Electrochemical modulation enhances the selectivity of peripheral neurostimulation in vivo.

Electrical nerve stimulation serves an expanding list of clinical applications, but it faces persistent challenges in selectively activating bundled nerve fibers. In this study, we investigated electrochemical modulation with an ion-selective membrane (ISM) and whether it, used together with electrical stimulation, may provide an approach for selective control of peripheral nerves. Guided by theoretical transport modeling and direct concentration measurements, we developed an implantable, multimodal ISM cuff capable of simultaneous electrical stimulation and focused Ca2+ depletion. Acutely implanting it on the sciatic nerve of a rat in vivo, we demonstrated that Ca2+ depletion could increase the sensitivity of the nerve to electrical stimulation. Furthermore, we found evidence that the effect of ion modulation would selectively influence functional components of the nerve, allowing selective activation by electrical current. Our results raise possibilities for improving functional selectivity of new and existing bioelectronic therapies, such as vagus nerve stimulation.

Separation of Activated T Cells Using Multidimensional Double Spiral (MDDS) Inertial Microfluidics for High-Efficiency CAR T Cell Manufacturing.

This study introduces a T cell enrichment process, capitalizing on the size differences between activated and unactivated T cells to facilitate the isolation of activated, transducible T cells. By employing multidimensional double spiral (MDDS) inertial sorting, our approach aims to remove unactivated or not fully activated T cells post-activation, consequently enhancing the efficiency of chimeric antigen receptor (CAR) T cell manufacturing. Our findings reveal that incorporating a simple, label-free, and continuous MDDS sorting step yields a purer T cell population, exhibiting significantly enhanced viability and CAR-transducibility (with up to 85% removal of unactivated T cells and approximately 80% recovery of activated T cells); we found approximately 2-fold increase in CAR transduction efficiency for a specific sample, escalating from ∼10% to ∼20%, but this efficiency highly depends on the original T cell sample as MDDS sorting would be more effective for samples possessing a higher proportion of unactivated T cells. This new cell separation process could augment the efficiency, yield, and cost-effectiveness of CAR T cell manufacturing, potentially broadening the accessibility of this transformative therapy and contributing to improved patient outcomes.

Comparative Analysis of Serum and Serum-Free Medium Cultured Mesenchymal Stromal Cells for Cartilage Repair.

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium's ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair.

Continuous Online Titer Monitoring in CHO Cell Culture Supernatant Using a Herringbone Nanofluidic Filter Array.

Online monitoring of monoclonal antibody product titers throughout biologics process development and production enables rapid bioprocess decision-making and process optimization. Conventional analytical methods, including high-performance liquid chromatography and turbidimetry, typically require interfacing with an automated sampling system capable of online sampling and fractionation, which suffers from increased cost, a higher risk of failure, and a higher mechanical complexity of the system. In this study, a novel nanofluidic system for continuous direct (no sample preparation) IgG titer measurements was investigated. Tumor necrosis factor α (TNF-α), conjugated with fluorophores, was utilized as a selective binder for adalimumab in the unprocessed cell culture supernatant. The nanofluidic device can separate the bound complex from unbound TNF-α and selectively concentrate the bound complex for high-sensitivity detection. Based on the fluorescence intensity from the concentrated bound complex, a fluorescence intensity versus titer curve can be generated, which was used to determine the titer of samples from filtered, unpurified Chinese hamster ovary cell cultures continuously. The system performed direct monitoring of IgG titers with nanomolar resolution and showed a good correlation with the biolayer interferometry assays. Furthermore, by variation of the concentration of the indicator (TNF-α), the dynamic range of the system can be tuned and further expanded.

Rapid and Label-Free Classification of Blood Leukocytes for Immune State Monitoring.

A fully automated and label-free sample-to-answer white blood cell (WBC) cytometry platform for rapid immune state monitoring is demonstrated. The platform integrates (1) a WBC separation process using the multidimensional double spiral (MDDS) device and (2) an imaging process where images of the separated WBCs are captured and analyzed. Using the deep-learning-based image processing technique, we analyzed the captured bright-field images to classify the WBCs into their subtypes. Furthermore, in addition to cell classification, we can detect activation-induced morphological changes in WBCs for functional immune assessment, which could allow the early detection of various diseases. The integrated platform operates in a rapid (<30 min), fully automated, and label-free manner. The platform could provide a promising solution to future point-of-care WBC diagnostics applications.

