WO2025199174A1

WO2025199174A1 - Flexible electronics for pancreatic islets and other applications

Info

Publication number: WO2025199174A1
Application number: PCT/US2025/020476
Authority: WO
Inventors: Jia Liu; Ren Liu; Qiang Li; Douglas A. Melton; Juan R. ALVAREZ-DOMINGUEZ
Original assignee: Harvard University
Current assignee: Harvard University
Priority date: 2024-03-19
Filing date: 2025-03-18
Publication date: 2025-09-25
Anticipated expiration: 2026-09-19

Abstract

Stretchable electronics implanted during tissue formation allow chronic studies of tissue-wide electrophysiology. This disclosure describes integrated, stretchable mesh nanoelectronics implanted during organogenesis of human stem cell-derived pancreatic islets, allowing for the long-term, stable recording of single-cell extracellular spike bursting dynamics. By employing a conventional spike sorting pipeline, two major electrical states for both α and β cells were identified based on their responses to glucose threshold for action potential firing. Continuous recording during further maturation by entrainment to 24-hour feeding cycles showed islet-level hormone secretion rhythms reflect increased and sustained coordination between α and β stimulated electrical activities, linked to cell-cell communication and exocytic network induction, revealing a role for circadian rhythms in coordinating system- level stimulus-coupled responses. These results highlight the ability for "cyborg" islets to advance efforts to generate fully functional in vitro islets for research and therapeutic applications.

Description

FLEXIBLE ELECTRONICS FOR PANCREATIC ISLETS AND OTHER APPLICATIONS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/567,332, filed March 19, 2024, entitled “Flexible Electronics for Pancreatic Islets and Other Applications,” by Liu, et al. incorporated herein by reference in its entirety.

GOVERNMENT SPONSORSHIP

This invention was made with government support under DK 130673 awarded by National Institutes of Health (NIH). The government has certain rights in this invention.

TECHNICAL FIELD

Flexible and/or stretchable and/or soft electronics for pancreatic islets and other applications are generally described.

BACKGROUND

Human pluripotent stem-cell-derived islets (SC-islets) hold great promise for advancing diabetes research, pharmacology, and transplantation therapy. However, SC- islets often exhibit immature physiology, marked by poor insulin stimulation capacity. To address this issue, various methods have been advanced to enhance SC-islet functional maturity, such as extended culture, cell enrichment, and diurnal feedingfasting entrainment. These manipulations trigger hallmarks of postnatal islet maturation that are typically reversed with the loss of glucose responsiveness in diabetics, improving the potential of transplanted SC-islets to mitigate diabetes. However, in vitro SC-islets still exhibit less mature phenotype compared to transplanted SC-islets, and both lack the precision, kinetics, and magnitude of hormone secretion shown by adult islets in vivo. Whether these limitations reflect poor coordination between (and/or within) populations of SC-islet cell types, or intrinsic heterogeneity in their maturation, remains unclear. Thus, a system- level, single-cell resolution understanding of how SC-islets turn specialized would fuel transformational opportunities to build fully functional SC-islets for research and regenerative medicine applications.

Islet specialization occurs after birth, as a/p cells raise the responsiveness of insulin/glucagon release to the influx of Ca2+ ions that results from membrane potential changes driven by glucose oxidation-generated ATP. It has not been possible to continuously trace these changes, as existing methods to study islet function either lack single-cell resolution across intact islets or are unable to simultaneously detect islet-wide a- and P-cell activities continuously. Remarkably, the pancreas and neurons share a developmental lineage, suggesting potential parallels in their electrophysiological behaviors. While substantial advancements in bioelectronics have transformed electrical recording techniques in the study of neural systems, the application of these innovations to the pancreas remains limited.

Accordingly, improved articles, systems, and methods are desired.

SUMMARY

Flexible and/or stretchable and/or soft electronics for pancreatic islets and other applications are generally described. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

In one aspect, an article comprising a mesh comprising a plurality of electrodes electrically connected to a plurality of interconnects; and a plurality of pancreatic cells in contact with at least one of the electrodes, wherein the electrodes and/or the interconnects are stretchable and/or flexible and/or soft is described.

In another aspect, a system, comprising a mesh comprising a plurality of electrodes electrically connected to a plurality of interconnects; and a plurality of pancreatic cells comprising a first pancreatic cell type and a second pancreatic cell type; and wherein the mesh is configured to distinguish the first pancreatic cell type from the second pancreatic cell type is described.

In another aspect, a method comprising applying a first electrical potential to a mesh in contact with a plurality of pancreatic cells comprising a first pancreatic cell type and a second pancreatic cell type, the mesh comprising a plurality of electrodes electrically connected to a plurality of interconnects, wherein the first electrical potential is applied at first concentration of glucose; and applying a second electrical potential to the mesh, wherein the second electrical potential is applied at a second concentration of glucose, different than the first concentration of glucose is described.

In a different aspect, an article is described, the article comprising a mesh comprising a plurality of wires, wherein at least a portion of the wires are electrically coupled to one or more interconnects; and a plurality of pancreatic cells adjacent to the mesh and in electrical communication with at least one interconnect of the one or more interconnects, wherein the plurality of wires and/or the one or more interconnects are stretchable and/or flexible and/or soft.

In yet another aspect, a system, comprising a mesh comprising a plurality of sensors electrically connected to a plurality of interconnects and a plurality of electrodes, wherein the mesh defines a processor configured to convert sensor measurements to electrical stimuli; and a plurality of pancreatic cells in electrical communication with the plurality of electrodes is described.

In yet another aspect, a method is described, the method comprising determining a concentration of a compound in an implant contained within a subject using a sensor configured to sense the compound; converting a sensor measurement to an electrical stimulus within a flexible and/or stretchable and/or soft mesh comprising a plurality of wires; and applying the electrical stimulus to one or more pancreatic cells.

In still another aspect, an article is described. According to some embodiments, the article comprises: a mesh comprising a plurality of electrodes electrically connected to a plurality of interconnects; and a plurality of pancreatic cells in contact with at least one of the electrodes, wherein the pancreatic cells have been entrained by circadian feeding-fasting entrainment.

Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

FIGs. 1A-1B illustrate cell scaffolds embedded within organoids, in accordance with certain embodiments. FIG. 2 illustrates a method of producing a cell scaffold in accordance with another embodiment.

FIGs. 3A-3F illustrate a method for building cyborg human SC-islets, according to some embodiments. FIG. 3 A includes schematics showing integration of stretchable mesh nanoelectronics into cyborg islets for long-term stable electrical recording. Human pluripotent stem cell-derived islet endocrine cells are embedded with stretchable mesh nanoelectronics on a Matrigel™ hydrogel substrate. Co-culture with mesenchymal stem cells stimulated self-assembly of cyborg islets within 48 hours. Electrical recordings were performed weekly over a 2-month time course of early maturation prompted by extended culture, and hourly upon further maturation induced by entrainment to circadian feeding cycles. FIG. 3B Left: Shows the three-dimensional (3D) rendering of fluorescence imaging of a cleared, immunostained cyborg islet at 2 months post device integration. Right: shows a zoom-in view of the white box inset in the left panel that shows intimate coupling of flexible interconnects from the device with SC-a and SC-P cells. The various shadings represent INS (Insulin); GCG (Glucagon); R6G (device); and DAPI. FIG. 3C, Left: Shows representative raw voltage traces showing spike bursting dynamics for a cyborg islet exposed to 2.8 mM and 20 mM glucose at 6 weeks post device integration. Middle: Shows single spikes from the regions highlighted in the box insets from 2.8 mM and 20 mM glucose treatments (the positions of the insets are identified with bold arrows). Right: Shows representative averaged single spike waveforms. EIG. 3D shows the data analysis pipeline for the spike analysis, according to some embodiments. Spike sorting was performed on 300-3000 Hz filtered voltage traces (i) to identify distinct waveforms (ii). 3D UMAP waveform clustering identifies cellspecific activity profiles (iii). Representative cell- specific spike trains were detected from 5-minute continuous recordings (iv). PIG. 3E shows 2D UMAP clustering of single-cell RNA expression profiles from cyborg islet (with device) and control islet (without device), according to some embodiments. Cells are indicated by sample of origin (left) and by cell type assignment (middle), with cell type compositions in cyborg and control islets shown to the right. PIG. 3P shows expression profiles of markers for each cell type identified in cyborg and control islets, shown as normalized z-scores, according to one set of embodiments.

PIGs. 4A-4F show simultaneous tracing of islet-wide cell type-specific and stimulus-coupled electrical dynamics, according to some embodiments. FIG. 4A shows schematics that demonstrated SC-a and SC-P cells present electrical activities, voltagegated glucagon and insulin secretion in response to low (2.8 mM) and high (20 mM) glucose stimulation, respectively, upon functional maturation. FIG. 4B shows cyborg islet cell type- specific electrical features analyzed, according to some embodiments. Spike firing rate, amplitude, duration, peak-trough ratio, width at half-maximum, repolarization slope, and recovery slope were compared between 2.8 mM and 20 mM glucose stimulation. FIG. 4C shows SC-a cell spike trains (i), average amplitude (ii), and average firing rates (iii) recorded at 2.8 mM and 20 mM glucose (bar plots show mean ± SEM of N = 19 cells pooled from n = 5 cyborg islets), according to some embodiments. Glucagon secretion into the medium measured by protein ELISA at 2.8 mM and 20 mM glucose (iv) (data are mean ± SEM from n = 2 replicate measurements). FIG. 4D shows SC-P cell spike trains (i), average amplitude (ii), and average firing rate (iii) recorded at 2.8 mM and 20 mM glucose (bar plots show mean ± SEM of N = 71 cells pooled from n = 5 cyborg islets), according to some embodiments. Insulin secretion into the medium measured by protein ELISA at 2.8 mM and 20 mM glucose (iv) (data are mean ± SEM from n = 2 replicate measurements). FIG. 4E shows a heatmap showing the firing rate of SC-a cells recorded at 2.8 mM glucose, 20 mM glucose, and 2.8 mM glucose + 100 pM tolbutamide (i) (N = 32 cells pooled from n = 3 cyborg islets). The firing rate per minute of SC-a cells was recorded at 2.8 mM glucose and 1 pM tetrodotoxin (ii) (bar plots show mean ± SEM of n = 12 pooled from n = 2 cyborg islets). Glucagon secretion into the medium measured by protein ELISA at 2.8 mM glucose and 2.8 mM glucose + 100 pM tolbutamide (iii) (data are mean ± SEM from n = 2 replicate measurements). FIG. 4F shows a heatmap showing the firing rate of SC-P cells recorded at 2.8 mM glucose, 20 mM glucose, and 20 mM glucose + 10 pM forskolin (i) (N = 15 cells pooled from n = 3 cyborg islets). Firing rate per minute of SC-P cells recorded at 20 mM glucose and 1 pM tetrodotoxin (ii) (bar plots show mean ± SEM of n = 27 pooled from n = 2 cyborg islets). Insulin secretion into the medium measured by protein ELISA at 20 mM glucose and 20 mM glucose + 10 pM forskolin (iv) (data are mean ± SEM from n = 2 replicate measurements), n.s., not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 from two-tailed, unpaired t tests, according to one set of embodiments.

FIGs. 5A-5L show long-term stable tracking of SC-a and SC-P electrical activities during early functional maturation, in accordance with some embodiments. FIG. 5 A shows representative electrical spike trains recorded over 5 minutes at 2.8 mM and 20 mM glucose from weeks 2 to 7 after cyborg islet device integration, according to some embodiments. Cyborg islets were cultured in serum-free medium without exogenous signaling factors. Data was collected from 6 cyborg islets. FIG. 5B shows clustering of firing rates during extended culture of cyborg islets identified cells with increased firing under 2.8 mM (A) or 20 mM glucose (B) as SC-a or SC-P, respectively (i). The percentage of SC-a, SC-P, and other cells (ii). FIG. 5C shows spike firing rates for SC-a 1 and SC-P 1 cells at 2.8 mM and 20 mM glucose during extended culture after device integration (bar plots show mean ± SEM of N = 33 SC-a 1 and 126 SC-P 1 cells pooled from n = 5 cyborg islets). FIG. 5D shows spike firing rates for SC-a 2 and SC-P 2 cells at 2.8 mM and 20 mM glucose during extended culture after device integration (bar plots show mean ± SEM of N = 57 SC-a 2 and 193 SC-P 2 cells pooled from n = 5 cyborg islets). FIG. 5E shows the number of electrically recorded SC-a 1 and SC-P 1 cells from week 2 to week 7 after device integration. Data are mean ± SEM of N = 33 SC-a 1 and 126 SC-P 1 cells pooled from n = 5 cyborg islets. FIG. 5F shows the number of electrically recorded SC-a 2 and SC-P 2 cells from week 2 to week 7 after device integration. Data are mean ± SEM of N = 57 SC-a 2 and 193 SC-P 2 cells pooled from n = 5 cyborg islets. FIG. 5G shows spike firing rate ratios for SC-a 2 (2.8 mM / 20 mM glucose) and SC-P 2 (20 mM / 2.8 mM glucose) cells from week 2 to week 7 after device integration. Data are mean ± SEM of N = 48 SC-a 2 and 126 SC-P 2cells pooled from n = 5 cyborg islets. FIG. 5H shows spike amplitude ratios for SC-a 2 (2.8 mM / 20 mM glucose) and SC-P 2 (20 mM / 2.8 mM glucose) cells from week 2 to week 7 after device integration. Data are mean ± SEM of N = 48 SC-a 2 and 126 SC-P 2 cells pooled from n = 5 cyborg islets. FIG. 51 shows early maturation trajectory of SC-a cells using 2D PHATE visualization, by month (top) or inferred pseudotime ordering (middle), and pseudotime distributions of month 1 and month 2 islet cells (bottom). FIG. 5 J shows SC- a gene expression programs along with their enriched pathways. Heatmap shows the row z-scored expression of selected dynamic genes along SC-a pseudotime. FIG. 5K shows early maturation trajectory of SC-P cells using 2D PHATE visualization, by month (top) or inferred pseudotime ordering (middle), and pseudotime distributions of month 1 and month 2 islet cells (bottom). FIG. 5L shows SC-P gene expression programs along with their enriched pathways. Heatmap shows the row z-scored expression of selected dynamic genes along SC-P pseudotime, according to one set of embodiments; and FIGs. 6A-6J show tracing of SC-a and SC-P electrical maturation triggered by circadian feeding entrainment, according to some embodiments. FIG. 6A shows a timeline for metabolic shock/recovery cycles conducted 4 times over 4 days, followed by functional assays, including electrical and insulin/glucagon secretion measurements at 2.8 mM and 20 mM glucose, conducted every 4 hours for 72 hours. FIGs. 6B-6C show spike trains recorded over 5 minutes at 2.8 mM (FIG. 6B) and 20 mM (FIG. 6C) glucose every 4 hours for 3 days following circadian feeding entrainment. FIGs. 6D-6E show the spike firing rate ratios for SC-a (FIG. 6D) and SC-P (FIG. 6E) cells recorded over 72 hours after cyborg islet diurnal feeding (entrained) or mock (control) entrainment. Data were detrended (baseline-subtracted) mean ± SEM of N = 210-420 SC-a and SC-P cells pooled from 5 independent entrained organoids and of N = 60-52 SC-a and SC-P cells from a control islet. FIGs. 6F-6G show glucose stimulation indexes for glucagon (FIG. 6F) and insulin (FIG. 6G) secretion into the medium measured by protein ELISA over 72 hours after cyborg islet diurnal feeding (entrained) or mock (control) entrainment. Data were detrended (baseline-subtracted) mean ± SEM of 5 independent entrained cyborg islets and a control islet, with n = 2 replicate measurements each. FIG. 6H shows 3D PHATE visualization of the averaged spike waveforms of N = 341 SC-P cells over 72 hours at 20 mM glucose from an entrained cyborg islet. FIGs. 6I-6J show MCODE analysis of biological functions enriched among genes upregulated in SC-a (FIG. 61) and SC-P (FIG. 6J) cells in circadian-entrained versus control cyborg islets. For SC-a cells, 6 modules (i.e., COPI complex | vesicle-mediated transport, respiratory electron transport, type 1 diabetes mellitus, metabolism of RNA, and circadian entrainment | cAMP-coupled GPCR signaling) were shown (FIG. 61); For SC-P cells, 5 modules (i.e., insulin signaling, COPI complex | vesicle-mediated transport, respiratory electron transport, and regulation of membrane potential | ion transport, etc.) were shown (FIG. 6J), according to one set of embodiments.

FIGs. 7A-7M show the design, fabrication, and integration of stretchable mesh electronics into cyborg islets, in accordance with some embodiments. FIG. 7A shows a schematic illustrating the multilayer structure of stretchable mesh electronics. FIG. 7B shows representative bright-field (BF) images of unreleased (i) and released (ii) 16- channel stretchable mesh electrodes. Zoom-in image (iii) shows the individual electrode, fluorescence bar code, and twisted stretchable interconnects. FIG. 7C shows representative BF images of representative unreleased (i) and released (ii) 64-channel high-density electrode arrays in stretchable mesh electronics. FIG. 7D shows average electrochemical impedance of electrodes at 1 kHz (n = 64 electrodes for each sample device, values are mean ± SEM). FIG. 7E shows average impedance of electrodes at 1 kHz as a function of incubation time in PBS at 37°C (n = 10 samples, values are mean ± SD). FIG. 7F shows the optical photograph of a representative culturing chamber with four cyborg islets. FIG. 7G shows BF phase images show a representative cyborg islet integrated with 16-channel stretchable mesh nanoelectronics. Inset shows the zoom-in view of box-highlighted region showing an electrode embedded in the islet. FIGs. 7H-7I show BF phase image of a representative cyborg islet integrated with 64-channel stretchable mesh electronics (FIG. 7H) and zoom-in view (FIG. 71) show the embedded high-density electrode array. FIGs. 7J-7K show fluorescence images of cleared, immunostained control SC-islets (without device integration) with 16-channel stretchable mesh nanoelectronics. Zoom-in views show cell morphologies and celldevice coupling, respectively. Various shadings represent Insulin (INS), glucagon (GCG), CD44, device, and DAPI. FIG. 7L-7M show fluorescence images of cleared, immunostained cyborg SC-islets with 16-channel stretchable mesh nanoelectronics (FIG. 7L) and 64-channel stretchable mesh nanoelectronics (FIG. 7M). Zoom-in views show cell morphologies and cell-device coupling, respectively. Various shadings represent Insulin (INS), CD44, glucagon (GCG), device, and DAPI.

