Systematic Analysis of Different Cell Spheroids with a Microfluidic Device Using Scanning Electrochemical Microscopy and Gene Expression Profiling.

The 3D cell spheroid is an emerging tool that allows better recapitulating of in vivo scenarios with multiple factors such as tissue-like morphology and membrane protein expression that intimately coordinates with enzyme activity, thus providing a psychological environment for tumorigenesis study. For analyzing different spheroids, conventional optical imaging may be hampered by the need for fluorescent labeling, which could cause toxicity side effects. As an alternative approach, scanning electrochemical microscopy (SECM) enables label-free imaging. However, SECM for cell spheroid imaging is currently suffering from incapability of systematically analyzing the cell aggregates from spheroid generation, electrochemical signal gaining, and the gene expression on different individual cell spheroids. Herein, we developed a top-removable microfluidic device for cell aggregate yielding and SECM imaging methodology to analyze heterotypic 3D cell spheroids on a single device. This technique allows not only on-chip culturing of cell aggregates but also SECM imaging of the spheroids after opening the chip and subsequent qPCR assay of corresponding clusters. Through employment of the micropit arrays (85 × 4) with a top withdrawable microfluidic layer, uniformly sized breast tumor cell and fibroblast spheroids can be simultaneously produced on a single device. By leveraging voltage-switching mode SECM at different potentials of dual mediators, we evaluated alkaline phosphatase without disturbance of substrate morphology for distinguishing the tumor aggregates from stroma. Moreover, this method also enables gene expression profiling on individual tumor or stromal spheroids. Therefore, this new strategy can seamlessly bridge SECM measurements and molecular biological analysis.

Extracellular Matrix Rigidities Regulate the Tricarboxylic Acid Cycle and Antibiotic Resistance of Three-Dimensionally Confined Bacterial Microcolonies.

As a major cause of clinical chronic infection, microbial biofilms/microcolonies in host tissues essentially live in 3D-constrained microenvironments, which potentially modulate their spatial self-organization and morphodynamics. However, it still remains unclear whether and how mechanical cues of 3D confined microenvironments, for example, extracellular matrix (ECM) stiffness, exert an impact on antibiotic resistance of bacterial biofilms/microcolonies. With a high-throughput antibiotic sensitivity testing (AST) platform, it is revealed that 3D ECM rigidities greatly modulate their resistance to diverse antibiotics. The microcolonies in 3D ECM with human tissue-specific rigidities varying from 0.5 to 20 kPa show a ≈2-10 000-fold increase in minimum inhibitory concentration, depending on the types of antibiotics. The authors subsequently identified that the increase in 3D ECM rigidities leads to the downregulation of the tricarboxylic acid (TCA) cycle, which is responsible for enhanced antibiotic resistance. Further, it is shown that fumarate, as a potentiator of TCA cycle activity, can alleviate the elevated antibiotic resistance and thus remarkably improve the efficacy of antibiotics against bacterial microcolonies in 3D confined ECM, as confirmed in the chronic infection mice model. These findings suggest fumarate can be employed as an antibiotic adjuvant to effectively treat infections induced by bacterial biofilms/microcolonies in a 3D-confined environment.

Copper Single-Atom Jellyfish-like Nanomotors for Enhanced Tumor Penetration and Nanocatalytic Therapy.

Single-atom catalysts with extraordinary catalytic activity have been receiving great attention in tumor therapy. However, most single-atom catalysts lack self-propulsion properties, restricting them from actively approaching cancer cells or penetrating the interior of tumors. Herein, we design N-doped jellyfish-like mesoporous carbon nanomotors coordinated with single-atom copper (Cu-JMCNs). It is a combination of single-atom nanocatalytic medicine and nanomotor self-propulsion for cancer therapy. The Cu single atom can catalyze H2O2 into toxic hydroxyl radical (•OH) for chemodynamic therapy (CDT). Near-infrared light triggers Cu-JMCNs to achieve self-thermophoretic motion because of the jellyfish-like asymmetric structure and photothermal property of carbon, which significantly improves the cellular uptake and the penetration of three-dimensional tumors. In vivo experiments indicate that the combination of single-atom Cu for CDT and near-infrared light propulsion can achieve over 85% tumor inhibition rate. This work sheds light on the development of advanced nanomotors with single-atom catalysts for biomedical applications.

Mini-pillar microarray for individually electrochemical sensing in microdroplets.

High throughput and high sensitivity are two important aspects in multiple biomarker recognition, drug discovery and relevant biochemical sensing. Here, we integrate mini-pillar microarray with the circuit components toward high-throughput individual electrochemical sensing in microdroplets. On such droplet-microarray-based electrochemical platform, the high adhesion of the mini-pillar can hold a microdroplet (hundreds nanoliter to a few microliter) regardless of rotation and deformation. Each pillar as a unit has a three-electrode to achieve individual electrochemical sensing, and electrodes are integrated on one side to achieve the sequential electrochemical read-out. Qualitative and quantitative electrochemical assessments of multiple glucose concentrations in individual microdroplets are also achieved. Such mini-pillar-based individual electrochemical platform shows great potential in high-throughput and high-sensitive biomolecular recognitions, provides an opportunity to develop miniaturized sensing platform for emerging biological and pathological applications.

A 3D Printed Hanging Drop Dripper for Tumor Spheroids Analysis Without Recovery.

Compared with traditional monolayer cell culture, the three-dimensional tumor spheroid has emerged as an essential in vitro model for cancer research due to the recapitulation of the architecture and physiology of solid human tumors. Herein, by implementing the rapid prototyping of a benchtop 3D printer, we developed a new strategy to generate and analyze tumor spheroids on a commonly used multi-well plate. In this method, the printed artifact can be directly mounted on a 96/384-well plate, enables hanging drop-based spheroid formation, avoiding the tedious fabrication process from micromechanical systems. Besides long-term spheroid culture (20 days), this method supports subsequent analysis of tumor spheroid by seamlessly dripping from the printed array, thereby eliminating the need for spheroids retrieval for downstream characterization. We demonstrated several tumor spheroid-based assays, including tumoroid drug testing, metastasis on or inside extracellular matrix gel, and tumor transendothelial (TEM) assay. Based on quantitative phenotypical and molecular analysis without any precarious retrieval and transfer, we found that the malignant breast cancer (MDA-MB-231) cell aggregate presents a more metastatic morphological phenotype than the non-malignant breast cancer (MCF-7) and colonial cancer (HCT-116) cell spheroid, and shows an up-regulation of epithelial-mesenchymal transition (EMT) relevant genes (fold change > 2). Finally, we validated this tumor malignancy by the TEM assay, which could be easily performed using our approach. This methodology could provide a useful workflow for expediting tumoroid modeled in vitro assay, allowing the "Lab-on-a-Cloud" scenario for routine study.