Portable Seawater Desalination System for Generating Drinkable Water in Remote Locations.

A portable seawater desalination system would be highly desirable to solve water challenges in rural areas and disaster situations. While many reverse osmosis-based portable desalination systems are already available commercially, they are not adequate for providing reliable drinking water in remote locations due to the requirement of high-pressure pumping and repeated maintenance. We demonstrate a field-deployable desalination system with multistage electromembrane processes, composed of two-stage ion concentration polarization and one-stage electrodialysis, to convert brackish water and seawater to drinkable water. A data-driven predictive model is used to optimize the multistage configuration, and the model predictions show good agreement with the experimental results. The portable system desalinates brackish water and seawater (2.5-45 g/L) into drinkable water (defined by WHO guideline), with the energy consumptions of 0.4-4 (brackish water) and 15.6-26.6 W h/L (seawater), respectively. In addition, the process can also reduce suspended solids by at least a factor of 10 from the source water, resulting in crystal clear water (<1 NTU) even from the source water with turbidity higher than 30 NTU (i.e., cloudy seawater by the tide). We built a fully integrated prototype (controller, pumps, and battery) packaged into a portable unit (42 × 33.5 × 19 cm3, 9.25 kg, and 0.33 L/h production rate) controlled by a smartphone, tested for battery-powered field operation. The demonstrated portable desalination system is unprecedented in size, efficiency, and operational flexibility. Therefore, it could address unique water challenges in remote, resource-limited regions of the world.

Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates.

Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-ß-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the 'HepaCometChip', enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.

Universal amplification-free molecular diagnostics by billion-fold hierarchical nanofluidic concentration.

Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g., PCR), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis. We report a universal biomolecule enrichment technique termed hierarchical nanofluidic molecular enrichment system (HOLMES) for amplification-free molecular diagnostics using massively paralleled and hierarchically cascaded nanofluidic concentrators. HOLMES achieves billion-fold enrichment of both nucleic acids and proteins within 30 min, which not only overcomes many inherent issues of nucleic acid amplification but also provides unprecedented enrichment performance for protein analysis. HOLMES features the ability to selectively enrich target biomolecules and simultaneously deplete nontargets directly in complex crude samples, thereby enormously enhancing the signal-to-noise ratio of detection. We demonstrate the direct detection of attomolar nucleic acids in urine and serum within 35 min and HIV p24 protein in serum within 60 min. The performance of HOLMES is comparable to that of nucleic acid amplification tests and near million-fold improvement over standard enzyme-linked immunosorbent assay (ELISA) for protein detection, being much simpler and faster in both applications. We additionally measured human cardiac troponin I protein in 9 human plasma samples, and showed excellent agreement with ELISA and detection below the limit of ELISA. HOLMES is in an unparalleled position to unleash the potential of protein-based diagnosis.

Multiplexed Single-Cell Leukocyte Enzymatic Secretion Profiling from Whole Blood Reveals Patient-Specific Immune Signature.

Enzymatic secretion of immune cells (leukocytes) plays a dominant role in host immune responses to a myriad of biological triggers, including infections, cancers, and cardiovascular diseases. Current tools to probe these leukocytes inadequately profile these vital biomarkers; the need for sample preprocessing steps of cell lysis, labeling, washing, and pipetting inevitably triggers the cells, changes its basal state, and dilutes the individual cell secretion in bulk assays. Using a fully integrated system for multiplexed profiling of native immune single-cell enzyme secretion from 50 µL of undiluted blood, we eliminate sample handling. With a total analysis time of 60 min, the integrated platform performs six tasks of leukocyte extraction, cell washing, fluorescent enzyme substrate mixing, single-cell droplet making, droplet incubation, and real-time readout for leukocyte secretion profiling of neutrophil elastase, granzyme B, and metalloproteinase. We calibrated the device, optimized the protocols, and tested the leukocyte secretion of acute heart failure (AHF) patients at admission and predischarge. This paper highlights the presence of single-cell enzymatic immune phenotypes independent of CD marker labeling, which could potentially elucidate the innate immune response states. We found that patients recovering from AHF showed a corresponding reduction in immune-cell enzymatic secretion levels and donor-specific enzymatic signatures were observed, which suggests patient-to-patient heterogeneous immune response. This platform presents opportunities to elucidate the complexities of the immune response from a single drop of blood and bridge the current technological, biological, and medical gap in understanding immune response and biological triggers.

