and a silicone substrate 907 with a hole 909. In some embodiments, the interlocking petals 905 are inserted through the hole 909 of the silicone substrate 907 and buckled together to form the pocket 901. For example, through controller buckling, the interlocking petals 905 may form an overall shape emulating a mesoscale pocket; through microfolding, the size of the pocket 901 may be finetuned. Additional details of microfolding are discussed in U.S. Patent Application No. 19 / 122,211, filed on April 17, 2025, the content of which is incorporated by reference in its entirety.

[0161] FIG. 24 illustrates experimental conformality. Time-lapse and multi-view images confirm the simulated behavior: the petals close uniformly around the organoid, with visual continuity of contact and preserved gross geometry. The absence of visible compression artifacts and the maintenance of spherical shape support the hypothesis that device-tissue interfaces are formed by gentle, distributed forces rather than adhesive or penetrative anchoring. The residual strains in the devices at their 3D buckled format are well below the threshold of fracturing, confirming the device robustness.Adaptivity to organoid size and preservation of viability

[0162] FIG. 10 illustrates size adaptivity of an example HotPocket in accordance with some embodiments of the present disclosure. In FIG. 10, side-view and bottom-view series show the same device design accommodating organoids spanning ~l-2 mm radius. Across this range, the pocket morphs to maintain wrap symmetry and surface contact without evident folding or crimping that could distort tissue. This adaptivity simplifies logistics across differentiation protocols and maturation stages, reducing the need for device-to-organoid matching.

[0163] FIG. 12A and FIG. 12D highlight viability and handling of example devices in accordance with some embodiments of the present disclosure. Brightfield images (as shown in FIG. 12A) acquired at 1 min, 1 day, and 12 days after enclosure show preserved overallmorphology, and a cytotoxicity assay (N = 6) (as shown in FIG. 12D) indicates no increase in cell death after 1 day of enclosure compared with unenclosed controls. These results are consistent with the intended non-destructive operation and support longitudinal studies in which organoids are repeatedly loaded, recorded, and returned to culture.Bidirectional electrophysiology and pharmacological sensitivity

[0164] As described above, FIG. 15C to FIG. 15E illustrate example electrical activity in the organoid detected by the electrodes and example spiking events (shown as vertical lines) associated with living neurons that are captured by the electrodes. The electrodes may also stimulate electrical activity in the organoid, as shown in FIG. 14D.

[0165] FIG. 25 A to FIG. 25D illustrate example recording / stimulation readiness and pharmacological modulation. The field-potential traces recorded around the enclosed organoid exhibit the expected pharmacology: low activity in base medium, increased multi -unit / spiking activity following glutamate exposure, and suppression upon TTX addition (as shown in FIG. 25A). Spike-calling with a threshold at 5x local standard deviation and noise-cluster exclusion yields condition-dependent raster patterns (as shown in FIG. 25B), while multichannel maps (as shown in FIG. 25C and FIG. 25D) show spatially coherent activation across neighboring electrodes. These data confirm that (i) HotPockets in accordance with some embodiments of the present disclosure achieve sufficient signal quality and signal-to-noise to detect compound-evoked changes; (ii) coverage of HotPockets is not restricted to a single contact patch; and (iii) the device supports bidirectional protocols where stimulation can be used alongside recording to probe network dynamics.Practical advantages and implications

[0166] Collectively, the figures illustrate that HotPockets provide:

[0167] (1) short preparation time by eliminating adhesive immobilization and leveraging elastic wrapping (as shown in at least FIG. 5 A to FIG. 5D and FIG. 9 A),

[0168] (2) reusability via robust, compliant microfabrication and gentle loading that tolerate repeated handling (as shown in at least FIG. 5A to FIG. 5D, FIG. 7B and FIG. 7C, and FIG. 22A to FIG. 22B),

[0169] (3) flexible, conformal attachment that preserves organoid geometry while maximizing electrode-tissue proximity (FIG. 10, FIG. 12A and FIG. 12D), and

[0170] (4) adaptivity to organoids of various sizes without retooling (FIG. 8A to FIG. 8D andFIG. 23A to FIG. 23B).

[0171] FIG. 24 illustrates viability data, indicating that this geometry-driven interface avoids acute toxicity and preserves morphology over days, while pharmacological tests (as shown integrated optical component FIG. 25 A to FIG. 25D) demonstrate functional sensitivity and spatial mapping capability across the organoid surface.

