Gradient Nanoconfinement Facilitates Binding of Transcriptional Factor NF-κB to Histone- and Protamine-DNA Complexes.

Mechanically induced chromosome reorganization plays important roles in transcriptional regulation. However, the interplay between chromosome reorganization and transcription activities is complicated, such that it is difficult to decipher the regulatory effects of intranuclear geometrical cues. Here, we simplify the system by introducing DNA, packaging proteins (i.e., histone and protamine), and transcription factor NF-κB into a well-defined fluidic chip with changing spatical confinement ranging from 100 to 500 nm. It is uncovered that strong nanoconfinement suppresses higher-order folding of histone- and protamine-DNA complexes, the fracture of which exposes buried DNA segments and causes increased quantities of NF-κB binding to the DNA chain. Overall, these results reveal a pathway of how intranuclear geometrical cues alter the open/closed state of a DNA-protein complex and therefore affect transcription activities: i.e., NF-κB binding.

From Chips-in-Lab to Point-of-Care Live Cell Device: Development of a Microfluidic Device for On-Site Cell Culture and High-Throughput Drug Screening.

Live cell assays provide real-time data of cellular responses. In combination with microfluidics, applications such as automated and high-throughput drug screening on live cells can be accomplished in small devices. However, their application in point-of-care testing (POCT) is limited by the requirement for bulky equipment to maintain optimal cell culture conditions. In this study, we propose a POCT device that allows on-site cell culture and high-throughput drug screening on live cells. We first observe that cell viabilities are substantially affected by liquid evaporation within the microfluidic device, which is intrinsic to the polydimethylsiloxane (PDMS) material due to its hydrophobic nature and nanopatterned surface. The unwanted PDMS-liquid-air interface in the cell culture environment can be eliminated by maintaining a persistent humidity of 95-100% or submerging the whole microfluidic device under water. Our results demonstrate that in the POCT device equipped with a water tank, both primary cells and cell lines can be maintained for up to 1 week without the need for external cell culture equipment. Moreover, this device is powered by a standard alkali battery and can automatically screen over 5000 combinatorial drug conditions for regulating neural stem cell differentiation. By monitoring dynamic variations in fluorescent markers, we determine the optimal doses of platelet-derived growth factor and epidermal growth factor to suppress proinflammatory S100A9-induced neuronal toxicities. Overall, this study presents an opportunity to transform lab-on-a-chip technology from a laboratory-based approach to actual point-of-care devices capable of performing complex experimental procedures on-site and offers significant advancements in the fields of personalized medicine and rapid clinical diagnostics.

A Stand-Alone Microfluidic Chip for Long-Term Cell Culture.

Live-cell microscopy is crucial for biomedical studies and clinical tests. The technique is, however, limited to few laboratories due to its high cost and bulky size of the necessary culture equipment. In this study, we propose a portable microfluidic-cell-culture system, which is merely 15 cm×11 cm×9 cm in dimension, powered by a conventional alkali battery and costs less than USD 20. For long-term cell culture, a fresh culture medium exposed to 5% CO2 is programmed to be delivered to the culture chamber at defined time intervals. The 37 °C culture temperature is maintained by timely electrifying the ITO glass slide underneath the culture chamber. Our results demonstrate that 3T3 fibroblasts, HepG2 cells, MB-231 cells and tumor spheroids can be well-maintained for more than 48 h on top of the microscope stage and show physical characters (e.g., morphology and mobility) and growth rate on par with the commercial stage-top incubator and the widely adopted CO2 incubator. The proposed portable cell culture device is, therefore, suitable for simple live-cell studies in the lab and cell experiments in the field when samples cannot be shipped.

Super-Resolution Reconstruction of Cytoskeleton Image Based on A-Net Deep Learning Network.

To date, live-cell imaging at the nanometer scale remains challenging. Even though super-resolution microscopy methods have enabled visualization of sub-cellular structures below the optical resolution limit, the spatial resolution is still far from enough for the structural reconstruction of biomolecules in vivo (i.e., ~24 nm thickness of microtubule fiber). In this study, a deep learning network named A-net was developed and shows that the resolution of cytoskeleton images captured by a confocal microscope can be significantly improved by combining the A-net deep learning network with the DWDC algorithm based on a degradation model. Utilizing the DWDC algorithm to construct new datasets and taking advantage of A-net neural network's features (i.e., considerably fewer layers and relatively small dataset), the noise and flocculent structures which originally interfere with the cellular structure in the raw image are significantly removed, with the spatial resolution improved by a factor of 10. The investigation shows a universal approach for exacting structural details of biomolecules, cells and organs from low-resolution images.