FIG. 8 shows the expression of gene markers in cyborg and control islets, according to some embodiments. 2D UMAP visualization of clustering of single-cell RNA expression profiles for the indicated marker genes in cyborg (with device) and control (without device) islets is presented, according to some embodiments. Cells are shaded by gene expression level.

FIGs. 9A-9D show the analysis of cell type-specific electrical features from cyborg islet recordings, in accordance with some embodiments. FIGs. 9A-9B show electrical features from SC-a (FIG. 9A) and SC-P (FIG. 9B) cell recordings. Spike duration, peak-trough ratio, width at half-maximum, repolarization slope, and recovery slope were analyzed and compared between 2.8 mM and 20 mM glucose incubations, n.s., not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 from two- tailed, unpaired t test. FIG. 9C shows SC-a cell spike trains recorded at 2.8 mM glucose, 20 mM glucose, and 2.8 mM glucose + 100 pM tolbutamide (N = 32 cells pooled from n = 3 cyborg islets). FIG. 9D shows SC-P cell spike trains recorded at 2.8 mM glucose, 20 mM glucose, and 20 mM glucose + 10 pM forskolin (N = 15 cells pooled from n = 3 cyborg islets).

FIGs. 10A-100 show analysis of cell type-specific electrical features from cyborg islet long-term recordings, in accordance with some embodiments. FIG. 10A shows the multiple of the median (MoM) as a measure of how far an individual SC-a (i) or -P (ii) cell firing rate ratio deviated from the median across the population for further classifying cell state 1 and 2, according to some embodiments. The number of electrically recorded SC-a, SC-P and other cells from week 2 to week 7 after device integration (iii). Data are mean ± SEM of N = 380 SC-a, 800 SC-P, and 1 6 other cells pooled from n = 6 cyborg islets. FIG. 10B shows the spike firing rates for SC-a 1 (i) and -a 2 (ii) cells at 2.8 mM and 20 mM glucose from week 2 to week 7 after device integration. FIG. 10C shows spike firing rates for SC-P 1 (i) and - P 2 (ii) cells at 2.8 mM and 20 mM glucose from week 2 to week 7 after device integration. FIG. 10D shows the amplitudes for SC-a 1 (i) and -a 2 (ii) cells at 2.8 mM and 20 mM glucose from week 2 to week 7 after device integration. FIG. 10E shows the amplitudes for SC-P 1 (i) and -P 2 (ii) cells at 2.8 mM and 20 mM glucose from week 2 to week 7 after device integration. FIGs. 10F-10O show electrical features including spike duration (FIGs. 10F-10G), peaktrough ratio (FIGs. 10H-10I), width at half-maximum (FIGs. 10J-10K), repolarization slope (FIGs. 10L-10M), and recovery slope (FIGs. 10N-10O) for SC-a 1 & P 1 (i) and SC-a 2 & P 2 (ii) cells were analyzed.

Plots show mean ± SEM of N = 33 SC-a 1 and 57 SC-a 2, 126 SC-P 1 and 193 SC- P 2 cells pooled from n = 5 cyborg islets. Two-tailed, unpaired Welch’s t-tests were conducted under the two glucose conditions.

FIGs. 11A-11C show early cyborg islet maturation characterized by sc-RNA seq, according to some embodiments. FIG. 11A shows a heatmap showing biological functions enriched among genes upregulated in SC-a, SC-P, or SC-EC cells in later (month 2) versus earlier (month 1) pseudotime. FIG. 11B shows early maturation trajectory of SC-EC cells using 2D PHATE visualization, by month (top) or inferred pseudotime ordering (middle), and pseudotime distributions of month 1 and month 2 islet cells (bottom). FIG. 11C shows SC-EC pseudotime-dependent gene expression programs along with their enriched pathways, which are also highlighted in bold in panel (FIG. 11 A). Heatmap shows the row z-scored expression of selected dynamic genes along SC- EC pseudotime. FIGs. 12A-12O show SC-a and SC-P cell maturation triggered by circadian entrainment, according to some embodiments. FIGs. 12A-12B show fluorescence images of tissue cleared, immunostained cyborg islets without (FIG. 12A) and with (FIG. 12B) circadian entrainment at month 2 after device integration. Zoom-in views show cell morphologies and cell-device coupling. Various shadings represent Insulin (INS), Glucagon (GCG), device, and DAPI. FIGs. 12C-12D show spike firing rates at 2.8 mM (FIG. 12C) and 20 mM (FIG. 12D) glucose recorded over 72 hours after cyborg islet diurnal feeding (entrained) or mock (control) entrainment. Data are mean ± SEM across N = 220 recording channels pooled from 5 independent entrained cyborg islets and across N = 50 recording channels from a control islet. FIGs. 12E-12H show spike firing rate rhythms of SC-a cells during mock (control) entrainment (FIG. 12E) and after diurnal feeding (entrained) (FIG. 12F), and SC-P cells during mock (control) entrainment (FIG. 12G) and after diurnal feeding (entrained) (FIG. 12H) at 2.8 mM and 20 mM glucose recorded over 72 hours. P-values, RAIN rhythmicity test. FIGs. 12I-12J show spike firing rates of SC-a (FIG. 121) and SC-P (FIG. 12J) cells at 2.8 mM and 20 mM glucose after cyborg islet diurnal feeding (entrained, “B” in the figures) or mock (control, “A” in the figures) entrainment. Data are mean ± SEM across N = 200 recording channels over 72 hours pooled from 5 independent entrained cyborg islets and across N = 50 recording channels over 72 hours from a control islet. FIG. 12K shows the biological functions enriched among protein-protein interaction complexes identified by the MCODE algorithm based on genes upregulated in SC-EC cells in circadian-entrained versus control cyborg islets. The top 4 interaction complexes identified (calcium ion transmembrane transport, respiratory electron transport, cell-cell adhesion, and enzyme- linked receptor signaling | cell migration) are highlighted. FIG. 12L shows a heatmap showing biological functions enriched among genes upregulated in SC-a, SC-P, or SC- EC cells in circadian-entrained versus control cyborg islets. FIGs. 12M-12N show network visualization of enriched biological pathways among genes upregulated in circadian-entrained versus control cyborg islets, shaded by enrichment cluster terms (FIG. 12M) or by cell type (FIG. 12N). FIG. 120 shows bar plots showing expression of selected genes in control versus circadian-entrained cyborg islets. Data are mean ± s.e.m., *** p<0.001, **** p<0.0001. DETAILED DESCRIPTION

The following disclosure describes articles, systems, and methods measuring and/or stimulating pancreatic cells (e.g., pancreatic organoids, pancreatic islets). By way of illustration, and not limitation, the articles, systems, and methods include a mesh comprising one or more electrodes and/or interconnects. The one or more electrodes may be in contact with one or more pancreatic cells, and the one or more pancreatic cells may be a part or a portion of a pancreatic organoid and/or a pancreatic islet. The pancreatic cells can be electrically stimulated by the electrodes. In some embodiments, the electrical stimulation is used to measure or determine a property of the pancreatic cells. In some embodiments, electrical stimulation is used to direct the growth or differentiation of the pancreatic cells. In some embodiments, electrical stimulation is used to measure a property of the pancreatic cells and direct the growth or differentiation of the pancreatic cells.

In many cases, the articles, systems, and methods include stretchable electronics (e.g., electrodes, interconnects) implanted during tissue formation (e.g., pancreatic cell growth) to allow tissue-wide electrophysiology. For example, in some embodiments, a stretchable mesh comprising nanoelectronics (e.g., nanoscale electrode, microscale electrodes and/or interconnects, etc.) during organogenesis of stem cell-derived pancreatic islets, allowing long-term, stable recording of single-cell extracellular spike bursting dynamics.

One aspect is now described with reference to FIG. 1A. In this figure, a cell scaffold is shown, some or all of which may be stretchable. The cell scaffold, for example, may comprise a plurality of nodes connected by various interconnects, e.g., forming a mesh structure, where the interconnects have a shape and/or are formed from materials that allow the interconnects to be manipulated or distorted without disrupting their connections, e.g., during stretching, compression, folding, or the like. For example, the interconnects may have an “S” or a serpentine shape. In some cases, the interconnects may be formed from materials that can be manipulated by the cells within the scaffold. For example, the cells may be pancreatic islets, and the scaffold may be used as at implantable article to diagnose and or control diabetes or other pancreatic diseases.

In some cases, the cells may be partially or completely embedded within the implantable article. For example, the implantable article may be a pancreatic implant which can be implanted into a subject in some embodiments. In some embodiments, the pancreatic implant may be formed around the cell scaffold, in contrast to techniques in which the cell scaffold is implanted into a biological structure (i.e., a pre-existing biological structure). In some cases, the biological structure (e.g., a pancreatic implant) is able to manipulate or distort the cell scaffold as part of the growth process, e.g., to cause the cell scaffold to form a more suitable embedded shape within the biological structure. For example, as is shown in FIG. 1A, a cell scaffold may initially be substantially planar and seeded with cells that form a substantially spherical organoid; as the organoid forms and develops into the implantable article, the cells cause the cell scaffold to adopt more of a spherical configuration, thereby resulting in an organoid embedding the cell scaffold.

In certain embodiments, the cell scaffold may also contain components, including nanoelectric components, that may form electrical circuits, or portions thereof. For instance, some or all of the interconnects may contain metal or other conductive pathways, and/or there may be sensors, stimulators, nanoscale wires, or the like within the cell scaffold. In some cases, the cell scaffold may define a self-contained electrical circuit, and/or a portion of the cell scaffold may be interfaceable or connectable with an external electrical device, such as a computer, using a suitable connector, such as a cable. Thus, for example, in some cases, the electrical activity of the pancreatic islets may be monitored, and in some embodiments, used to control the pancreatic islets.

In various embodiments, the cell scaffold within the implantable article can be connected to electronic circuits extending externally of the cell scaffold. The nanoscale wires may form an integral part of the biological structure, and can be determined or controlled, e.g., using an external electrical device. This allows for the creation of different types of functionalized biological structure, e.g., due to the high degree of electronic control. Accordingly, such implantable articles can be determined and/or controlled at high resolutions, e.g., spatial and/or temporal resolutions. In some cases, such implantable article can be used in a wide variety of applications, e.g., for modeling cells or implantation into a subject.

The above discussion is a non-limiting example of one embodiment of the present disclosure that are directed to cell scaffolds, including those embedded (partially or completely) within biological structures, such as tissues, organoids, organs, organisms, and the like. However, other embodiments are also possible. Accordingly, more generally, various aspects of the disclosure are directed to various cell scaffolds and other devices.

For example, in various aspects, cells may be cultured on a cell scaffold and allowed to grow to become an article, such as an implantable article (for example, a pancreatic implant). The cell scaffold can become partially or completely embedded within the structure, e.g., during growth of structure.

A variety of cells may be used with the scaffold. The cell may be an isolated cell, a cell aggregate, in a tissue construct containing cells, or the like. If the cell is from a multicellular organism, the cell may be from any part of the organism. For instance, the cells may include pancreatic cells, such as pancreatic islets. In certain embodiments, the cells are stem cells, e.g., pluripotent stem cells. In some cases, the cells may also be exposed to other compounds, such as drugs, to determine their effects on the growth of the cells into organoids, organs, or organisms. This may be useful, for example, for drug testing.

For example, in one set of embodiments, cells such as pancreatic cells may be seeded on a cell scaffold, and allowed to grow or self-assemble into a biological structure, e.g., to form an implantable article. In some cases, the article may comprise a mesh or a network, e.g., comprise one or more nodes that are connected by various interconnects, e.g., forming a mesh or a network. The article may also comprise one or more electrodes, e.g., such as those described herein. In some cases, the electrodes and/or the interconnects may be flexible, stretchable, soft, or have other properties such as any of those described in more detail herein. In some cases, the electrode may be used to determine a property of the cell scaffold (e.g., an electrical property, a chemical property, a mechanical property, etc.), and/or to apply a stimulus (e.g., an electrical stimulus) to the biological structure. In some cases, for example, the mesh may comprise a plurality of electrodes electrically connected to a plurality of interconnects. In some embodiments, the article is a device comprising electrodes and/or various interconnects.

In certain embodiments, the article may be in contact with one or more cells. Examples of cells include any of those described herein. In certain cases, the cells may include pancreatic cells, for example, pancreatic islets. Non-limiting examples of islet cells include alpha, beta, gamma, delta, epsilon, or PP cells. In addition, in some cases, the pancreatic cells may include immature pancreatic cells and/or mature pancreatic cells. One or more than one type of pancreatic cell may be present, e.g., a first pancreatic cell type and a second pancreatic cell type, etc. In some cases, the electrodes within a mesh may be in physical contact and/or in electrical communication with one or more of the pancreatic cells. In some cases, different electrodes may be in physical contact and/or in electrical communication with different pancreatic cells. In some cases, the pancreatic cells may form an organ or an organoid, e.g., partially or fully containing the mesh or network.

In one non-limiting set of embodiments, such articles containing one or more pancreatic cell types may be used to sense glucose, and/or to treat diabetes. For example, an article such as any of those described herein may be implanted into a subject. In some cases, the article may be used to determine and/or control glucose levels in a subject, e.g., after implantation. The subject may be human or a non-human mammal. Diabetes may include, for example, type 1 diabetes and/or type 2 diabetes.

In some cases, the article may be used to administer, to the subject, an effective amount of insulin, for example, in response to glucose within the subject, e.g., within the subject’s bloodstream. For example, in one set of embodiments, a glucose concentration may be determined, processed within the article, and used to stimulate one or more pancreatic islets to produce insulin. In some cases, different glucose concentrations may result in different stimulations of pancreatic islets. For example, a first glucose concentration may cause a first electrical potential to be applied, e.g., to the mesh, or to one or more pancreatic cells, etc., while a second glucose concentration, different from the first glucose concentration, may cause a second electrical potential to be applied, e.g., to the mesh, or to one or more pancreatic cells, etc.

In some cases, an effective amount of insulin may be administered to the subject in response to a glucose concentration, or a change in glucose concentration. An “effective amount” of an agent is based at least in part, on the tissue and/or cell type targeted, the means of administration, characteristics of the agent, etc. Other determinants include the body weight, age, height, sex and general health of the subject. As used herein, “treat” to either therapeutic treatment or prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described herein (e.g., diabetes). Thus, in some embodiments, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. In some embodiments, “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.

In some case, the article may define a special purpose computer system that may be specially programmed to be used in connection with any of the embodiments of the disclosure provided herein. The article may include one or more processors and one or more articles of manufacture that comprise non-transitory computer-readable storage media. The processor may control writing data to and reading data from the memory and the non-volatile storage device in any suitable manner. To perform any of the functionality described herein (e.g., determining glucose concentrations, stimulating pancreatic cells in response, etc.), the processor may execute one or more processorexecutable instructions e.g., stored within the article. In one set of embodiments, the processor may be able to build or use predictive models (for example, artificial intelligence or machine learning) and/or return probabilistic outputs, e.g., to be applied to the pancreatic cells, for example, based on glucose concentrations.

The terms “program” or “software” or “app” are used herein in a generic sense to refer to any type of computer code or set of processor-executable instructions that can be employed to program a computer or other processor to implement various aspects of embodiments as discussed above. Additionally, it should be appreciated that according to one aspect, one or more computer programs that when executed perform methods of the disclosure provided herein need not reside on a single computer or processor, but may be distributed in a modular fashion among different computers or processors to implement various aspects of the disclosure provided herein.

Processor-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

Also, data structures may be stored in one or more non-transitory computer- readable storage media, e.g., within the article, in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a non-transitory computer-readable medium that convey relationships between the fields. However, any suitable mechanism may be used to establish relationships among information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationships among data elements.

Also, various inventive concepts may be embodied as one or more processes, of which examples have been provided. The acts performed as part of each process may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

In some embodiments an effective amount of the agent is administered to a subject (e.g., a mammalian subject, such as a human or a non-human subject) to treat a cancer or multiple cancers. The agent may be administered by any route that results in a therapeutically effective outcome, including but not limited to intradermal, intramuscular, intranasal, and/or subcutaneous administration. An “effective amount” of an agent is based at least in part, on the tissue and/or cell type targeted, the means of administration, characteristics of the agent. Other determinants include the body weight, age, height, sex and general health of the subject. Typically, an effective amount of an agent treats a cancer. As used herein, “treat” to either therapeutic treatment or prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described herein (e.g., cancer). Thus, in some embodiments, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. In some embodiments, “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.

In one set of embodiments, glucose concentration may be determined based on enzymatic reaction. For example, the mesh, such as any of those described herein, may contain one or more enzymes, e.g., on a surface of an electrode, and/or elsewhere within the article. In some cases, the enzyme may react with an analyte (e.g., glucose) and produce a species that can be determined electrochemically, e.g., using one or more electrodes within the mesh. Non-limiting examples of glucose-sensitive enzymes include, but are not limited to, glucose oxidase (GOx), glucose dehydrogenase nicotinamide adenine dinucleotide (GDH-NAD), glucose dehydrogenase flavin adenine dinucleotide (GDH-FAD), glucose dehydrogenase pyrroloquinoline quinone (GDH- PQQ), etc. Concentrations of glucose may be related to electrical activity (e.g., voltage and/or current signals).

Various embodiments described here include one or more pancreatic cells (e.g., a first pancreatic cell type, a second pancreatic cell type, a plurality of pancreatic cells). Depending on the embodiment, an electrode (e.g., a nanoscale electrode, a microscale electrode) may be in contact with one or more pancreatic cells, and the electrode can provide a current to the pancreatic cell and/or a first electrode and a second electrode provide an electrical potential across the pancreatic cell.

In some embodiments, the one or more pancreatic cells contacted with at least one electrode and in electrical communication through at least one interconnect. In some embodiments, the method comprises comparing a response of the first pancreatic cell type to the first electrical potential to a response of the second pancreatic cell type to second electrical potential. In some embodiments, the first pancreatic cell type comprises immature pancreatic cells. In some embodiments, the second pancreatic cell type comprises mature pancreatic cells. In some embodiments, wherein the pancreatic cells form a portion of a pancreatic organoid. In some such embodiments, the first pancreatic cell type and the second pancreatic cell type (or some other types of cells) can be distinguished based on the responsive activity of the two cells types to different concentrations of the glucose (or some other metabolite).