[0172] From a methodological perspective, these attributes resolve several long-standing limitations of planar MEAs and penetrating arrays. Planar interfaces undersample a spherical surface and typically require irreversible attachment steps that complicate repeated measures, while penetrating electrodes risk tissue disruption and drift. In contrast, HotPockets’ wholesurface, adhesive-free contact favors longitudinal phenotyping and swap-in / swap-out testing across cohorts, which is crucial for scalable discovery (e.g., genotype-phenotype associations, drug-response profding). Mechanically, tunable pre-stretch provides a simple control knob for laboratories to optimize conformality for organoids with different radii and stiffness, while the polymer / metal stack demonstrated here is compatible with established cleanroom processes and can be extended to higher channel counts or alternative coatings for impedance reduction.

[0173] Functionally, the pharmacological sensitivity to glutamate and TTX confirms that HotPockets deliver stable recordings with expected neuropharmacology, and the multi-site maps support analyses of network-level behavior (such as synchrony and propagation). Paired with stimulation, the platform can interrogate plasticity and evoked dynamics without compromising specimen health, enabling longitudinal, bidirectional studies that are difficult to realize with immobilized or invasive alternatives. These capabilities in accordance with some embodiments of the present disclosure can accelerate applications ranging from developmental neurobiology to disease modeling (e.g., epilepsy-relevant hyperexcitability assays) and medium-throughput screening paradigms that require non-destructive, repeatable measurements.

[0174] In summary, HotPockets provide a practical, non-destructive interface for long-term, whole-surface electrophysiology and stimulation of intact human brain organoids. By leveraging a petal-like, elastically morphable architecture, the devices establish conformal contact without adhesives or penetration, enabling rapid minutes-scale preparation, reusability across sessions andspecimens, stable, flexible attachment that preserves organoid geometry, and adaptivity to a broad range of organoid sizes. Across benchtop and pharmacological tests, HotPockets captured spatially distributed spontaneous and evoked activity with expected modulation by glutamate and TTX, while preserving viability over days, thereby supporting longitudinal phenotyping on the same specimen. These attributes directly address key limitations of planar and penetrating electrodes and create a path toward scalable studies of development, disease mechanisms, and drug response in human-relevant models. Additional aspects include increasing channel density, integrating low- impedance coatings and compact packaging for bioreactor culture, and combining electrical readout with optical or microfluidic modalities for closed-loop interrogation. With these advances, HotPockets can enable standardized, high-content neural assays that bridge basic neurobiology and translational screening in organoid platforms.

[0175] It is to be understood that the disclosure is not to be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation, unless described otherwise.

Claims

CLAIMS1. A devi ce compri si ng : biocompatible material; and electrodes fabricated onto the biocompatible material, wherein the device, through controlled buckling and microfolding, forms a pocket for an organoid.

2. The device of claim 1 further comprising a platinum coating on the electrodes.

3. The device of claim 1, wherein the electrodes record electrical activity in the organoid.

4. The device of claim 1, wherein the electrodes stimulate electrical activity in the organoid.

5. The device of claim 1, wherein the electrodes capture spiking events associated with living neurons.

6. The device of claim 1 further comprising petals.

7. The device of claim 6 further comprising a silicone substrate with a hole.

8. The device of claim 7, wherein the petals are inserted through the hole of the silicone substrate to form the pocket.

9. The device of claim 1, wherein the biocompatible material comprises biocompatible polymer.

10. The device of claim 9, wherein the biocompatible polymer comprises parylene.

11. The device of claim 10, wherein the parylene is associated with a thickness of 8 pm.

12. The device of claim 1, wherein the electrodes comprise sensing electrodes.

13. The device of claim 12, wherein the sensing electrodes comprise gold nanomembranes.

14. The device of claim 13, wherein the gold nanomembranes are associated with a thickness of 200 nm.

15. The device of claim 13, wherein the gold nanomembranes are embedded in biocompatible polymer.

16. The device of claim 1, wherein the electrodes are associated with a density of approximately 40 electrodes / mm2.

17. The device of claim 1, wherein the electrodes are associated with a density of approximately 70 electrodes / mm2.

18. The device of claim 1, wherein the electrodes are associated with a density of more than 100 electrodes / mm2.

19. The device of claim 1, further comprising an encapsulation layer, a gold layer, a chrome layer, and a substrate layer.

20. The device of claim 19, wherein the gold layer is between the encapsulation layer and the chrome layer, wherein the chrome layer is between the gold layer and the substrate layer.

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