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Intracellular electrophysiology is essential in neuroscience, cardiology, and pharmacology for studying cells' electrical properties. Traditional methods like patch-clamp are precise but low-throughput and invasive. Nanoelectrode Arrays (NEAs) offer a promising alternative by enabling simultaneous intracellular and extracellular action potential (iAP and eAP) recordings with high throughput. However, accessing intracellular potentials with NEAs remains challenging. This study presents an AI-supported technique that leverages thousands of synchronous eAP and iAP pairs from stem-cell-derived cardiomyocytes on NEAs. Our analysis revealed strong correlations between specific eAP and iAP features, such as amplitude and spiking velocity, indicating that extracellular signals could be reliable indicators of intracellular activity. We developed a physics-informed deep learning model to reconstruct iAP waveforms from extracellular recordings recorded from NEAs and Microelectrode arrays (MEAs), demonstrating its potential for non-invasive, long-term, high-throughput drug cardiotoxicity assessments. This AI-based model paves the way for future electrophysiology research across various cell types and drug interactions.

Nanoscale surface topography is an effective approach in modulating cell-material interactions, significantly impacting cellular and nuclear morphologies, as well as their functionality. However, the adaptive changes in cellular metabolism induced by the mechanical and geometrical microenvironment of the nanotopography remain poorly understood. In this study, we investigated the metabolic activities in cells cultured on engineered nanopillar substrates by using a label-free multimodal optical imaging platform. This multimodal imaging platform, integrating two photon fluorescence (TPF) and stimulated Raman scattering (SRS) microscopy, allowed us to directly visualize and quantify metabolic activities of cells in 3D at the subcellular scale. We discovered that the nanopillar structure significantly reduced the cell spreading area and circularity compared to flat surfaces. Nanopillar-induced mechanical cues significantly modulate cellular metabolic activities with variations in nanopillar geometry further influencing these metabolic processes. Cells cultured on nanopillars exhibited reduced oxidative stress, decreased protein and lipid synthesis, and lower lipid unsaturation in comparison to those on flat substrates. Hierarchical clustering also revealed that pitch differences in the nanopillar had a more significant impact on cell metabolic activity than diameter variations. These insights improve our understanding of how engineered nanotopographies can be used to control cellular metabolism, offering possibilities for designing advanced cell culture platforms which can modulate cell behaviors and mimic natural cellular environment and optimize cell-based applications. By leveraging the unique metabolic effects of nanopillar arrays, one can develop more effective strategies for directing the fate of cells, enhancing the performance of cell-based therapies, and creating regenerative medicine applications.

The development of biomaterials capable of regulating cellular processes and guiding cell fate decisions has broad implications in tissue engineering, regenerative medicine, and cell-based assays for drug development and disease modeling. Recent studies have shown that three-dimensional (3D) nanoscale physical cues such as nanotopography can modulate various cellular processes like adhesion and endocytosis by inducing nanoscale curvature on the plasma and nuclear membranes. Two-dimensional (2D) biochemical cues such as protein micropatterns can also regulate cell function and fate by controlling cellular geometries. Development of biomaterials with precise control over nanoscale physical and biochemical cues can significantly influence programming cell function and fate. In this study, we utilized a laser-assisted micropatterning technique to manipulate the 2D architectures of cells on 3D nanopillar platforms. We performed a comprehensive analysis of cellular and nuclear morphology and deformation on both nanopillar and flat substrates. Our findings demonstrate the precise engineering of single cell architectures through 2D micropatterning on nanopillar platforms. We show that the coupling between the nuclear and cell shape is disrupted on nanopillar surfaces compared to flat surfaces. Furthermore, our results suggest that cell elongation on nanopillars enhances nanopillar-induced endocytosis. We believe our platform serves as a versatile tool for further explorations into programming cell function and fate through combined physical cues that create nanoscale curvature on cell membranes and biochemical cues that control the geometry of the cell.

Materials with engineered nano‐scale surface topographies, such as nanopillars, nanoneedles, and nanowires, mimic natural structures like viral spike proteins, enabling them to bypass biological barriers like the plasma membrane. These properties have led to applications in nanoelectronics for intracellular sensing and drug delivery platforms, some of which are already in clinical trials. Here, evidence is present that nanotopographic materials can induce transient openings in the nuclear membranes of various cell types without penetrating the cells, breaching the nucleo‐cytoplasmic barrier, and allowing uncontrolled molecular exchange across the nuclear membrane. These openings, induced by nanoscale curvature, are temporary and repaired through the Endosomal Sorting Complexes Required for Transport (ESCRT)‐mediated mechanisms. The findings suggest a potential for nano\topographic materials to temporarily breach the nuclear membrane with potential applications in direct nuclear sensing and delivery. [ABSTRACT FROM AUTHOR]