According to some embodiments, the one or more pancreatic cells have been entrained by circadian feeding-fasting entrainment. Entrainment by circadian feedingfasting may comprise subjecting pancreatic cells (e.g., the cells of pancreatic islets) to shock and recovery cycles (e.g., glucose and/or forskolin shock and recovery cycles) at regular intervals. The intervals may have any of a variety of appropriate durations. In some embodiments, cells are entrained with feeding-fasting cycles having a duration of greater than or equal to 12 h, greater than or equal to 24 h, or greater than or equal to 36 h. In some embodiments, cells are entrained with feeding-fasting cycles having a duration of less than or equal to 48 h, less than or equal to 36 h, or less than or equal to 24 h. Combinations of these ranges are also possible (e.g., greater than or equal to 12 h and less than or equal to 48 h, or greater than or equal to 24 h and less than or equal to 36 h). For example, in some embodiments, the shock and recovery cycles are diurnal.

Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

Entrainment of the cells may be associated with more consistent functioning of the pancreatic cells, and may, in some cases, be associated with long term electrical activity in the presence of glucose, relative to unentrained pancreatic cells. For example, entrained pancreatic cells exhibit detectable electrical activity for durations of greater than or equal to 48 h, 60 h, 72 h, or more. Such entrained pancreatic cells may, advantageously, be more functionally mature and/or potent than unentrained pancreatic cells.

In some embodiments, the method includes culturing the plurality of pancreatic cells. In some embodiments, the method includes dissociating the plurality of pancreatic cells into single cells.

As noted above, some embodiments include applying an electrical potential across one or more pancreatic cells. In some embodiments, the first electrical potential is applied at first concentration of glucose. In some embodiments, the first electrical potential is applied at first concentration of glucose. In some embodiments, a second electrical potential is applied to the mesh, wherein the second electrical potential is applied at a second concentration of glucose.

In some embodiments, an electrical potential is applied across one or more pancreatic cells (e.g., one or more pancreatic cell types). For example, in some embodiments, a first concentration of glucose is less than or equal to 10 mM and/or a second concentration of glucose is greater than or equal to 10 mM. In some embodiments, a concentration of glucose of or surrounding the cells (e.g. pancreatic cells), is less than or equal to 10 mM, less than or equal to 5 mM, or less than or equal to 1 mM. In some embodiments, a concentration of glucose of or surrounding the cells is greater than or equal to 1 mM, greater than or equal to 5 mM, or greater than or equal to 10 mM. Combinations of the foregoing range are possible (e.g., greater than or equal to 1 mM and less than or equal to 10 mM). Other ranges are possible.

As mentioned, certain aspects are generally directed to flexible and/or stretchable and/or soft meshes or networks that can be used as cell scaffolds to grow structures such as organoids, tissues, or even organisms. In general, cell scaffolds are structures that cells can attach to and grow on, e.g., to form biological tissues and other biological structures, for example, implantable articles such as pancreatic implants. The cell scaffold may comprise biocompatible and/or biodegradable materials, and may in some embodiments also contain growth factors such as growth hormones, extracellular matrix proteins, specific metabolites or nutrients, or the like. The cell scaffold typically is porous, e.g., to facilitate cell seeding therein, and/or diffusion into and out of the cell scaffold, for example, of nutrients, waste products, etc.

In one set of embodiments, the scaffold may have a shape and/or may be formed from one or more materials that allow the scaffold to be flexible and/or stretchable and/or soft. For example, the scaffold may be formed of shapes, such as serpentine shapes, that can be extended. In some cases, the cell scaffold can be formed of components that are not straight, and can be extended, e.g., when pulled on. For instance, the cell scaffold may comprise one or more nodes that are connected by various interconnects, e.g., forming a mesh or a network. The nodes may be evenly or nonevenly distributed within the cell scaffold, and the interconnects may connect them in a regular pattern (for example, in rectangular or triangular arrays of nodes), or in an irregular pattern. As a non-limiting example, FIG. 1A, panel I shows a mesh of nodes (dots) in a square array connected by a plurality of interconnects between pairs of nodes (shown as wiggly lines). The nodes may represent points of connectivity, or there may be one or more electronic components at some or all of the nodes, such as conductive pathways, nanoscale wires, sensors, or the like. The same or different electronic components may independently be present at different nodes within a mesh or network.

The interconnects connecting two (or more) nodes together may have the same or different shapes or structure within a mesh or network, and different interconnects within the mesh may independently have the same or different shapes. In some cases, an interconnect may have a shape that is extendible. For example, an interconnect may have a straight-line or linear shape, or have shapes that are non-linear, such as S shapes, serpentine shapes (e.g., having two, three, four, or more bends or inflection points), zigzag shapes (e.g., having two, three, four, or more vertices), coiled shapes, or the like. Such interconnect shapes may allow various manipulations to occur without disrupting the connection of the interconnect to the nodes, e.g., during stretching, compression, folding, etc.

In one set of embodiments, an interconnect may comprise one or metal leads and one or more polymers, such as those discussed below. The polymers can include photoresist polymers (such as SU-8), and/or biocompatible polymers (such as polyimide, parylene, Matrigel™, as non-limiting example). Other examples of photoresist polymers include, but are not limited to, those described below.

In some cases, the cell scaffold may have an overall filling ratio or area of less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, etc. The filling ratio or area is the area of the physical components of the cell scaffold, compared to the overall area of the cell scaffold (including void spaces). Thus, this is a measure of the “porosity” in two dimensions of the cell scaffold. For example, in some cases, the cell scaffold may have a mesh structure or layout as described above, where the mesh is relatively open. Cell scaffolds with smaller filling ratios thus would have greater “open space,” for example, to allow cells to penetrate.

In some cases, the cell scaffold can be defined by one or more pores. Pores that are too small can hinder or restrict cell access. Thus, in some embodiments, the cell scaffold may have an average pore size of at least about 100 micrometers, at least about 200 micrometers, at least about 300 micrometers, at least about 400 micrometers, at least about 500 micrometers, at least about 600 micrometers, at least about 700 micrometers, at least about 800 micrometers, at least about 900 micrometers, or at least about 1 mm. However, in other embodiments, pores that are too big may prevent cells from being able to satisfactorily use or even access the pore volume. Thus, in some cases, the cell scaffold may have an average pore size of no more than about 1.5 mm, no more than about 1.4 mm, no more than about 1.3 mm, no more than about 1.2 mm, no more than about 1.1 mm, no more than about 1 mm, no more than about 900 micrometers, no more than about 800 micrometers, no more than about 700 micrometers, no more than about 600 micrometers, or no more than about 500 micrometers. Combinations of these are also possible, e.g., in one embodiment, the average pore size is at least about 100 micrometers and no more than about 1.5 mm. In addition, larger or smaller pores than these can also be used in a cell scaffold in certain cases. Pore sizes may be determined using any suitable technique, e.g., through visual inspection, BET measurements, or the like.

In some cases, an interconnect may have a smallest dimension or a maximum cross-sectional dimension of less than about 100 micrometers, less than about 50 micrometers, less than about 40 micrometers, less than about 30 micrometers, less than about 20 micrometers, less than about 10 micrometers, less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 80 nm, less than about 50 nm, less than about 30 nm, less than about 10 nm, less than about 5 nm, less than about 2 nm, etc. An interconnect may also have any suitable cross-sectional shape, e.g., circular, square, rectangular, polygonal, elliptical, regular, irregular, etc.

In addition, in one set of embodiments, one or more materials within the cell scaffold are flexible and/or stretchable and/or soft. For example, in some cases, a cell scaffold may comprise a mesh or portions thereof (e.g., interconnects) that can be flexible and/or stretchable and/or soft, or can be manipulated or distorted in some fashion. It should be understood that the flexibility of a material is not purely an intrinsic material propriety; a thinner piece of material may offer more flexibility than a comparably thicker piece of the same material. In addition, in some cases, the flexibility of the material may also be a function of its shape, e.g., as discussed above.

Stretchability and/or flexibility can be measured by bending stiffness. For example, in some embodiments, a stiffness of a material within the cell scaffold (e.g., nanoelectrodes, interconnects) has a stiffness between 0.09 to 20 nNm. In some embodiments, a stiffness of a material within the cell scaffold is greater than or equal to 0.09 nNm, greater than or equal to 1 nNm, greater than or equal to 5 nNm, greater than or equal to 10 nNm, greater than or equal to 15 nNm, or greater than or equal to 20 nNm. In some embodiments, a stiffness of a material within the cell scaffold is less than or equal to 20 nNm, less than or equal to 15 nNm, less than or equal to 10 nNm, less than or equal to 5 nNm, less than or equal to 1 nNm, or less than or equal to 0.09 nNm. Combinations of the foregoing range are also possible (e.g., greater than or equal to 0.09 nNm and less than or equal to 20 nNm). Other ranges are possible.

In certain instances, a cell scaffold may have components, such as interconnects, that are sufficiently flexible or stretchable such that the cell scaffold (or a component thereof, such as an interconnect) may be stretchable in a linear direction by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, etc. (e.g., by length), for example, before catastrophic failure of the cell scaffold, breakage, disruption of the connection of the interconnect to the nodes, loss of electrical connections, or the like.

In addition, in certain cases, the cell scaffold may also exhibit some degree of elasticity, e.g., such that the cell scaffold may return (at least partially) to its original structure prior to stretching. For instance, the cell scaffold (or a component thereof, such as an interconnect) may return at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% (perfectly elastic) back to its original structure, measured from when stretching of the material is stopped. Thus, for example, a 1 cm material stretched to 2 cm experiences a 100% stretch in a linear direction, and if it afterwards contracts to 1.5 cm, it exhibits a 50% recovery to its original structure (returning 0.5 cm from its stretch of 1 cm). However, it should be understood that in some embodiments, the cell scaffold is not elastic.

In some embodiments, the scaffold may have components, such as interconnects, that are sufficiently flexible or stretchable such that the cell scaffold (or a component thereof, such as an interconnect) may have an effective bending stiffness of at least 0.01 n-Nm, at least 0.02 n-Nm, at least 0.03 n-Nm, at least 0.04 n-Nm, at least 0.05 n-Nm, at least 0.06 n-Nm, at least 0.07 n-Nm, at least 0.08 n-Nm, at least 0.09 n-Nm, at least 0.1 n-Nm, at least 0.2 n-Nm, at least 0.3 n-Nm, at least 0.4 n-Nm, at least 0.5 n-Nm, at least 0.7 n-Nm, at least 1 n-Nm, at least 1.5 n-Nm, at least 2 n-Nm, at least 2.5 n-Nm, at least 3 n-Nm, at least 3.5 n-Nm, at least 4 n-Nm, at least 4.5 n-Nm, at least 5 n-Nm, etc. In some embodiments, the interconnects may have an effective bending stiffness of less than 5 n-Nm, less than 4.5 n-Nm, less than 4 n-Nm, less than 3.5 n-Nm, less than 3 n-Nm, less than 2.5 n-Nm, less than 2 n-Nm, less than 1.9 n-Nm, less than 1.8 n-Nm, less than 1.5 n-Nm, less than 1.3 n-Nm, less than 1 n-Nm, less than 0.9 n-Nm, less than 0.8 n-Nm, less than 0.5 n-Nm, less than 0.3 n-Nm, etc. Combinations of any of these are also possible; for example, the cell scaffold or interconnect may exhibit an effective bending stiffness of between 0.090 n-Nm and 1.9 n-Nm. See the examples below for an example of determining effective bending stiffness of a material.

In certain embodiments, the cell scaffold may have components, such as interconnects, that are sufficiently flexible or stretchable such that the cell scaffold (or a component thereof, such as an interconnect) can be compressed in a linear direction by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, etc., without catastrophic failure of the cell scaffold, breakage, disruption of the connection of the interconnect to the nodes, loss of electrical connections, or the like.

In addition, in certain embodiments, the cell scaffold may have components, such as interconnects, that are sufficiently flexible or stretchable such that the cell scaffold (or a component thereof, such as an interconnect) is foldable by at least 30°, at least 45°, at least 90°, at least 135°, at least 150°, at least 180°, etc. from an initial planar structure.

Cell scaffolds may comprise a variety of materials in different embodiments. For example, the cell scaffold may comprise one or more polymers, such as photoresists, that define interconnects or other components within the cell scaffold. In some cases, one or more portions of the cell scaffold may comprise components, such as nanoelectric components, that may form electrical circuits within the cell scaffold. For example, the cell scaffold may contain metal or other conductive pathways, e.g., which define an electrical circuit, and/or can be connected to an external electrical device.

Thus, in certain embodiments, the cell scaffold contains one or more polymers, e.g., photoresists, biocompatible polymers, biodegradable polymers, etc., as is discussed herein. For example, in various embodiments, one or more of the polymers may be a photoresist. While not commonly used in cell scaffolds, photoresists are typically used in lithographic techniques, which can be used as discussed herein. For example, the photoresist may be chosen for its ability to react to light to become substantially insoluble (or substantially soluble, in some cases) to a photoresist developer.

Photoresists that can be used include, but are not limited to, SU-8, SI 805, LOR 3A, poly(methyl methacrylate), poly(methyl glutarimide), phenol formaldehyde resin (diazonaphthoquinone/novolac), diazonaphthoquinone (DNQ), Hoechst AZ 4620, Hoechst AZ 4562, Shipley 1400-17, Shipley 1400-27, Shipley 1400-37, or the like. These and many other photoresists are available commercially. Other examples of photoresist polymers include, but are not limited to, those described below, and those described in Int. Pat. Apl. Pub. No. WO 2019/084498, incorporated herein by reference. In some cases, the photoresist may be a soft material, for example, a hydrogel. In some embodiments, the photoresist comprises a polymer formed by photo-curing a fluorinated monomer including cross-linkable function groups using a photoinitiator. One example of such a polymer is perfluoropoly ether dimethacrylate (PFPE-DMA). Other examples are discussed in more detail below. As used herein, the term “soft” may refer to a material or a composite of materials having a relatively low elastic modulus and/or a relatively low yield strength. For example, in some embodiments, a soft material may have an elastic modulus of less than or equal to 5 GPa, less than or equal to 4 GPa, less than or equal to 3 GPa, less than or equal to 2 GPa, or less than or equal to 1 GPa, and/or a yield strength of less than or equal to 100 MPa, less than or equal to 70 MPa, less than or equal to 50 MPa, less than or equal to 40 MPa, less than or equal to 30 MPa, less than or equal to 20 MPa, or less.

In some cases, one or more of the polymers may be biocompatible and/or biodegradable. Examples of such biocompatible and/or biodegradable polymers include, but are not limited to, polyimide, parylene, poly(lactic-co-glycolic acid), polylactic acid, poly glycolic acid, poly (methyl methacrylate), poly (trimethylene carbonate), collagen, fibrin, polysaccharidic materials such as chitosan or glycosaminoglycans, hyaluronic acid, polycaprolactone, and the like. Certain photoresists are also biocompatible and/or biodegradable in some cases.

Typically, a biocompatible material is one that does not illicit an immune response, or elicits a relatively low immune response, e.g., one that does not impair the cell scaffold or the cells therein from continuing to function for its intended use. In some embodiments, the biocompatible material is able to perform its desired function without eliciting any undesirable local or systemic effects in a subject, e.g., when present within a subject. In some cases, the material is present without eliciting any undesirable local or systemic effects, or such that any biological response by the subject does not substantially affect the ability of the material from continuing to function for its intended use. For example, in a cell scaffold, the cell scaffold may be able to support appropriate cellular or tissue activity when implanted within a subject, e.g., including the facilitation of molecular and/or mechanical signaling systems, without substantially eliciting undesirable effects in those cells, or undesirable local or systemic responses, or without eliciting a response that causes the cell scaffold to cease functioning for its intended use.

A biodegradable material typically degrades over time when exposed to a biological system, e.g., through oxidation, hydrolysis, enzymatic attack, phagocytosis, or the like. For example, a biodegradable material can degrade over time when exposed to water (e.g., hydrolysis) or enzymes. In some cases, a biodegradable material is one that exhibits degradation (e.g., loss of mass and/or structure) when exposed to physiological conditions for at least about a month, at least about 6 months, or at least about a year. For example, the biodegradable material may exhibit a loss of mass of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In certain cases, some or all of the degradation products may be resorbed or metabolized, e.g., into cells or tissues. For example, certain biodegradable materials, during degradation, release substances that can be metabolized by cells or tissues.

In some embodiments, the cell scaffold may also contain other materials in addition to the polymers described herein. Non-limiting examples include other polymers, growth hormones, extracellular matrix protein, specific metabolites or nutrients, or the like. For example, in one of embodiments, one or more agents able to promote cell growth can be added to the cell scaffold, e.g., hormones such as growth hormones, extracellular matrix protein, pharmaceutical agents, vitamins, or the like. Many such growth hormones are commercially available, and may be readily selected by those of ordinary skill in the art based on the specific type of cell or tissue used or desired. Similarly, non-limiting examples of extracellular matrix proteins include gelatin, laminin, fibronectin, heparan sulfate, proteoglycans, entactin, hyaluronic acid, collagen, elastin, chondroitin sulfate, keratan sulfate, Matrigel™, or the like. Many such extracellular matrix proteins are available commercially, and also can be readily identified by those of ordinary skill in the art based on the specific type of cell or tissue used or desired.

As another example, in some embodiments, additional scaffold materials can be added to the cell scaffold, e.g., to control the size of pores within the cell scaffold, to promote cell adhesion or growth within the cell scaffold, to increase the structural stability of the cell scaffold, to control the flexibility of the cell scaffold, etc. For instance, additional fibers or other suitable polymers may be added to the cell scaffold, e.g., electrospun fibers can be used as a secondary scaffold. The additional scaffold materials can be formed from any of the materials described herein in reference to cell scaffolds, e.g., photoresists or biocompatible and/or biodegradable polymers, or other polymers described herein. As another non-limiting example, a glue such as a silicone elastomer glue can be used to control the shape of the cell scaffold.