Feature Extraction of 3T3 Fibroblast Microtubule Based on Discrete Wavelet Transform and Lucy-Richardson Deconvolution Methods.

Accompanied by the increasing requirements of the probing micro/nanoscopic structures of biological samples, various image-processing algorithms have been developed for visualization or to facilitate data analysis. However, it remains challenging to enhance both the signal-to-noise ratio and image resolution using a single algorithm. In this investigation, we propose a composite image processing method by combining discrete wavelet transform (DWT) and the Lucy-Richardson (LR) deconvolution method, termed the DWDC method. Our results demonstrate that the signal-to-noise ratio and resolution of live cells' microtubule networks are considerably improved, allowing the recognition of features as small as 120 nm. The method shows robustness in processing the high-noise images of filament-like biological structures, e.g., the cytoskeleton networks captured by fluorescent microscopes.

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

Dynamic intracellular mechanical cues facilitate collective signaling responses.

Collective behavior emerges in diverse life machineries, e.g., the immune responses to dynamic stimulations. The essential questions that arise here are that whether and how cells in vivo collectively respond to stimulation frequencies higher than their intrinsic natural values, e.g., the acute inflammation conditions. In this work, we systematically studied morphological and signaling responses of population fibroblasts in an interconnected cell monolayer and uncovered that, besides the natural NF-κB oscillation frequency of 1/90 min-1, collective signaling response emerges in the cell monolayer at 1/20 min-1 TNF-α input periodicity as well. Using a customized microfluidic device, we independently induced dynamic chemical stimulation and cytoskeleton reorganization on the stand-alone cells to exclude the effect of cell-cell communication. Our results reveal that, at this particular frequency, chemical stimulation is translated into dynamic intracellular mechanical cues through RAC1-medicated induction of dynamic cell-cell connections and cytoskeleton reorganizations, which synergize with chemical input to facilitate collective signaling responses.

Air-Bubble Induced Mixing: A Fluidic Mixer Chip.

In this study, we report the design and fabrication of a novel fluidic mixer. As proof-of-concept, the laminar flow in the main channel is firstly filled with small air-bubbles, which act as active stirrers inducing chaotic convective turbulent flow, and thus enhance the solutes mixing even at a low input flow rate. To further increase mixing efficiency, a design of neck constriction is included, which changes the relative positions of the inclusion bubbles significantly. The redistribution of liquid volume among bubbles then causes complex flow profile, which further enhances mixing. This work demonstrates a unique approach of utilizing air bubbles to facilitate mixing in bulk solution, which can find the potential applications in microfluidics, fast medical analysis, and biochemical synthesis.

Proinflammatory S100A9 Regulates Differentiation and Aggregation of Neural Stem Cells.

Inflammation is the primary pathological feature of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease. Proinflammatory molecules (e.g., S100A9) play important roles during the progression of the diseases by regulating behavior and fate of multiple cell types in the nervous system. Our earlier studies reveal that S100A9 is toxic to neurons, and its interaction with Aß peptides leads to the formation of large nontoxic amyloidogenic aggregates, suggesting a protective role of coaggregation with Aß amyloids. We herein demonstrate that S100A9 interacts with neural stem cells (NSCs) and causes NSC differentiation. In the brain of transgenic AD mouse models, we found large quantities of proinflammatory S100A9, which colocalizes with the differentiated NSCs. NSC sphere formation, which is a representative character of NSC stemness, is also substantially inhibited by S100A9. These results suggest that S100A9 is a representative marker for the inflammatory conditions in AD, and it promotes NSC differentiation. Intriguingly, in contrast to the death of both stem and differentiated NSCs caused by high S100A9 doses, S100A9 at a moderate concentration is toxic only to the early differentiated NSCs but not the stem cells. We therefore postulate that, at the early stage of AD, the expression of S100A9 leads to NSC differentiation, which remedies the neuron damage. The application of drugs, which help maintain NSC stemness (e.g., the platelet-derived growth factor, PDGF), may help overcome the acute inflammatory conditions and improve the efficacy of NSC transplantation therapy.