In one set of embodiments, the cell scaffold may contain metal or other conductive pathways, e.g., within interconnects or nodes within the cell scaffold. Examples of metals for metal leads or pathways that can be used include, but are not limited to platinum, aluminum, gold, silver, copper, molybdenum, tantalum, titanium, nickel, tungsten, chromium, palladium, or the like, as well as any combinations of these and/or other metals. Other examples include conductive polymers such as platinum black, poly (3, 4-ethylenedioxy thiophene) (PEDOT), poly acetylene, polyphenylene vinylene, polypyrrole, polythiophene (for example poly(3,4-ethylenedioxythiophene)), polyphenylene sulfide, etc.

In some cases, the material can be chosen to be one that is readily introduced into the cell scaffold, e.g., using techniques compatible with lithographic techniques. For example, in one set of embodiments, lithographic techniques such as e-beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc. may be used to layer or deposit one or more metals on a substrate. Additional processing steps can also be used to define or register the pathways in some cases.

In some embodiments, more than one metal can be used within a pathway. For example, two, three, or more metals may be used within a pathway. The metals may be deposited in different regions or alloyed together, or in some cases, the metals may be layered on top of each other, e.g., layered on top of each other using various lithographic techniques. If dissimilar metals are layered on top of each other, they may be layered in some embodiments in a “stressed” configuration (although in other embodiments, they may not necessarily be stressed). For example, a chromium/palladium/chromium deposition process, in some embodiments, may form a pre-stressed arrangement that is able to spontaneously form a 3-dimensional structure after release from the substrate. See, e.g., U.S. Pat. Nos. 9,457,128 or 9,786,850, each incorporated herein by reference in its entirety.

In some embodiments, the conductive pathway may be relatively narrow. For example, the conductive pathway may have a smallest dimension or a largest cross- sectional dimension of less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 80 nm, less than about 50 nm, less than about 30 nm, less than about 10 nm, less than about 5 nm, less than about 2 nm, etc. The conductive pathway may have any suitable cross-sectional shape, e.g., circular, square, rectangular, polygonal, elliptical, regular, irregular, etc. As - 1 - is discussed in detail below, such conductive pathways may be achieved using lithographic or other techniques.

In some cases, the conductive pathways may define an electrical circuit that is internally contained within the cell scaffold, and/or that extends externally of the cell scaffold, e.g., such that the electrical circuit is in electrical communication with an external electrical system, such as a computer or a transmitter (for instance, a radio transmitter, a wireless transmitter, an Internet connection, etc.). The cell scaffold, in some embodiments, may contain components such as nanoelectric components. Nonlimiting examples of such components include nanoscale wires, sensors such as nanosensors, transistors such as field effect transistors, resistors, capacitors, inductors, diodes, integrated circuits, batteries, power sources, RFID tags, antennae, transmitter, or the like, which may be present in one or more electrical circuit within the cell scaffold. It should, of course, be appreciated that the cell scaffold may contain components (e.g., wires) in some embodiments that are non-nanoelectronic, e.g., microscale wires.

In addition, in certain cases, a component within the cell scaffold may comprise an electrode. The electrode may comprise any suitable material, for example, carbon, or metals such as gold, platinum, silver, or the like. In some cases, the electrode may be used to determine a property of the cell scaffold (e.g., an electrical property, a chemical property, a mechanical property, etc.), and/or to apply a stimulus (e.g., an electrical stimulus) to the biological structure. In some cases, a conductive polymer may also be used with the electrode. Non-limiting examples of conductive polymers include poly(3,4-ethylenedioxythiophene) (PEDOT), polyacetylene, polyphenylene vinylene, polypyrrole, poly thiophene (for example poly (3, 4-ethylenedioxy thiophene)), polyphenylene sulfide, or other conductive polymers such as those described herein.

The electrodes may be of any suitable size, and different electrodes may independently be the same or different sizes. In some cases, the electrodes may have a width of less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 80 nm, less than about 60 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, less than about 10 nm, less than about 8 nm, less than about 6 nm, less than about 4 nm, or less than about 2 nm, etc. In some cases, the components may be used to determine a property of the cell scaffold, e.g., when it is embedded within a biological structure, such as discussed herein. For example, one or more locations within a cell scaffold (for example, an electrode or a nanoscale wire, etc.) may be determined to determine a property, such as a chemical property, an electrical property, a mechanical property, or the like. Other examples include sensing Ca²⁺ spikes, voltage changes, cell signaling pathways, ion concentrations, pH changes, sensing of biomolecules or reaction entities, etc. In some cases, the locations are defined as one or more nodes within the cell scaffold, some or all of which may be individually addressable. For example, a node within a cell scaffold may comprise a nanoscale wire, such as those discussed in more detail below.

In certain embodiments, the conductive pathways may define an electrical circuit that is interfaceable or connectable with an external electrical device, such as a computer, using a suitable connector, or using wireless method, such as Bluetooth, WiFi, etc. For example, the cell scaffold may be directly connected to an external device (for instance, using an interface such as described in U.S. Pat. Apl. Pub. No. 2018/0328884, incorporated herein by reference in its entirety). In some cases, a suitable connector, such as a cable, may be used to make such a connection between an electrical circuit within a cell scaffold and the external device. Non-limiting examples of cables include those commercially available, such as ribbon cables, flexible flat cable, 8-pin cables, 16- pin cables, etc., or other electrical cables. However, an external connection is not always required, and in some cases, the scaffold may be a self-contained electrical circuit. For example, the circuit may be able to transmit information wirelessly to an external device, store information for later access (e.g., after sacrificing the organoids, organs, or organisms), or the like.

In addition, in certain embodiments, the cell scaffold may be able to communicate with an external device using wireless communications, e.g., in addition to and/or instead of an electrical connection. For example, the cell scaffold may contain a transmitter (for instance, a radio transmitter, a wireless transmitter, an Internet connection, etc.) and/or a receiver, e.g., which may be in communication with a transmitter and/or a receiver on an external device.

In some embodiments, more than one electrical circuit and/or more than one conductive pathway may be used within a cell scaffold. For example, multiple conductive pathways or circuits can be used such that some or all of the nodes may be individually electronically addressable within the cell scaffold. However, in other embodiments, more than one node may be addressable by a particular conductive pathway.

In some cases, as discussed cells such as pancreatic cells may be cultured on a cell scaffold and allowed to grow to become an article, such as an implantable article (for example, a pancreatic implant). For instance, cells such as pancreatic cells may be seeded on a cell scaffold, and allowed to grow or self-assemble into a biological structure. In some cases, the cells may form a “ball” shape as it forms a biological structure, and the flexibility of the scaffold may be such that the cells are able to cause the cell scaffold to stay embedded within the biological structure, thereby resulting in a cell scaffold embedded within the biological structure. For example, the cell scaffold may be sufficiently flexible and/or stretchable and/or soft such that the cells within the biological structure are able to manipulate or distort the cell scaffold as it forms a biological structure. For instance, the cells may be able to distort the cell scaffold by at least 20% in a linear direction, or other distortions as described herein. In contrast, many other scaffolds lack this flexibility, and thus cannot become embedded within the biological structure as it forms. Accordingly, in certain embodiments, the cell scaffold may be embedded within a biological structure without being inserting or injected into the biological structure, e.g., after it has been formed.

In some cases, the cell scaffold may contain at least part of the biological structure. For example, the cell scaffold may be manipulated or distorted, e.g., by the cells, to form a 3-dimensional structure defining an internal volume (e.g., such that the cell scaffold is not a 2-dimensional planar structure), where the cell scaffold is embedded at least partially within the biological structure.

The cell scaffold may be partially or completely embedded within the biological structure. For example, the cell scaffold may be completely embedded inside of the biological structure, such that no portion of the cell scaffold is exposed externally of the biological structure. In another set of embodiments, the cell scaffold is only partially embedded within the biological structure, and at least a portion of the cell scaffold is exposed externally of the biological structure. For instance, an external portion of the cell scaffold may be used to electrically connect the cell scaffold to an external electrical device, such as a computer. For instance, a suitable connector may be connected to the exposed portion of the cell scaffold, e.g., to form a connection between an electrical circuit within a cell scaffold, and the external device.

In some cases, the cell scaffold contains a relatively large part of the biological structure. For instance, the cell scaffold may contain at least 30 vol%, at least 40 vol%, at least 50 vol%, at least 60 vol%, at least 70 vol%, at least 80 vol%, at least 90 vol%, or at least 95 vol% of the biological structure. In addition, in some cases, the cell scaffold may be distributed within a relatively large part of the biological structure. For example, the cell scaffold may be manipulated or distorted such that at least 10 vol% of the biological structure is no more than 5, 10, or 30 micrometers from the cell scaffold embedded therein, and in some cases, at least 20 vol%, at least 30 vol%, at least 40 vol%, at least 50 vol%, at least 60 vol%, at least 70 vol%, at least 80 vol%, at least 90 vol%, or at least 95 vol% of the biological structure is no more than 5, 10, or 30 micrometers from the cell scaffold.

In some cases, the cell scaffold may be manipulated or distorted, e.g., by the cells, to stretch the cell scaffold. For example, the biological structure may grow and or expand, and the embedded cell scaffold may be stretched along with the biological structure as it expands. In certain embodiments, the cell scaffold may exhibit a lower filling ratio as it is expanded by the biological structure. For instance, the cell scaffold may have an first, initial filling ratio (e.g., prior to adding cells) of less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, etc., and a second filling ratio, after expansion, that is less than the initial filling ratio. For example, the second filling ratio may be less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the initial filing ratio. In some cases, the second filing ratio may be less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, etc. In addition, in some cases, the cells may case strain to the cell scaffold during expansion. For example, at least a portion of the cell scaffold may exhibit a tensile strain of at least 10% or at least 20%.

In some embodiments, one or more cells (e.g., pancreatic cells) are grown or embedded in a hydrogel. The hydrogel can provide a medium for cell growth, while also providing support to one or more adjacent electrodes and/or interconnects. For example, in some embodiments, a hydrogel surrounding at least a portion of the pancreatic cells. In one set of embodiments, the cell scaffold may further comprise a hydrogel, e.g., surrounding at least a portion of the scaffold and/or cells within the scaffold. In some cases, the hydrogel may be helpful, for example, to help stabilize the structure, to add additional agents to enhance its biocompatibility, to cause it to form a suitable 3- dimension structure, to control pore sizes, etc. Non-limiting examples of suitable hydrogel structures include Matrigel™, polyacrylamide, agarose, alginic acid that can be gelled by the addition of calcium, etc.

In some embodiments, the biological structure may be an organoid, e.g., a pancreatic organoid. An organoid, in some cases, is a miniaturized and simplified version of an organ produced in vitro. They can be derived from various sources, such as one or a few cells from a tissue, embryonic stem cells, induced pluripotent stem cells, or the like. In some cases, such cells are able to self-organize in three-dimensional culture, e.g., owing to their self-renewal and differentiation capacities, for example, as in a pancreatic implant. Thus, in certain embodiments, such cells may be added to a cell scaffold, and the cells may form an organoid that embeds the cell scaffold.

In addition, in one set of embodiments, a cell scaffold (with or without a biological structure) may be implanted into an organism. For example, an organoid or an organ containing a cell scaffold can be implanted within an organism. The organism may be a human or non-human mammal, such as a monkey, cow, sheep, goat, horse, rabbit, pig, mouse, rat, dog, or cat. The organoid or organ may be from the same or different species as the organism, and may be from the same individual or a different one. In one embodiment, the cell scaffold is an implantable article such as a pancreatic implant.

Certain aspects generally directed to systems and methods for preparing an implantable article comprising a cell scaffold. Implantable articles include any of those described herein. The cell scaffolds can be fabricated, for example, using well-known lithographic techniques such as those discussed below. Additional details of cell scaffold fabrication, in accordance with certain embodiments, may be seen in U.S. Pat. Apl. Pub. No. 2022-0213425 or Int. Pat. Apl. Pub. No. WO 2020/263772, each incorporated herein by reference in its entirety.

In various embodiments, a cell scaffold is constructed by assembling various polymers, metals, and other components (for example, nanoscale wires) together on a substrate. For example, lithographic techniques such as e-beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc. may be used to pattern polymers, metals, etc. on the substrate. After assembly, at least a portion of the substrate (e.g., a sacrificial material) may be removed, allowing the scaffold to be partially or completely removed from the substrate. Other materials may also be added to the scaffold, e.g., to help stabilize the structure, to add additional agents to enhance its biocompatibility, etc. The scaffold can be used in vivo, e.g., by implanting it in a subject, and/or in vitro, e.g., by seeding cells, etc. on the scaffold. In addition, in some cases, cells may initially be grown or cultured on the scaffold, e.g., to form a biological structure, such as tissues, organoids, organs, organisms, and the like. In some cases, as discussed, the cell scaffold may be sufficiently flexible and/or stretchable and/or soft such that the cell scaffold becomes embedded within the biological structures as it forms.

For example, in one set of embodiments, a cell scaffold may be constructed by providing a substrate, depositing a sacrificial layer on the substrate, then patterning a first photoresist on the sacrificial layer, a conductive pathway on the first photoresist, and a second photoresist on the conductive pathway, and removing the sacrificial layer to produce the cell scaffold. See, e.g., FIG. 2. The first and second photoresists may comprise the same or different materials. Optionally, other components can also be added to the cell scaffold, before or during formation, such as electrode components, nanoscale wires, connectors such as cables, or the like.

The substrate may be chosen to be one that can be used for lithographic techniques such as e-beam lithography or photolithography, or other lithographic techniques including those discussed herein. For example, the substrate may comprise or consist essentially of a semiconductor material such as silicon, although other substrate materials (e.g., a metal) can also be used. Typically, the substrate is one that is substantially planar, e.g., so that polymers, metals, and the like can be patterned on the substrate. In some cases, a portion of the substrate can be oxidized, e.g., forming SiC and/or ShN4 on a portion of the substrate, which may facilitate subsequent addition of materials (metals, polymers, etc.) to the substrate.

In certain embodiments, one or more polymers can also be deposited or otherwise formed prior to depositing the sacrificial material. In some cases, the polymers may be deposited or otherwise formed as a layer of material on the substrate. Deposition may be performed using any suitable technique, e.g., using lithographic techniques such as e- beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc. In some cases, some or all of the polymers may be biocompatible and/or biodegradable. The polymers that are deposited may also comprise methyl methacrylate and/or poly(methyl methacrylate), in some embodiments.

Next, a sacrificial material may be deposited. The sacrificial material can be chosen to be one that can be removed without substantially altering other materials (e.g., polymers, other metals, nanoscale wires, etc.) deposited thereon. For example, in one embodiment, the sacrificial material may be a metal, e.g., one that is easily etchable. For instance, the sacrificial material can comprise germanium or nickel, which can be etched or otherwise removed, for example, using a peroxide (e.g., H2O2) or a nickel etchant (many of which are readily available commercially). In some cases, the sacrificial material may be deposited on oxidized portions or polymers previously deposited on the substrate. In some cases, the sacrificial material is deposited as a layer. The layer can have a thickness of less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, etc.

In some embodiments, a first photoresist can be deposited, e.g., on the sacrificial material. The photoresist may include one or more polymers, which may be deposited as one or more layers. Examples of photoresist include, but are not limited to, SU-8, SI 805, LOR 3A, poly(methyl methacrylate), poly(methyl glutarimide), phenol formaldehyde resin (diazonaphthoquinone/novolac), diazonaphthoquinone (DNQ), Hoechst AZ 4620, Hoechst AZ 4562, Shipley 1400-17, Shipley 1400-27, Shipley 1400- 37, etc., as well as any others discussed herein.

The photoresist can be used to at least partially define a cell scaffold. In one set of embodiments, the photoresist may be deposited as a layer of material, such that portions of the photoresist may be subsequently removed. For example, the photoresist can be deposited using lithographic techniques such as e-beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc., or using other techniques for removing polymer that are known to those of ordinary skill in the art. In some cases, more than one photoresist is used, e.g., deposited as more than one layer (e.g., sequentially), and each layer may independently have a thickness of less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, etc. For example, in some embodiments, portions of the photoresist may be exposed to light (visible, UV, etc.), electrons, ions, X- rays, etc. (e.g., projected onto the photoresist), and the exposed portions can be etched away (e.g., using suitable etchants, plasma, etc.) to produce the pattern.

Accordingly, the photoresist may be formed into a particular pattern, e.g., in a grid or a mesh, e.g., as discussed herein. For instance, the pattern may include a mesh and interconnects that have a shape that allow the interconnects to be manipulated or distorted without disrupting their connections, e.g., during stretching, compression, folding, or the like. The pattern can be regular or irregular.

Next, a metal or other conductive material can be deposited e.g., on one of the previous materials, to form conductive pathways within the cell scaffold. More than one metal can be used, which may be deposited as one or more layers. For example, a first metal may be deposited, and a second metal may be deposited on at least a portion of the first metal. Optionally, more metals can be used, e.g., a third metal may be deposited on at least a portion of the second metal, and the third metal may be the same or different from the first metal. In some cases, each metal may independently have a thickness of less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 80 nm, less than about 60 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, less than about 10 nm, less than about 8 nm, less than about 6 nm, less than about 4 nm, or less than about 2 nm, etc., and the layers may be of the same or different thicknesses.

Any suitable technique can be used for depositing metals, and if more than one metal is used, the techniques for depositing each of the metals may independently be the same or different. For example, in one set of embodiments, deposition techniques such as sputtering can be used. Other examples include, but are not limited to, physical vapor deposition, vacuum deposition, chemical vapor deposition, cathodic arc deposition, evaporative deposition, e-beam PVD, pulsed laser deposition, ion-beam sputtering, reactive sputtering, ion-assisted deposition, high-target-utilization sputtering, high-power impulse magnetron sputtering, gas flow sputtering, or the like.

The metals can be chosen in some cases such that the deposition process yields a pre-stressed arrangement, e.g., due to atomic lattice mismatch, which causes the subsequent metal leads to warp or bend, for example, once released from the substrate. Although such processes were typically undesired in the prior art, in certain embodiments of the present disclosure, such pre-stressed arrangements may be used to cause the resulting cell scaffold to form a 3-dimensional structure, in some cases spontaneously, upon release from the substrate. See, e.g., U.S. Pat. Apl. Pub. Nos. 2014/0073063, 2014/0074253, 2017/0069858, 2017/0072109, each of which is incorporated herein by reference in its entirety. However, it should be understood that in other embodiments, the metals may not necessary be deposited in a pre-stressed arrangement.

Examples of metals that can be deposited (stressed or unstressed) include, but are not limited to, aluminum, gold, silver, copper, molybdenum, tantalum, titanium, nickel, tungsten, chromium, palladium, as well as any combinations of these and/or other metals. For example, a chromium/gold/chromium deposition process can be used, as is shown in FIG. 2.

In certain embodiments, a second photoresist can be deposited on the previous materials. The second photoresist may be the same or different from the first photoresist, and may include any of the photoresist materials discussed herein, including any of those described with reference to the first photoresist. The second photoresist may include one or more polymers, which may be deposited as one or more layers. In some embodiments, the second photoresist may be deposited on one or more portions of a substrate, e.g., as a layer of material such that portions of the second photoresist can be subsequently removed, e.g., using lithographic techniques such as e-beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc., or using other techniques for removing photoresist that are known to those of ordinary skill in the art. In some cases, more than one photoresist may be used, e.g., deposited as more than one layer (e.g., sequentially), and each layer may independently have a thickness of less than about 5 micrometers, less than about 4 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, etc.

After formation of the cell scaffold, some or all of the sacrificial material may then be removed in some cases. In one set of embodiments, for example, at least a portion of the sacrificial material is exposed to an etchant able to remove the sacrificial material. For example, if the sacrificial material is a metal such as nickel, a suitable etchant (for example, a metal etchant such as a nickel etchant, acetone, etc.) can be used to remove the sacrificial metal. Many such etchants may be readily obtained commercially. In addition, in some embodiments, the cell scaffold can also be dried, e.g., in air (e.g., passively), by using a heat source, by using a critical point dryer, etc.

Other materials may be also added to the cell scaffold, e.g., before or after it forms a 3-dimensional structure, for example, to help stabilize the structure, to add additional agents to enhance its biocompatibility (e.g., growth hormones, extracellular matrix protein, Matrigel™, etc.), to cause it to form a suitable 3-dimension structure, to control pore sizes, etc. Non-limiting examples of such materials have been discussed above, and include other polymers, growth hormones, extracellular matrix protein, specific metabolites or nutrients, additional scaffold materials, or the like.

In addition, in some cases, the cell scaffold is exposed to cells, which can be cultured or allowed to grow, e.g., to form a biological structure. In some cases, the cells are plated or seeded as individual cells, although in certain cases, larger cell assemblies (tissues, embryos, etc.) may be used. In one set embodiments, the cell scaffold may be exposed to cells in vitro, and/or the cell scaffold may be exposed or even submerged within a suitable cell growth medium. Such media are widely available commercially. In some embodiments, the cell scaffold can be subsequently implanted in vivo into a subject, e.g., upon the growth of tissue, an organ, an organoid, etc. However, it should be understood that implantation is not required in all embodiments, for example, in cases where an entire organism develops from the cells.

In addition, it should be understood that exposure to cells is not necessarily required in all embodiments. For instance, in one set of embodiments, the cell scaffold may be prepared without the presence of cells. For example, the cell scaffold may be sold as part of a kit, and the user may expose the cell scaffold to cells (or use it for other purposes). In addition, the cell scaffold can be interfaced in some embodiments with one or more electronics, e.g., an external electrical system such as a computer or a transmitter (for instance, a radio transmitter, a wireless transmitter, etc.), e.g., as discussed herein. The interfacing may occur at any suitable time, e.g., before or after exposure to cells, before or after a biological structure (e.g., an organoid or an organism) has formed, before or after sale to a user, or the like.

For instance, in some cases, electronic testing of the cell scaffold may be performed. The cell scaffold, or a portion thereof, can be connected to an external electrical circuit, e.g., to electronically interrogate or otherwise determine the electronic state of the cell scaffold. For example, the cell scaffold may comprise one or more nanoscale wires, or other nanoelectronic components, that can be used as sensors. Such determinations may be performed quantitatively and/or qualitatively, depending on the application, and can involve all, or only a portion, of the cell scaffold, e.g., as discussed herein.

Thus, as mentioned, in some aspects, the cell scaffold can comprise one or more nanoscale wires. For instance, one or more nodes may contain nanoscale wires, and/or nanoscale wires may be contained within interconnects, or the like. In some cases, the cell scaffold within the organoids, organs, or organisms ay include one or more sensors or stimulators, interconnected with stretchable mesh interconnects, to form a network, e.g., as is shown in FIGs. 1A-1B. The sensors or stimulators may, in some embodiments, comprise nanoscale wires, such as those described herein. Such sensors may be monitored, e.g., individually or collectively.

Non-limiting examples of suitable nanoscale wires include carbon nanotubes, nanorods, nanowires, organic and inorganic conductive and semiconducting polymers, metal nanoscale wires, semiconductor nanoscale wires (for example, formed from silicon), and the like. If carbon nanotubes are used, they may be single-walled and/or multi-walled, and may be metallic and/or semiconducting in nature. Other conductive or semiconducting elements that may not be nanoscale wires, but are of various small nanoscopic-scale dimension, also can be used within the cell scaffold.

In general, a “nanoscale wire” (also known herein as a “nanoscopic-scale wire” or “nanoscopic wire”) generally is a wire or other nanoscale object, that at any point along its length, has at least one cross-sectional dimension and, in some embodiments, two orthogonal cross-sectional dimensions (e.g., a diameter) of less than 1 micrometer, less than about 500 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 70, less than about 50 nm, less than about 20 nm, less than about 10 nm, less than about 5 nm, than about 2 nm, or less than about 1 nm. In some embodiments, the nanoscale wire is generally cylindrical. In other embodiments, however, other shapes are possible; for example, the nanoscale wire can be faceted, i.e., the nanoscale wire may have a polygonal cross-section. The cross-section of a nanoscale wire can be of any arbitrary shape, including, but not limited to, circular, square, rectangular, annular, polygonal, or elliptical, and may be a regular or an irregular shape. The nanoscale wire can also be solid or hollow.

In some cases, the nanoscale wire has one dimension that is substantially longer than the other dimensions of the nanoscale wire. For example, the nanoscale wire may have a longest dimension that is at least about 1 micrometer, at least about 3 micrometers, at least about 5 micrometers, or at least about 10 micrometers or about 20 micrometers in length, and/or the nanoscale wire may have an aspect ratio (longest dimension to shortest orthogonal dimension) of greater than about 2:1, greater than about 3:1, greater than about 4:1, greater than about 5:1, greater than about 10:1, greater than about 25:1, greater than about 50:1, greater than about 75:1, greater than about 100:1, greater than about 150:1, greater than about 250:1, greater than about 500:1, greater than about 750:1, or greater than about 1000:1 or more in some cases.

In some embodiments, a nanoscale wire is substantially uniform, or the nanowire may have a variation in average diameter of the nanoscale wire of less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. For example, the nanoscale wires may be grown from substantially uniform nanoclusters or particles, e.g., colloid particles. See, e.g., U.S. Patent No. 7,301,199, issued November 27, 2007, entitled “Nanoscale Wires and Related Devices,” incorporated herein by reference in its entirety. In some cases, the nanoscale wire may be one of a population of nanoscale wires having an average variation in diameter, of the population of nanowires, of less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%.

In some embodiments, a nanoscale wire has a conductivity of or of similar magnitude to any semiconductor or any metal. The nanoscale wire can be formed of suitable materials, e.g., semiconductors, metals, etc., as well as any suitable combinations thereof. In some cases, the nanoscale wire will have the ability to pass electrical charge, for example, being electrically conductive. For example, the nanoscale wire may have a relatively low resistivity, e.g., less than about 10'³ Ohm m, less than about 10'⁴ Ohm m, less than about 10'⁶ Ohm m, or less than about 10'⁷ Ohm m. The nanoscale wire can, in some embodiments, have a conductance of at least about 1 microsiemens, at least about 3 microsiemens, at least about 10 microsiemens, at least about 30 microsiemens, or at least about 100 microsiemens.

The nanoscale wire can be solid or hollow, in various embodiments. As used herein, a “nanotube” is a nanoscale wire that is hollow, or that has a hollowed-out core, including those nanotubes known to those of ordinary skill in the art. As another example, a nanotube may be created by creating a core/shell nanowire, then etching away at least a portion of the core to leave behind a hollow shell. Accordingly, in one set of embodiments, the nanoscale wire is a non-carbon nanotube. In contrast, a “nanowire” is a nanoscale wire that is typically solid (i.e., not hollow). Thus, in one set of embodiments, the nanoscale wire may be a semiconductor nanowire, such as a silicon nanowire.

For example, in one embodiment, a nanoscale wire may comprise or consist essentially of a metal. Non-limiting examples of potentially suitable metals include aluminum, gold, silver, copper, molybdenum, tantalum, titanium, nickel, tungsten, chromium, or palladium. In another set of embodiments, a nanoscale wire comprises or consists essentially of a semiconductor. Typically, a semiconductor is an element having semiconductive or semi-metallic properties (i.e., between metallic and non-metallic properties). An example of a semiconductor is silicon. Other non-limiting examples include elemental semiconductors, such as gallium, germanium, diamond (carbon), tin, selenium, tellurium, boron, or phosphorous. In other embodiments, more than one element may be present in the nanoscale wire as the semiconductor, for example, gallium arsenide, gallium nitride, indium phosphide, cadmium selenide, etc. Still other examples include a Group II- VI material (which includes at least one member from Group II of the Periodic Table and at least one member from Group VI, for example, ZnS, ZnSe, ZnSSe, ZnCdS, CdS, or CdSe), or a Group III-V material (which includes at least one member from Group III and at least one member from Group V, for example GaAs, GaP, GaAsP, InAs, InP, AlGaAs, or InAsP). ff

In certain embodiments, the semiconductor can be undoped or doped (e.g., p-type or n-type). For example, in one set of embodiments, a nanoscale wire may be a p-type semiconductor nanoscale wire or an n-type semiconductor nanoscale wire, and can be used as a component of a transistor such as a field effect transistor (“FET”). For instance, the nanoscale wire may act as the “gate” of a source-gate-drain arrangement of a FET, while metal leads or other conductive pathways (as discussed herein) are used as the source and drain electrodes.

In some embodiments, a dopant or a semiconductor may include mixtures of Group IV elements, for example, a mixture of silicon and carbon, or a mixture of silicon and germanium. In other embodiments, the dopant or the semiconductor may include a mixture of a Group III and a Group V element, for example, BN, BP, BAs, AIN, A1P, AlAs, AlSb, GaN, GaP, GaAs, GaSb, InN, InP, InAs, or InSb. Mixtures of these may also be used, for example, a mixture of BN/BP/BAs, or BN/A1P. In other embodiments, the dopants may include alloys of Group III and Group V elements. For example, the alloys may include a mixture of AlGaN, GaPAs, InPAs, GalnN, AlGalnN, GalnAsP, or the like. In other embodiments, the dopants may also include a mixture of Group II and Group VI semiconductors. For example, the semiconductor may include ZnO, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, BeS, BeSe, BeTe, MgS, MgSe, or the like. Alloys or mixtures of these dopants are also be possible, for example, (ZnCd)Se, or Zn(SSe), or the like. Additionally, alloys of different groups of semiconductors may also be possible, for example, a combination of a Group II-Group VI and a Group III-Group V semiconductor, for example, (GaAs)_x(ZnS)i-_x. Other examples of dopants may include combinations of Group IV and Group VI elemnts, such as GeS, GeSe, GeTe, SnS, SnSe, SnTe, PbO, PbS, PbSe, or PbTe. Other semiconductor mixtures may include a combination of a Group I and a Group VII, such as CuF, CuCl, CuBr, Cui, AgF, AgCl, AgBr, Agl, or the like. Other dopant compounds may include different mixtures of these elements, such as BeSiN2, CaCN2, ZnGeP2, CdSnAs2, ZnSnSb2, CuGeP₃, CuSi2P₃, Si₃N₄, Ge₃N₄, A1₂O₃, (Al, Ga, In)₂(S, Se, Te)₃, A1₂CO, (Cu, Ag)(Al, Ga, In, Tl, Fe)(S, Se, Te)2 and the like.

The doping of the semiconductor to produce a p-type or n-typc semiconductor may be achieved via bulk-doping in certain embodiments, although in other embodiments, other doping techniques (such as ion implantation) can be used. Many such doping techniques that can be used will be familiar to those of ordinary skill in the art, including both bulk doping and surface doping techniques. A bulk-doped article (e.g. an article, or a section or region of an article) is an article for which a dopant is incorporated substantially throughout the crystalline lattice of the article, as opposed to an article in which a dopant is only incorporated in particular regions of the crystal lattice at the atomic scale, for example, only on the surface or exterior. For example, some articles are typically doped after the base material is grown, and thus the dopant only extends a finite distance from the surface or exterior into the interior of the crystalline lattice. It should be understood that “bulk-doped” does not define or reflect a concentration or amount of doping in a semiconductor, nor does it necessarily indicate that the doping is uniform. “Heavily doped” and “lightly doped” are terms the meanings of which are clearly understood by those of ordinary skill in the art. In some embodiments, one or more regions comprise a single monolayer of atoms (“deltadoping”). In certain cases, the region may be less than a single monolayer thick (for example, if some of the atoms within the monolayer are absent). As a specific example, the regions may be arranged in a layered structure within the nanoscale wire, and one or more of the regions can be delta-doped or partially delta-doped.

Accordingly, in one set of embodiments, the nanoscale wires may include a heterojunction, e.g., of two regions with dissimilar materials or elements, and/or the same materials or elements but at different ratios or concentrations. The regions of the nanoscale wire may be distinct from each other with minimal cross-contamination, or the composition of the nanoscale wire can vary gradually from one region to the next. The regions may be both longitudinally arranged relative to each other, or radially arranged (e.g., as in a core/shell arrangement) on the nanoscale wire. Each region may be of any size or shape within the wire. The junctions may be, for example, a p/n junction, a p/p junction, an n/n junction, a p/i junction (where i refers to an intrinsic semiconductor), an n/i junction, an i/i junction, or the like. The junction can also be a Schottky junction in some embodiments. The junction may also be, for example, a semiconductor/semiconductor junction, a semiconductor/metal junction, a semiconductor/insulator junction, a metal/metal junction, a metal/insulator junction, an insulator/insulator junction, or the like. The junction may also be a junction of two materials, a doped semiconductor to a doped or an undoped semiconductor, or a junction between regions having different dopant concentrations. The junction can also be a defected region to a perfect single crystal, an amorphous region to a crystal, a crystal to another crystal, an amorphous region to another amorphous region, a defected region to another defected region, an amorphous region to a defected region, or the like. More than two regions may be present, and these regions may have unique compositions or may comprise the same compositions. As one example, a wire can have a first region having a first composition, a second region having a second composition, and a third region having a third composition or the same composition as the first composition. Non-limiting examples of nanoscale wires comprising heterojunctions (including core/shell heterojunctions, longitudinal heterojunctions, etc., as well as combinations thereof) are discussed in U.S. Patent No. 7,301,199, issued November 27, 2007, entitled “Nanoscale Wires and Related Devices,” incorporated herein by reference in its entirety.

In some embodiments, a nanoscale wire is a bent or a kinked nanoscale wire. A kink is typically a relatively sharp transition or turning between a first substantially straight portion of a wire and a second substantially straight portion of a wire. For example, a nanoscale wire may have 1, 2, 3, 4, or 5 or more kinks. In some cases, the nanoscale wire is formed from a single crystal and/or comprises or consists essentially of a single crystallographic orientation, for example, a <110> crystallographic orientation, a <112> crystallographic orientation, or a <1 120> crystallographic orientation. It should be noted that the kinked region need not have the same crystallographic orientation as the rest of the semiconductor nanoscale wire. In some embodiments, a kink in the semiconductor nanoscale wire may be at an angle of about 120° or a multiple thereof. The kinks can be intentionally positioned along the nanoscale wire in some cases. For example, a nanoscale wire may be grown from a catalyst particle by exposing the catalyst particle to various gaseous reactants to cause the formation of one or more kinks within the nanoscale wire. Non-limiting examples of kinked nanoscale wires, and suitable techniques for making such wires, are disclosed in International Patent Application No. PCT/US2010/050199, filed September 24, 2010, entitled “Bent Nanowires and Related Probing of Species,” published as WO 2011/038228 on March 31, 2011, incorporated herein by reference in its entirety.

In one set of embodiments, the nanoscale wire is formed from a single crystal, for example, a single crystal nanoscale wire comprising a semiconductor. A single crystal item may be formed via covalent bonding, ionic bonding, or the like, and/or combinations thereof. While such a single crystal item may include defects in the crystal in some cases, the single crystal item is distinguished from an item that includes one or more crystals, not ionically or covalently bonded, but merely in close proximity to one another. In some embodiments, the nanoscale wires used herein are individual or freestanding nanoscale wires. For example, an “individual” or a “free-standing” nanoscale wire may, at some point in its life, not be attached to another article, for example, with another nanoscale wire, or the free-standing nanoscale wire may be in solution. This is in contrast to nanoscale features etched onto the surface of a substrate, e.g., a silicon wafer, in which the nanoscale features are never removed from the surface of the substrate as a free-standing article. This is also in contrast to conductive portions of articles which differ from surrounding material only by having been altered chemically or physically, in situ, i.e., where a portion of a uniform article is made different from its surroundings by selective doping, etching, etc. An “individual” or a “free-standing” nanoscale wire is one that can be (but need not be) removed from the location where it is made, as an individual article, and transported to a different location and combined with different components to make a functional device such as those described herein and those that would be contemplated by those of ordinary skill in the art upon reading this disclosure.

In various embodiments, more than one nanoscale wire may be present within the cell scaffold. The nanoscale wires may each independently be the same or different. For example, the cell scaffold can comprise at least 5 nanoscale wires, at least about 10 nanoscale wires, at least about 30 nanoscale wires, at least about 50 nanoscale wires, at least about 100 nanoscale wires, at least about 300 nanoscale wires, at least about 1000 nanoscale wires, etc. The nanoscale wires may be distributed uniformly or non- uniformly throughout the cell scaffold. In some cases, the nanoscale wires may be distributed at an average density of at least about 10 nanoscale wires/mm³, at least about 30 nanoscale wires/mm³, at least about 50 nanoscale wires/mm³, at least about 75 nanoscale wires/mm³, or at least about 100 nanoscale wires/mm³. In certain embodiments, the nanoscale wires are distributed within the cell scaffold such that the average separation between a nanoscale wire and its nearest neighboring nanoscale wire is less than about 2 mm, less than about 1 mm, less than about 500 micrometers, less than about 300 micrometers, less than about 100 micrometers, less than about 50 micrometers, less than about 30 micrometers, or less than about 10 micrometers.

Within the cell scaffold, some or all of the nanoscale wires may be individually electronically addressable. For instance, in some cases, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially all of the nanoscale wires within the cell scaffold may be individually electronically addressable. In some embodiments, an electrical property of a nanoscale wire can be individually determinable (e.g., being partially or fully resolvable without also including the electrical properties of other nanoscale wires), and/or such that the electrical property of a nanoscale wire may be individually controlled (e.g., by applying a desired voltage or current to the nanoscale wire, for instance, without simultaneously applying the voltage or current to other nanoscale wires). In other embodiments, however, at least some of the nanoscale wires can be controlled within the same electronic circuit (e.g., by incorporating the nanoscale wires in series and/or in parallel), such that the nanoscale wires can still be electronically controlled and/or determined.

The nanoscale wire, in some embodiments, may be responsive to a property external of the nanoscale wire, e.g., a chemical property, an electrical property, a physical property, etc. Such determination may be qualitative and/or quantitative. For example, in one set of embodiments, the nanoscale wire may be responsive to voltage. For instance, the nanoscale wire may exhibits a voltage sensitivity of at least about 5 microsiemens/V; by determining the conductivity of a nanoscale wire, the voltage surrounding the nanoscale wire may thus be determined. In other embodiments, the voltage sensitivity can be at least about 10 microsiemens/V, at least about 30 microsiemens/V, at least about 50 microsiemens/V, or at least about 100 microsiemens/V. Other examples of electrical properties that can be determined include resistance, resistivity, conductance, conductivity, impendence, or the like.

As another example, a nanoscale wire may be responsive to a chemical property of the environment surrounding the nanoscale wire. For example, an electrical property of the nanoscale wire can be affected by a chemical environment surrounding the nanoscale wire, and the electrical property can be thereby determined to determine the chemical environment surrounding the nanoscale wire. As a specific non-limiting example, the nanoscale wires may be sensitive to pH or hydrogen ions. Further nonlimiting examples of such nanoscale wires are discussed in U.S. Patent No. 7,129,554, filed October 31, 2006, entitled “Nanosensors,” incorporated herein by reference in its entirety.

As an example, the nano scale wire may have the ability to bind to an analyte indicative of a chemical property of the environment surrounding the nanoscale wire (e.g., hydrogen ions for pH, or concentration for an analyte of interest), and/or the nanoscale wire may be partially or fully functionalized, i.e. comprising surface functional moieties, to which an analyte is able to bind, thereby causing a determinable property change to the nanoscale wire, e.g., a change to the resistivity or impedance of the nanoscale wire. The binding of the analyte can be specific or non-specific. Functional moieties may include simple groups, selected from the groups including, but not limited to, -OH, -CHO, -COOH, -SO3H, -CN, -NH₂, -SH, -COSH, -COOR, halide; biomolecular entities including, but not limited to, amino acids, proteins, sugars, DNA, antibodies, antigens, and enzymes; grafted polymer chains with chain length less than the diameter of the nanowire core, selected from a group of polymers including, but not limited to, polyamide, polyester, polyimide, polyacrylic; a shell of material comprising, for example, metals, semiconductors, and insulators, which may be a metallic element, an oxide, an sulfide, a nitride, a selenide, a polymer and a polymer gel.

In some embodiments, a reaction entity may be bound to a surface of the nanoscale wire, and/or positioned in relation to the nanoscale wire such that the analyte can be determined by determining a change in a property of the nanoscale wire. The “determination” may be quantitative and/or qualitative, depending on the application. The term “reaction entity” refers to any entity that can interact with an analyte in such a manner to cause a detectable change in a property (such as an electrical property) of a nanoscale wire. The reaction entity may enhance the interaction between the nanowire and the analyte, or generate a new chemical species that has a higher affinity to the nanowire, or to enrich the analyte around the nanowire. The reaction entity can comprise a binding partner to which the analyte binds. The reaction entity, when a binding partner, can comprise a specific binding partner of the analyte. For example, the reaction entity may be a nucleic acid, an antibody, a sugar, a carbohydrate or a protein. Alternatively, the reaction entity may be a polymer, catalyst, or a quantum dot. A reaction entity that is a catalyst can catalyze a reaction involving the analyte, resulting in a product that causes a detectable change in the nanowire, e.g. via binding to an auxiliary binding partner of the product electrically coupled to the nanowire. Another exemplary reaction entity is a reactant that reacts with the analyte, producing a product that can cause a detectable change in the nanowire. The reaction entity can comprise a shell on the nanowire, e.g. a shell of a polymer that recognizes molecules in, e.g., a gaseous sample, causing a change in conductivity of the polymer which, in turn, causes a detectable change in the nanowire.

The term “binding partner” refers to a molecule that can undergo binding with a particular analyte, or “binding partner” thereof, and includes specific, semi-specific, and non-specific binding partners as known to those of ordinary skill in the art. The term “specifically binds,” when referring to a binding partner (e.g., protein, nucleic acid, antibody, etc.), refers to a reaction that is determinative of the presence and/or identity of one or other member of the binding pair in a mixture of heterogeneous molecules (e.g., proteins and other biologies). Thus, for example, in the case of a receptor/ligand binding pair the ligand would specifically and/or preferentially select its receptor from a complex mixture of molecules, or vice versa. An enzyme would specifically bind to its substrate, a nucleic acid would specifically bind to its complement, an antibody would specifically bind to its antigen. Other examples include, nucleic acids that specifically bind (hybridize) to their complement, antibodies specifically bind to their antigen, and the like. The binding may be by one or more of a variety of mechanisms including, but not limited to ionic interactions, and/or covalent interactions, and/or hydrophobic interactions, and/or van der Waals interactions, etc.

Additionally, as discussed, a cell scaffold in some aspects may include a photoresist, such as a soft photoresist. For example, in some embodiments, the photoresist may comprise a polymer formed by photo-curing a fluorinated monomer including cross -linkable function groups using a photoinitiator. This may, for examlpe, facilitate stretchability of the cell scaffold. One example of such a polymer is perfluoropolyether dimethacrylate (PFPE-DMA). In addition, in some cases, the photoresist may be a photo-curable composition. In some embodiments, a photo-curable composition includes: a fluorinated monomer including cross-linkable functional groups; and a photoinitiator. Additional non-limiting examples of photoresist may be found in Int. Pat. Apl. Pub. No. WO 2019/084498, incorporated herein by reference in its entirety.

Some embodiments of this disclosure are directed to a photo-curable composition that can be cured to form an elastomer exhibiting high stretchability and that is chemically orthogonal to various development solvents used in photolithography and, hence, compatible with photolithography. Further, the elastomer can be patterned with fine feature resolution, and can be used as a photoresist for patterning various materials, including electrically (or electronically) active materials. Examples of applications of such photo-patternable composition include forming stretchable and transparent substrates, stretchable and transparent dielectric/passivation/encapsulation films or layers for elastic or stretchable microelectronics, and photoresists for patterning of materials, such as in the context of implantable medical devices, wearable electronic devices, and soft electronic devices; other biomedical devices; cosmetics; prosthetics; and other applications involving an interface with a human body, an animal body, or other biological tissue where matching of mechanical properties with the biological tissue is desired.

In some embodiments, a kit may be provided, e.g., comprising a cell scaffold as is discussed herein. Cells may or may not be provided with the kit. The kit may include a package or an assembly including the cell scaffold, and optionally other components associated with the cell scaffold, such as cells. Examples of other components include, but are not limited to, solvents, surfactants, diluents, salts, buffers, emulsifiers, chelating agents, fillers, antioxidants, binding agents, bulking agents, preservatives, drying agents, antimicrobials, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, dishes, frits, filters, rings, clamps, wraps, patches, containers, and the like, for example, for using, administering, modifying, assembling, storing, packaging, preparing, mixing, diluting, and/or preserving the cell scaffold.

A kit may include instructions in any form that are provided in connection with the components of the kit in such a manner that one of ordinary skill in the art would recognize that the instructions are to be associated with those components. For instance, the instructions may include instructions for the use, modification, mixing, diluting, preserving, administering, assembly, storage, packaging, and/or preparation of the cell scaffold. The instructions may be provided in any form recognizable by one of ordinary skill in the art as a suitable vehicle for containing such instructions, for example, written or published, verbal, audible (e.g., telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) or electronic communications (including Internet or web-based communications), provided in any manner.

The following documents are incorporated herein by reference in their entireties: U.S. Provisional Patent Application Serial No. 62/865,648, filed June 24, 2019, entitled “Organoids Containing Electronics, and Methods Thereof,” by Liu, et al. U.S. Provisional Patent Application Serial No. 62/872,031, filed July 9, 2019, entitled “Organoids Containing Electronics, and Methods Thereof,” by Liu, et al. U.S. Patent No. 7,211,464, issued May 1, 2007, entitled “Doped Elongated Semiconductors, Growing Such Semiconductors, Devices Including Such Semiconductors, and Fabricating Such Devices”; U.S. Patent No. 7,301,199, issued November 27, 2007, entitled “Nanoscale Wires and Related Devices”; and International Patent Application No. PCT/US2010/050199, filed September 24, 2010, entitled “Bent Nanowires and Related Probing of Species,” published as WO 2011/038228 on March 31, 2011. In addition, the following are each incorporated herein by reference in their entireties: U.S. Pat. Nos. 9,786,850 and 9,457,128; U.S. Pat. Apl. Pub. Nos. 2017/0069858, 2014/0073063, 2017/0072109, and 2014/0074253; and Int. Pat. Apl. Pub. No. WO 2019/084498.

Also incorporated herein by reference in their entireties are U.S. Pat. Apl. Pub. No. 2022-0213425, Int. Pat. Apl. Pub. No. WO 2020/263772, and U.S. Pat. Apl. Ser. No. 63/567,332.

The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

EXAMPLE 1

The following example describes the integration of a mesh comprising nanoscale electrodes and interconnects within stem cell-derived pancreatic islets.

As described in more detail below in this example, soft materials and nanoelectronics were synergized to build tissue-like nanoelectronics with subcellular feature size, tissue-level flexibility, and mesh-like networks, enabling seamless integration throughout 3D tissue. Implanting and distributing such nanoelectronics across some, more, or the entire organoid bodies as “cyborg” organoids thus allows studying cell-level electrophysiology dynamics throughout organogenesis.

Here, cyborg SC-islets were developed, enabling continuous single-cell electrophysiology analysis across the intact 3D islet network during functional maturation. Using embedded electrodes, stable extracellular spike dynamics from SC- islets were successfully captured. Robust sorting of single-unit action potentials over months of chronic recording were obtained using a conventional spike sorting algorithm. The sorted action potentials allowed for the distinguishing and isolation of islet-wide a and P cell-specific electrical activities based on their distinct low/high glucose threshold for action potential firing, and two major electrical states were identified for both a and P cells. The evolution and coordination of these cell type- specific electrical states were traced over months of extended culture, during which SC-islets developed specialized hormone responses. By analyzing population dynamics of cell type-specific electrical activities and widespread heterogeneity in a and P stimulus-coupled dynamics, it was shown that improved glucose responsiveness during this early maturation time course reflects changing proportions of the two a/p electrical states, towards more cells with low basal firing rates.

Through single-cell RNA sequencing (scRNA-seq) of cyborg islets, these physiological changes were linked to broad induction of energy and hormone metabolism gene pathways across SC-islet cell types. Finally, it was demonstrated that entrainment to 24-hour feeding-fasting cycles elicits glucose-coupled insulin/glucagon secretion rhythms via increased and sustained coordination of stimulated firing rates between a and P cells. These changes are underpinned by induced cell-cell communication and exocytic gene networks, revealing a role for circadian rhythms in synchronizing islet-wide a-P activities to generate coordinated stimulus-coupled hormone responses. Together, these findings demonstrate the power of so-called “cyborg” islets as a platform to dissect the principles of electrical islet cell maturation, which may inform efforts to build fully mature SC-islets for research and generative medicine applications, among other applications.

Building cyborg human SC-islets. Cyborg human SC-islets were created by integrating stretchable mesh nanoelectronics with SC-islet cells, enabling chronically stable electrophysiology throughout in vitro maturation (FIG. 3A). SC-islet cells were seeded with stretchable mesh nanoelectronics on a Matrigel™ hydrogel substrate, followed by condensation of the cell-nanoelectronics structure by cell-cell attraction forces. 3D morphogenesis is triggered by co-culture with mesenchymal stem cells (MSCs), which promote a self-organized 3D folding process. Electrical recordings were performed weekly over a 2-month time course of early maturation prompted by extended culture, and hourly upon further maturation induced by entrainment to circadian feeding cycles (FIG. 3A). The stretchable mesh nanoelectronics were designed to allow localized and cell level electrophysiology recordings while minimizing noise interference (FIGs. 7A-7C). To evaluate their performance, electrode impedance was assessed (FIG. 7D), which showed consistent and stable performance across different samples. Additionally, long-term experiments were conducted in a physiological solution. The results indicate that the electrodes maintain a stable performance across 10 weeks, ensuring long term- reliable electrical recordings (FIG. 7E). These results demonstrated suitability and reliability of the stretchable mesh nanoelectronics for long-term electrical recordings.

To generate SC-islet cells, a stepwise 3D differentiation approach was employed involving controlled administration of signaling factors and small molecules in a series of growth media over a 20-day protocol. The resulting SC-islets gain glucose-responsive insulin secretion function upon 3-4 weeks of extended culture (in medium without serum or exogenous factors). The terminal differentiation-stage SC-islets were then dissociated into single cells and subsequently embedded with stretchable mesh nanoelectronics to form cyborg SC-islets (FIGs. 7F-7I).

Next, immunostaining, electrical recordings, and single-cell RNA sequencing (scRNA-seq) was conducted to validate the feasibility of cyborg islets. First, immuno staining results demonstrated uniform embedding of stretchable mesh nanoelectronics throughout the 3D volume of cyborg SC-islets (FIG. 3B and FIGs. 71- 7M). Compared to control SC-islets (FIGs. 7J-7K), cyborg SC-islets showed the same composition and distribution of a, P, and MSC cells (FIG. 3B, FIG. 7M). Zoomed-in images demonstrated effective coupling of the nanoelectronics with SC-a and SC-P cells (FIG. 3B, FIG. 7M), which was beneficial for successful cell-level electrical recordings. Second, using the embedded electrodes, voltage spike bursting dynamics in response to low (2.8 mM) and high (20 mM) glucose concentrations were captured, as depicted by representative voltage traces (FIG. 3C, left and middle). The raw electrical traces were subjected to bandpass filtering, limiting the frequencies to the range of 300 to 3,000 Hz. Then, a threshold for spike detection was implemented for the extraction of spike waveforms. Finally, spike sorting was carried out employing the MountainSort and Spikeinterface algorithms that have been widely applied for neural signal analysis. Specifically, averaged single spike waveforms were assessed through spike detection (FIG. 3C, right). A stepwise approach was then developed (FIG. 3D) to analyze the electrical recording data. Spike sorting was performed on recorded voltage traces to separate distinct waveforms (FIG. 3D i, D ii). Subsequently, a Uniform Manifold Approximation and Projection (UMAP) was performed using a waveform clustering method to identify cell-specific activity profiles (FIG. 3D iii). Finally, cell-specific spike trains from the continuous electrical recordings were extracted (FIG. 3D iv). This methodology allowed the continuous monitoring of single-cell electrical activities within SC-islets, offering valuable insights into their maturation dynamics. Finally, scRNA-seq was conducted on both cyborg and control SC-islets to further investigate the impact of the embedded mesh nanoelectronics on cell-type composition and gene expression. Unsupervised clustering analysis was then performed and, subsequently, visualized the gene expression profile with UMAP clustering analysis (FIG. 3E), which revealed two major cell clusters: endocrine and non-endocrine cells. Cell types were then further annotated within each of the major cell clusters by comparing with previously established gene markers. For example, endocrine cells include SC-a cells expressing GCG and ARX, SC-P cells expressing INS and NKX6-1 , SC-EC cells expressing TPH1 and FEV, SC-8 cells expressing SST and HHEX, and a small population of epsilon cells expressing GHRL andACSLl (FIGs. 3E-3F, FIG. 8). Notably, the continuous spectrum was observed between SC-P and SC-EC as well as between SC-a and SC-8, which suggested that they emerged from a common endocrine induction intermediate. Within the non-endocrine cluster, ductal cells were observed expressing KRT17 and KRT19, acinar cells expressing CP Al and CPA2, and a replicating population expressing TOP2A and MK167. Importantly, by comparing gene expression and cell type compositions between cyborg and control SC-islets, highly consistent patterns were observed across all cell types within both endocrine and non- endocrine cells, indicating negligible effect of nanoelectronics implantation on the SC- islet landscape. Collectively, these results demonstrated the reliability and robustness of the cyborg SC-islet platform for studying SC-islet maturation dynamics in vitro.

Capturing cell type-specific electrical dynamics. Using the cyborg islet platform, islet-wide a and P cellular electrophysiology could be distinguished (FIG. 4A). Human P cells were characterized by hyperpolarization and electrical inactivity under physiologically low glucose levels. When the glucose concentration rises, however, ATP generated from glucose oxidation lead to KATP channel closure, causing membrane depolarization and the initiation of electrical activity that triggered insulin exocytosis. Human a cells, conversely, exhibited action potentials under low glucose levels, reflecting activation of voltage-gated Na⁺ and Ca²⁺ and inhibition of KATP channels. SC- islets also exhibited voltage-gated glucagon and insulin secretion from SC-a and SC-P cells, respectively, in response to glucose concentrations of 2.8 mM and 20 mM (FIG. 4A). To identify cell type-specific activities, the features of electrical activity were analyzed and recorded from cyborg SC-islet cells, including spike firing rate, amplitude, duration, peak-trough ratio, width at half-maximum, repolarization slope, and recovery slope (FIG. 4B i). Additionally, changes in these features were evaluated between 2.8 mM and 20 mM glucose stimulation and used them to discern SC-a and SC-P cells (FIG. 4B ii, FIGs. 9A-9B).

Two distinct clusters of cells were observed exhibiting differential electrical activity in response to low/high glucose. One cluster showed greater electrical activity under 2.8 mM glucose (FIG. 4C), while the other was more active under 20 mM glucose (FIG. 4D). Specifically, the first cell cluster showed elevated spike firing under 2.8 mM relative to 20 mM glucose (FIG. 4C i), with significantly higher average amplitude and average firing rate (FIG. 4C ii-iii). Accordingly, glucagon secretion into the medium was also higher under 2.8 mM glucose (FIG. 4C iv). The second cluster, by contrast, displayed greater spike firing under 20 mM compared to 2.8 mM glucose, (FIG. 4D i), accompanied by an increased average amplitude and average firing rate (FIG. 4D ii-4D iii). Moreover, insulin secretion into the medium was higher under 20 mM glucose (FIG. 4D iv). Based on the known glucose-dependent behaviors of a and P cells, the first cluster was classified as SC-a cells and the second as SC-P cells.

To validate the cell type-specific electrical activities of SC-a and SC-P cells, electrical responses were measured upon exposure to additional secretion modulators, including tolbutamide (KATP channel inhibitor), forskolin (cyclic adenosine monophosphate / protein kinase A [cAMP/PKA] activator), and tetrodotoxin (TTX, Na⁺ channel blocker) (FIGs. 4E-4F). SC-a and SC-P cells were first classified based on their electrical activities in 2.8 mM and 20 mM glucose, and then examined the cell typespecific responses to specific drugs to confirm their identity. For SC-a cells under tolbutamide, a decrease in firing rate compared to 2.8 mM glucose was observed, but no significant change in the amplitude of firing (FIG. 4E i-4E iii, FIG. 9C). Glucagon secretion was lower in the presence of tolbutamide relative to 2.8 mM glucose (FIG. 4 iv), consistent with the inhibitory effect of tolbutamide on glucagon release in a cells. In contrast, for SC-P cells exposed to forskolin, an increase in firing rate was observed relative to 20 mM glucose only, with no significant difference in firing amplitude (FIG. 4F i- 4F iii, FIG. 7D). Accordingly, insulin secretion was higher in the presence of forskolin compared to 20 mM glucose only (FIG. 4F iv), consistent with forskolin enhancing electrical activity and insulin secretion in P cells. Finally, the cyborg islets were exposed to TTX, which effectively inhibited electrical activity in both SC-a and P cells (FIGs. 4E i-4F i).

In summary, utilizing the cyborg islets platform, simultaneous SC-a and SC-P specific electrical activities were measured and validated their distinct responses to glucose and various secretion modulators.

Long-term stably tracking maturing SC-a and SC-[> electrical activities. Cyborg SC-islets offer a platform to study islet-wide SC-a and P electrical activities and their stimulus-coupled dynamics as they evolve during functional maturation. Using embedded electrodes, glucose-stimulated electrical activities were monitored for an extended culture from week 2 to week 7 following the device integration (FIG. 5A). The results revealed that electrical signals significantly increased after 2 weeks extended culture, demonstrating that a significant number of recorded islet cells acquire electrophysiological functionality (FIG. 5A). A spike sorting approach was then used to trace the electrical maturation trends of individual SC-a and SC-P cells. Clustering of cell-level firing rates identified SC-a and P cells based on increased firing under 2.8 mM or 20 mM glucose, respectively, throughout the extended culture time course (FIG. 5B, FIG. 10A iii).

To characterize maturing SC-a and P electrical activities, their spike firing rate, amplitude, duration, peak-trough ratio, width at half-maximum, repolarization slope, and recovery slope were analyzed. The analysis uncovered significant changes in spike firing rate and amplitude during in vitro maturation (FIG. 5C). By contrast, no significant differences in the other waveform features was observed (FIG 5C), indicating that extracellular spike waveforms remain largely unchanged during in vitro maturation (FIGs. 10F-10O). By analyzing the spike firing rate and amplitude under different glucose concentrations (2.8 mM and 20 mM), two distinct states of SC-a and SC-P cells were identified (FIG. lOAi, ii), which can be further classified into al and a2, pi and P2 cells. Specifically, basal spike firing rates for al/pi cells were 0 or consistently below 0.1 spikes/second at 20 mM/2.8 mM glucose, respectively (FIG. 5D, FIGs. 10B-10E), which increased significantly under stimulatory 2.8 mM/20 mM glucose, respectively. a2/p2 cells, on the other hand, exhibited significantly elevated spike firing rates under stimulatory 2.8 mM/20 mM glucose, respectively, as well as high basal firing rates (above 0.1 spikes/second) at 20 mM/2.8 mM glucose, respectively (FIG. 5F). Moreover, the proportion of cells in al/pi states increased from week 2 to week 7 in all recorded SC-islets (FIG. 5E). Cells in a2/p2 states, on the contrary, decreased in proportion from week 2 to week 7 (FIG. 5G). As a metric of stimulation capacity, the ratio of spike firing rates and amplitudes between stimulatory and basal glucose conditions was calculated (2.8 mM/20 mM glucose for a, 20 mM/2.8 mM glucose for P). Firing rate and amplitude ratios for both a2 and P2 cells were found to be gradually increased from week 2 to week 7 (FIGs. 5H-5I). The longitudinal electrical recording studies thus revealed the following findings: First, SC-a and SC-P cells can be further classified into more mature (al and pi) and less mature (a2 and P2) cell states. These states were distinguished by the glucose threshold for action potential firing, with less mature cells firing more under non-stimulatory glucose conditions. Second, a shift in the proportion of cells in these states occurs over a 2-month extended culture period, with the percentage of more mature cells increasing, while the percentage of less mature cells decreases. Despite these changes, the spike waveform features of both SC-a and SC-P cells remained relatively stable during long-term culture. Third, cells in less mature states (a2 and P2) showed gradually increasing glucose stimulation capacity during the culture period, indicating ongoing specialization of glucose responsiveness.

In sum, these findings revealed population-level changes in the proportions of islet cell states, as well as cell-level maturational changes in frequency and amplitude of spike firing, while other electrical spike waveform features remained relatively consistent over time.

To investigate transcriptional changes associated with electrical maturation during the extended in vitro culture, scRNA-seq was performed on cyborg islets at month 1 and month 2 after device integration (FIGs. 5J-5M, FIGs. 11A-11C). Specifically, pseudotime analysis was performed focusing on endocrine populations and reconstructed continuous early maturation trajectories of SC-a, SC-P, and SC-EC cells. To explore the transcriptional changes along the differentiation trajectory, a two-step analysis was employed. First, the expression profile was projected into a potential of heat diffusion for affinity-based transition embedding (PHATE), followed by the application of the Slingshot algorithm to infer pseudotime. A functional enrichment analysis of upregulated genes was then conducted along the inferred pseudotime trajectory to examine how the transcriptional profile evolved across the endocrine subtypes (FIGs. 5K-5M). The analysis revealed a multifaceted functional landscape, illuminating the transcriptional maturation process (FIGs. 11A-11B). Shared enrichments in peptide chain elongation, ribosomal components (e.g., RPL3, RPL4, RPL5f and signaling by receptor tyrosine kinases (e.g., JUNB. JUND, and CLTA) across cell types underscored a conserved cellular machinery for protein synthesis and signal transduction. This conservation is important for the functionality of SC-islets along the maturation trajectory.

Additionally, enrichments of genes within SC-a and SC-P cells involved in vesicle-mediated transport (e.g., VAMP2 and ATP6AP1) suggests a coordinated mechanism for trafficking hormone-containing vesicles, central to hormone secretion. The presence of genes associated with the lysosomal pathway (e.g., CD63, CTSD, and LAM Pl) reflected a shared function in cellular degradation and recycling, suggesting the cells’ ability to efficiently break down and recycle cellular components, further underscoring the complex regulatory processes within SC-a and SC-P cells (FIGs. 5K-5 M). Furthermore, the enrichment of mTOR pathway-associated genes (e.g., E1F4B, RPS6, and LAMTOR1) within SC-a cells emphasizes a potentially crucial regulatory role in cell growth, energy balance, and metabolism. This pathway, known for its influence on nutrient sensing and autophagy, may modulate glucagon synthesis and secretion within SC-a cells (FIG. 5K).

Collectively, these findings demonstrated a correlation between physiological changes and the activation of energy and hormone metabolism gene pathways. They provided perspective on the early maturation process within SC-islet cells, shedding light on the multifaceted mechanisms that orchestrate their development.

Tracing SC-a and SC-/3 maturation triggered by circadian feeding-fasting entrainment. Entrainment to circadian feeding-fasting cycles enhances in vitro maturity of SC-islets. However, the specific role of circadian rhythms on electrical maturation of SC-a and SC-P cells remains poorly understood. Thus, cyborg islets were subjected to 24-hour glucose and forskolin shock and recovery cycles over a four-day timeframe, followed by electrical recording in a constant environment, to dissect their influence on SC-islet electrical maturation (FIG. 6A). Immunostaining of cyborg islets at 2 months after device integration with/without circadian entrainment showed the presence of insulin-positive P cells and glucagon-positive a cells (FIGs. 12A-12B). Following circadian entrainment, electrical measurements were conducted at 2.8 mM and 20 mM glucose every 4 hours for 72 hours, thereby capturing circadian electrical trends (FIGs. 6B-6C). In samples without circadian entrainment, a gradual loss of electrical activities was evident after three days of electrical recording. In contrast, circadian-entrained samples exhibited consistent electrical activities throughout the 3-day electrical recording (FIGs. 6D-6E). These results suggested that diurnal feeding-fasting enhanced in vitro SC-islet function within a relatively short period of four days.

Following entrainment, both SC-a cells and SC-P cells exhibited circadian rhythms in their glucose-coupled firing rates (FIGs. 6F-6G, FIGs. 12F-12H). In contrast, control samples (without entrainment) did not display firing rhythmicity and gradually lost electrical activities after 2 days of recording (FIGs. 12E-12G). Moreover, glucagon and insulin levels in the medium of entrained samples demonstrated clear circadian oscillations (FIGs. 6F-6G), while control samples did not exhibit any rhythmicity (FIGs. 6F-6G). These results indicated that diurnal feeding-fasting entrains electrical activities of both a and P cells within SC-islets to elicit circadian hormone secretion rhythms.

To further investigate the transcriptional impact of circadian entrainment, proteinprotein interaction analysis and functional enrichment analysis were conducted on SC- islet samples with/without circadian entrainment (FIGs. 6J-6K, FIGs. 12K-12O). Among genes upregulated in SC-a cells, overrepresented biological functions included respiratory electron transport, COPI complex-mediated vesicle transport, circadian entrainment, and cAMP-coupled GPCR signaling (FIG. 6J, FIGs. 12K-12O), suggesting greater/synchronized metabolism and hormone exocytosis under circadian regulation. In SC-P cells, upregulated genes were associated with respiratory electron transport, membrane potential regulation, ion transport, and insulin signaling, as well as cell junction establishment, evidencing a circadian influence on glucose-coupled insulin exocytosis and on communication and coordination among P cells (FIG. 6K). The EC cells demonstrated an enrichment in the calcium ion transmembrane transport, suggesting an important role of circadian rhythms in regulating calcium signaling in these cells, which could impact their secretory function.

These results demonstrated that feeding-fasting cycles triggered hormone secretion rhythms via increased and sustained coordination of islet-wide a-P stimulated activities, linked to cell-cell communication and exocytic network induction, revealing a role for circadian rhythms in synchronizing islet- wide a-P activities to generate coordinated stimulus-coupled hormone responses, potentially contributing to their coordinated function in maintaining glucose homeostasis. Discussion. How a and P cell activities within human islets evolve and become coordinated during the period of functional maturation, when they reach their full secretory capacity, has remained largely unexplored, both in their natural environment and in in vitro settings. Like neurons, islet a/p cells release insulin/glucagon in response to membrane potential changes, which are driven by glucose oxidation-generated ATP. In this example, a new approach had been introduced to investigate the physiological maturation of human SC-islets using embedded electrodes, allowing capture of the membrane potential changes that underlie hormone release. This approach offered an opportunity for 1) real-time electrophysiological recording of simultaneous a and P cell activities across the entire islet and 2) continuous tracking of islet-wide single-cell activities during the maturation process. These capabilities opened new avenues for studies of the interplay between changes in islet physiology associated with maturation. Moreover, they offered quantitative readouts for genetic and drug screenings aimed at identifying maturation-modulating factors. The cyborg islet platform was employed and electrical activities of a and P cells were characterized within SC-islets as their function specializes during extended in vitro culture and upon daily feeding entrainment. The data revealed that 1) specialized a and P cell states, marked by low basal firing rates, existed from the earliest maturation stages, 2) refinement of the glucose threshold for hormone release involved transitions in membrane depolarization activity, upstream of calcium handling, and 3) circadian rhythms improved coordinated stimulus-coupled hormone responses by synchronizing islet-wide a-P electrical activities. These findings expanded the understanding of islet physiology, complementing observations from calcium flux and hormone secretion studies about P-cell maturation and heterogeneity. Moreover, they explained how circadian rhythms lead to improved islet function.

Device fabrication, assembly, and. characterization. Stretchable mesh nanoelectronics were fabricated . First, a 500-pm-thick glass wafer was cleaned with acetone, isopropyl alcohol, and deionized (DI) water. Next, a 100-nm- thick nickel (Ni) sacrificial layer was deposited using a thermal evaporator (Sharon). The SU-8 precursor (SU-8 2000.5, MicroChem) was spin-coated to achieve a thickness of either 800 or 400 nm, followed by pre-baking at 65 °C and 95 °C for 2 minutes each. The SU-8 was then exposed to 365 nm UV for 200 mJ/cm², post-baked at 65 °C and 95 °C for 2 minutes each, developed using SU-8 developer (MicroChem) for 60 seconds, and baked at 180°C for 40 minutes to define mesh-like SU-8 patterns for bottom encapsulation. Then, the L0R3A photoresist (MicroChem) was spin-coated at 4000 rpm, pre-baked at 180 °C for 5 minutes, and the SI 805 photoresist (MicroChem) was spin-coated at 4000 rpm, prebaked at 115 °C for 1 minute, exposed to 405 nm UV for 40 mJ/cm², and developed using CD-26 developer (Micropost) for 70 seconds to define interconnect patterns. Chromium/gold/chromium (Cr/Au/Cr) with a thickness of 5/40/5 nm was deposited using an electron-beam evaporator (Denton), followed by a standard lift-off procedure in remover PG (MicroChem) to define the interconnects. Electrode patterns were defined in the LOR3A/S1805 bilayer photoresists as described above. Chromium/platinum (Cr/Pt) with a thickness of 5/50 nm was deposited using an electron-beam evaporator (Denton), followed by a standard lift-off procedure in remover PG (MicroChem) to define the electrodes. Finally, the top SU-8 encapsulation layer was defined as described above.

Multi-channel flexible flat cables (FFCs, Molex) were soldered onto the input/output (VO) pads using a flip-chip bonder (Finetech Fineplacer). Subsequently, a custom-made cell culture chamber was affixed to the substrate wafer with a biocompatible adhesive (Kwik-Sil, WPI). To achieve Pt black electroplating on the Pt electrode array, a 0.08 wt% chloroplatinic acid (H PtCV) precursor solution in DI water was drop-casted onto the device. A direct current (DC) electrical current density of 1 mA/cm² was then applied for 3 minutes using the electrodes as anodes and an external Pt wire as the cathode. The device was thoroughly rinsed with DI water and dried with N2 before undergoing treatment with oxygen plasma (Anatech 106 oxygen plasma barrel asher). Finally, a 1 mF Ni etchant (type TFG, Transene) was introduced into the chamber for 2 to 4 hours to completely release the stretchable mesh nanoelectronics from the glass substrate.

To determine the electrochemical impedance spectrum of electrodes in each device, a three-electrode setup was employed. The counter electrode comprised a platinum wire (300 pm in diameter, 1.5 cm in length immersed), while a standard silver/silver chloride electrode served as the reference electrode. For the measurements, the SP-150 potentiostat (BIOLOGIC®) was utilized and its commercial software EC-lab. To establish statistical significance, at least three frequency sweeps for each measurement were performed, ranging from 1 MHz to 1 Hz. A sinusoidal voltage of 100 mV peak-to-peak was applied, and the response to ten consecutive sinusoids (spaced out by 10% of the period duration) was accumulated and averaged for each data point. Additionally, crosstalk between electrodes was evaluated at 1 kHz using a Blackrock CerePlex Direct voltage amplifier. To perform cell culture, the devices were rinsed in DI water three times, followed by sterilization through immersion in 70% ethanol for 15 minutes. Then, the devices were washed with DPBS and incubated with Poly-D-lysine hydrobromide (0.01% w/v) and Matrigel™ solution (100 pg/mL). Finally, 60 pL liquid Matrigel™ (10 mg/mL) (hESC-Qualified Matrix, CORNING®) was added to the cell culture chamber to form a Matrigel™ hydrogel substrate before cell culture.

Cell culture and differentiation. Human embryonic pluripotent stem cells (HUES8 NIH hESC registry #09-0021) were seeded at a density of 0.6 million/ml in mTeSRl medium (STEMCELL TECHNOLOGIES®) supplemented with 10 pM ROCK inhibitor Y27632 (DNSK International) for directed differentiation toward islet organoids. After seeding, a half-feed with mTeSRl was performed after 24 hours, followed by a full feed with mTeSRl after 48 hours. At 72 hours post-seeding, the following stepwise differentiation protocol was initiated:

Stage 1 (3 days in SI medium):

• Day 1: 100 ng/mL ActivinA + 14 pg/mL CHIR99201

• Day 2: 100 ng/mL ActivinA

• Day 3: no media change.

Stage 2 (3 days in S2 medium) with 50 ng/mL KGF, feeding every other day.

Stage 3 (2 days in S3 medium):

• Day 1: 50 ng/ml KGF + 0.25 pM Santl + 2 pM Retinoic acid (RA) + 500 nM PDBU + 10 pM ROCK inhibitor + 200 nM LDN193189

• Day 2: Same factors as Day 1, except LDN193189 is omitted.

Stage 4 (5 days in S3 medium) with 50 ng/ml KGF + 0,25 uM Santl + 0,1 uM Retinoic acid (RA) + 10 uM ROCK inhibitor + 5 ng/mL Activin A, feeding every other day. Stage 5 (7 days in BE5 medium):

• Day 1-4: 0.25 nM Santl + 20 ng/mL Betacellulin + 1 pM XXi + 10 pM Alk5i II + 1 pM T3 + 0.1 pM RA. Media changed every other day.

• Day 5-7: Media containing no Sant 1 and 0.025 pM RA. Media changed every other day.

Stage 6: Extended culture in S3 medium, with feeding every other day. Human mesenchymal stem cells (hMSCs; PT-2501) obtained from LONZA® (Walkersville, MD, USA) were maintained in 6-well plates using MSCGM BULLETKIT® medium (cat # PT-3238 & PT-4105, LONZA®).

All methods involving human cells were approved by the Harvard University and University of Pennsylvania IRB and ESCRO committees.

Cyborg human SC-islet. Human SC-islet organoids were dissociated into a single cell suspension. Briefly, upon completion of differentiation, organoids were collected in suspension medium, washed with an equal volume of DPBS, and then incubated in 6.5 mL DPBS and 8 mL Accutase (STEMCELL TECHNOLOGIES®) for 7 minutes at room temperature. After washing, the organoids were dissociated into single cells in PBS + 10 pM ROCK inhibitor by mechanical pipetting up and down 40-50 times with a P1000 set to 1 mL. For MSC dissociation, trypsin-EDTA 0.05% was used, and trypan blue was used to count the cells. To integrate cells with stretchable mesh nanoelectronics, SC-islet single cells (IxlO⁶ cells per 16-channel device and 4xl0⁶ cells per 64-channel device) and hMSCs (0.5xl0⁵ cells per device) were suspended in a mixture (3:1) of S3 medium and MSCGM medium. The cell mixture was then transferred into the device-containing cell culture chamber as described above and cultured at 37 °C with 5% CO2.

In vitro circadian entrainment. Cyborg human SC-islets were subjected to circadian feeding entrainment. Specifically, they were entrained to 24-hour metabolic shock / recovery cycles as follows: cells were cultured in S3 medium containing 20 mM Glucose (SIGMA®; G7528) + 10 pM Forskolin (STEMGENT®; 04-0025) for 1 hour, then washed and cultured in S3 medium containing 20 mM Glucose for the remaining 11 hours. Afterward, they were washed and cultured in basal S3 medium for a 12-hour recovery period. The shock / recovery treatments were repeated four times, with a total duration of four days. Stringent washes were performed between media switches to ensure complete removal of any remaining added factors. The same washing and incubation times were used for control samples mock treated with basal S3 medium only.

Electrical recordings. Electrical activity was recorded using a RHD 64-channel headstage (INTAN TECHNOLOGIES®) connected to the Intan 1024 ch recording controller (INTAN TECHNOLOGIES®). A Pt electrode was employed for grounding the culture medium, while another Pt electrode served as the reference electrode. The samples were placed on a battery-powered warming plate to maintain a thermostatic 37 °C during electrical measurements. The entire measurement setup was enclosed in a Faraday cage. The electrical recording was performed at a sampling rate of 20,000 Hz. For long-term electrical recordings, the samples were recorded for 5 minutes weekly at both 2.8 mM and 20 mM glucose concentrations. For samples subjected to circadian feeding entrainment, upon completion of metabolic shock/recovery cycles, electrical recordings were performed every 4 hours for 72 hours at 2.8 mM and 20 mM glucose concentrations.

Glucose-stimulated insulin/glucagon secretion assays. Cyborg human SC-islets were washed twice with Krebs buffer containing 2.8 mM glucose, followed by a one- hour incubation in 2.8 mM glucose Krebs buffer to remove residual insulin. Next, samples were washed with 2.8 mM glucose Krebs buffer, and sequentially exposed to Krebs buffer containing 2.8 mM glucose and 20 mM glucose, with a one-hour incubation time for each concentration. An additional wash was carried out between the 2.8 mM and 20 mM glucose incubations to remove residual glucose. All incubations were conducted at 37 °C, and supernatant samples were collected at the end of each incubation. The levels of human insulin and glucagon in the collected supernatants were determined using a Human Ultrasensitive Insulin ELISA KIT (ALPCO Diagnostics; 80-INSHUU- E10) and a Glucagon ELISA KIT (MERCODIA®; 10-1271-01), respectively, according to the manufacturer’s instructions. Briefly, all collected supernatants were thawed and mixed well before use, and duplicate 25 pL samplings were assayed to ensure reliability of measurements. Samplings were mixed with detection antibodies in Kit-provided 96- well plates, which were then sealed and incubated at 750 rpm on a plate shaker at the indicated temperatures and incubation times. Unbound antibodies were removed by washing plates 6 times avoiding the formation of bubbles and ensuring removal of all buffers from the last wash. Bound conjugates were detected by incubation at 750 rpm with 3,3’,5,5’-tetramethylbenzidine (TMB), which was allowed to proceed for the indicated incubation time (or for a shorter time if the color of the more concentrated Kit insulin/glucagon concentration standard was strong enough). Reactions were then stopped by adding Kit-provided acidic stop solution to the plate, which was placed on a plate shaker for about 10 s to homogenize reactions. Colorimetric endpoints were read in a CLARIOstar microplate reader (BMG LABTECH®) using 450 nm excitation, and insulin/glucagon concentration was quantified for each sample based on the Kit-provided concentration standards. Immunostaining and imaging. Cyborg and control SC-islets were subjected to immuno staining and clearing procedures. The primary antibodies used in the staining process included Rat anti-INS (Cat# GN-ID4, RRID: AB_2255626, DSHB, 1:100); Mouse anti-GCG (SC-514592, SANTA CRUZ BIOTECH®. 1:300); and Mouse anti- CD44 (ab6124, ABCAM®, 1:250). Primary antibodies were incubated for 4 days at 4°C, followed by application of secondary antibodies (anti-mouse 647, Cat#A32787, RRID: AB_2762830, INVITROGEN®; anti-rat 594, A21209, RRID: AB_2535795, INVITROGEN®) and another 4-day incubation at 4°C. Finally, 4’,6-diamidino-2- phenylindole (DAPI, D9542, SIGMA-ALDRICH®) was added and stained for 1 day. Samples were then submerged in an optical clearing solution overnight and embedded in a 1% agarose gel for imaging with a Leica TCS SP8 confocal microscope.

Single-cell RNA sequencing. Cyborg and control SC-islets were dissociated into single cells. Then, the single cells were suspended in DPBS (without Ca²⁺ and Mg²⁺) with 0.04% bovine serum albumin (SIGMA®) at a concentration of 1000 cells per microliter. Library preparation and sequencing were carried out at the Bauer Sequencing Core facility at Harvard University. The 10X GENOMICS® Chromium Single Cell 3' v3 Reagent Kit was employed to prepare samples following the experimental protocol, as guided by the 10X Genomics Single Cell Protocols Cell Preparation Guide. Subsequently, the prepared samples underwent sequencing on the Illumina NovaSeq platform, with the following sequencing specifications: platform - NovaSeq S4 full flow cell, read length - 50, and read type - paired end.

Single-cell RNA-seq data analysis. The read alignments were first performed with Cell Ranger (lOx GENOMICS®) to the reference human genome GRCh38. Then the R package Seurat was used to analyze the scRNA-seq data. The cells were first filtered for quality control (mitochondrial reads <20%, genes detected >800 and < 12500). Then the cell gene expression matrices were normalized and scaled using the “NormalizedDataO”, “FindVariableFeatures()” and “ScaleData()” functions. Next, dimensional and clustering analysis was performed with “RunPCAQ”, “FindNeighbors()” and “FindClusters()” functions, followed by “RunUMAPQ” function to visualize the data. To annotate the cell-type, the marker genes for each cluster was compared with known values.

For functional enrichment and protein-protein interaction analysis before and after circadian entrainment experiment, the differentially expressed genes for each cell- type (e.g., SC-a, SC-P, and SC-EC) were used as input for the Metascape software. The graph visualizations were then performed with the Cytoscape software.

For pseudotime analysis, gene expression from each cell subtype was first subsetted and then the PHATE algorithm was performed to project the gene expression into 2D PHATE space using the “phate()” function. The Slingshot algorithm was then performed to infer the pseudotime trajectory with “slingshot()”.

Spike Sorting. The raw electrical recordings underwent bandpass filtering within the frequency range of 300-3,000 Hz. To extract the spike waveforms, a spike detection threshold was applied, set at 5 times the standard deviation away from the mean. Subsequently, spike sorting was performed using the MountainSort and Spikeinterface algorithms.

Cell type annotation. Averaged electrical firing rates of spike- sorted units were computed for each minute recorded under 2.8 mM and 20 mM glucose. Two-tailed, unpaired Welch's t-tests were then conducted on the per-minute firing rates under the two glucose conditions for each unit. A unit was annotated as an SC-a cell if it exhibits higher firing rates in 2.8 mM glucose with a p-value < 0.05, or as a SC-P cell if it exhibits higher firing rates in 20 mM glucose with a p-value < 0.05. If a unit did not exhibit significantly different firing rates between 2.8 mM and 20 mM glucose, it was labeled as “other”. To further classify an SC-a cell into al or a2 states, the ratio of its averaged per-minute firing rate in 2.8 mM glucose was calculated over its rate in 20 mM glucose. Then the multiple of the median (MoM) was computed as a measure of how far an individual SC-a cell firing rate ratio deviated from the median across the population. SC-a cells with MoM > 2 were assigned to an al state and those with MoM < 2 to an a2 state. To further classify an SC-P cell into pi or P2 states, the ratio of its averaged firing rate in 20 mM glucose over that in 2.8 mM glucose was computed. Then SC-P cells with MoM > 2 were assigned to a pi state and those with MoM < 2 to a P2 state. A small firing rate offset was added to the denominator when calculating firing rate ratios to avoid zero-division errors. All computations were carried out using Python 3.8 and Scipy 1.10.1.

Rhythmicity Analysis. Rhythmicity of hormone level and voltage spike firing rate time series measurements was evaluated with the RAIN R package, which uses nonparametric Mann- Whitney U tests to compare the ranks of measured values against those of alternative waveforms without assumptions of waveform shape or symmetry, and calculates a Benjamini-Hochberg corrected p value. RAIN was implemented with default parameters and “period = 24, deltat = 4”. The “method = longitudinal” was also used for hormone level measurements, representing a time series sampled from the same bulk cell culture, and specified “measure. sequence” for measurements with varying number of replicates and “na.rm = TRUE” for those with NAs. Comparable trends and statistical significance estimates were obtained using harmonic regression with the Harmonic Regression R library or using the eJTK Rhythmicity test. For plotting, time series measurements were detrended using BioDare2 to subtract a linear regression fit from time series measurements, considering both measurement means and SEMs to normalize within the same scale.

Waveform analysis. Waveform analysis was performed using Scanpy 1.9.5 and PHATE. Spike waveforms of all concerned units were first aligned by inverting waveforms with troughs preceding peaks. All waveforms from the same measured timepoint were averaged to obtain template waveforms for the 18 measurements over 72 hours. Templates in between two timepoints were linearly interpolated. Then the original waveforms, averaged template waveforms, and interpolated waveforms were jointly projected onto the same 3D PHATE space.

While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is/are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and/or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMS What is claimed is:

1. An article, comprising: a mesh comprising a plurality of electrodes electrically connected to a plurality of interconnects; and a plurality of pancreatic cells in contact with at least one of the electrodes, wherein the electrodes and/or the interconnects are stretchable and/or flexible.

2. An article, comprising: a mesh comprising a plurality of wires, wherein at least a portion of the wires are electrically coupled to one or more interconnects; and a plurality of pancreatic cells adjacent to the mesh and in electrical communication with at least one interconnect of the one or more interconnects, wherein the plurality of wires and/or the one or more interconnects are stretchable and/or flexible.

3. The article of claim 2, wherein at least some of the wires are nanoscale wires.

4. The article of any one of the preceding claims, further comprising a hydrogel surrounding at least a portion of the mesh.

5. The article of any one of the preceding claims, further comprising a hydrogel surrounding at least a portion of the pancreatic cells.

6. The article of any one of the preceding claims, wherein the mesh is flexible, stretchable, and/or soft.

7. The article of any one of the preceding claims, wherein the electrodes and/or the interconnects are stretchable.

8. The article of claim 7, wherein the electrodes and/or the interconnects are stretchable in a linear direction by at least 10% without failure.

9. The article of any one of the preceding claims, wherein at least some of the wires have flexibility determined by a bending stiffness of at least 0.09 n-Nm.

10. The article any one of the preceding claims, wherein the electrodes are nanoscale.

11. The article of any one of the preceding claims, wherein the electrodes are microscale.

12. The article of any one of the preceding claims, wherein the pancreatic cells form a portion of a pancreatic organoid.

13. The article of any one of the preceding claims, wherein the pancreatic cells have been entrained by circadian feeding-fasting entrainment.

14. A system, comprising: a mesh comprising a plurality of electrodes electrically connected to a plurality of interconnects; and a plurality of pancreatic cells comprising a first pancreatic cell type and a second pancreatic cell type; and wherein the mesh is configured to distinguish the first pancreatic cell type from the second pancreatic cell type.

15. A system, comprising: a mesh comprising a plurality of sensors electrically connected to a plurality of interconnects and a plurality of electrodes, wherein the mesh defines a processor configured to convert sensor measurements to electrical stimuli; and a plurality of pancreatic cells in electrical communication with the plurality of electrodes.

16. The system of claim 15, wherein at least some of the sensors comprise glucose sensors, glucagon sensors, insulin sensors, somatostatin sensors, lactate sensors, adenosine triphosphate (ATP) sensors, pancreatic polypeptide (PP) sensors, and/or amylin sensors.

17. The system of any one of claims 15-16, wherein at least some of the sensors are glucose sensors.

18. The system of any one of claims 15-17, wherein at least some of the plurality of sensors comprises electrical, chemical, and/or electrochemical sensors.

19. The system of any one of claims 14-18, further comprising a hydrogel surrounding at least a portion of the mesh.

20. The system of any one of claims 14-19, wherein the first pancreatic cell type comprises immature pancreatic cells.

21. The system of any one of claims 14-20, wherein the second pancreatic cell type comprises mature pancreatic cells.

22. The system of any one of claims 14-21, further comprising a hydrogel surrounding at least a portion of the pancreatic cells.

23. The system of any one of claims 14-22, wherein the mesh is flexible, stretchable, and/or soft.

24. The system of any one of claims 14-23, wherein the electrodes are nanoscale.

25. The system of any one of claims 14-24, wherein the electrodes are microscale.

26. The system of any one of claims 14-25, wherein the pancreatic cells form a portion of a pancreatic organoid.

27. The system of any one of claims 14-26, wherein the pancreatic cells have been entrained by circadian feeding-fasting entrainment.

28. A method comprising: applying a first electrical potential to a mesh in contact with a plurality of pancreatic cells comprising a first pancreatic cell type and a second pancreatic cell type, the mesh comprising a plurality of electrodes electrically connected to a plurality of interconnects, wherein the first electrical potential is applied at first concentration of glucose; and applying a second electrical potential to the mesh, wherein the second electrical potential is applied at a second concentration of glucose, different than the first concentration of glucose.

29. A method, comprising: determining a concentration of a compound in an implant contained within a subject using a sensor configured to sense the compound; converting a sensor measurement to an electrical stimulus within a flexible mesh comprising a plurality of wires; and applying the electrical stimulus to one or more pancreatic cells.

30. The method of claim 29, wherein at least some of the wires are nanoscale wires.

31. The method of any one of claims 29-30, wherein the compound is glucose, glucagon, insulin, somatostatin, lactate, adenosine triphosphate (ATP), a pancreatic polypeptide (PP), and/or an amylin.

32. The method of any one of claims 29-31, wherein the compound is glucose.

33. The method of any one of claims 28-32, further comprising a hydrogel surrounding at least a portion of the mesh.

34. The method of any one of claims 28 and 30-33, wherein the first pancreatic cell type comprises immature pancreatic cells.

35. The method of any one of claims 28 and 30-34, wherein the second pancreatic cell type comprises mature pancreatic cells.

36. The method of any one of claims 28-35, further comprising a hydrogel surrounding at least a portion of the pancreatic cells.

37. The method of any one of claims 28-36, wherein the mesh is flexible, stretchable, and/or soft.

38. The method any one of claims 28-and 30-37, wherein the electrodes are nanoscale.

39. The method of any one of claims 28 and 30-38, wherein the electrodes are microscale.

40. The method of any one of claims 28-39, wherein the pancreatic cells form a portion of a pancreatic organoid.

41. The method of any one of claims 28 and 30-40, wherein the first concentration of glucose is less than or equal to 10 mM.

42. The method of any one of claims 28 and 30-41, wherein the second concentration of glucose is greater than or equal to 10 mM.

43. The method of any one of claims 28-42, further comprising culturing the plurality of pancreatic cells.

44. The method of any one of claims 28-43, further comprising dissociating the plurality of pancreatic cells into single cells.

45. The method of any one of claims 28-44, further comprising comparing a response of the first pancreatic cell type to the first electrical potential to a response of the second pancreatic cell type to second electrical potential.

46. The method of any one of claims 28-45, wherein the pancreatic cells have been entrained by circadian feeding-fasting entrainment.

47. An article, comprising: a mesh comprising a plurality of electrodes electrically connected to a plurality of interconnects; and a plurality of pancreatic cells in contact with at least one of the electrodes, wherein the pancreatic cells have been entrained by circadian feeding-fasting entrainment.

48. The article of claim 47, wherein the circadian feeding-fasting entrainment comprises subjecting the pancreatic cells to diurnal circadian feeding-fasting cycles.

49. The article of claim 48, wherein the diurnal circadian feeding-fasting cycles comprise 24-hour glucose and forskolin shock and recovery cycles.