Separation of Ultra-High-Density Cell Suspension via Elasto-Inertial Microfluidics.

Separation of high-density suspension particles at high throughput is crucial for many chemical, biomedical, and environmental applications. In this study, elasto-inertial microfluidics is used to manipulate ultra-high-density cells to achieve stable equilibrium positions in microchannels, aided by the inherent viscoelasticity of high-density cell suspension. It is demonstrated that ultra-high-density Chinese hamster ovary cell suspension (>26 packed cell volume% (PCV%), >95 million cells mL-1 ) can be focused at distinct lateral equilibrium positions under high-flow-rate conditions (up to 10 mL min-1 ). The effect of flow rates, channel dimensions, and cell densities on this unique focusing behavior is studied. Cell clarification is further demonstrated using this phenomenon, from 29.7 PCV% (108.1 million cells mL-1 ) to 8.3 PCV% (33.2 million cells mL-1 ) with overall 72.1% reduction efficiency and 10 mL min-1 processing rate. This work explores an extreme case of elasto-inertial particle focusing where ultra-high-density culture suspension is efficiently manipulated at high throughput. This result opens up new opportunities for practical applications of high-particle-density suspension manipulation.

Label-Free Biophysical Markers from Whole Blood Microfluidic Immune Profiling Reveal Severe Immune Response Signatures.

Disease manifestation and severity from acute infections are often due to hyper-aggressive host immune responses which change within minutes. Current methods for early diagnosis of infections focus on detecting low abundance pathogens, which are time-consuming, of low sensitivity, and do not reflect the severity of the pathophysiology appropriately. The approach here focuses on profiling the rapidly changing host inflammatory response, which in its over-exuberant state, leads to sepsis and death. A 15-min label-free immune profiling assay from 20 µL of unprocessed blood using unconventional L and Inverse-L shaped pillars of deterministic lateral displacement microfluidic technology is developed. The hydrodynamic interactions of deformable immune cells enable simultaneous sorting and immune response profiling in whole blood. Preliminary clinical study of 85 donors in emergency department with a spectrum of immune response states from healthy to severe inflammatory response shows correlation with biophysical markers of immune cell size, deformability, distribution, and cell counts. The speed of patient stratification demonstrated here has promising impact in deployable point-of-care systems for acute infections triage, risk management, and resource allocation at emergency departments, where clinical manifestation of infection severity may not be clinically evident as compared to inpatients in the wards or intensive care units.

Miniature auto-perfusion bioreactor system with spiral microfluidic cell retention device.

Medium perfusion is critical in maintaining high cell concentration in cultures. The conventional membrane filtration method for medium exchange has been challenged by the fouling and clogging of the membrane filters in long-term cultures. In this study, we present a miniature auto-perfusion system that can be operated inside a common-size laboratory incubator. The system is equipped with a spiral microfluidic chip for cell retention to replace conventional membrane filters, which fundamentally overcomes the clogging and fouling problem. We showed that the system supported continuous perfusion culture of Chinese hamster ovary (CHO) cells in suspension up to 14 days without cell retention chip replacement. Compared to daily manual medium change, 25% higher CHO cell concentration can be maintained at an average auto-perfusion rate of 196 ml/day in spinner flask at 70 ml working volume (2.8 VVD). The auto-perfusion system also resulted in better cell quality at high concentrations, in terms of higher viability, more uniform and regular morphology, and fewer aggregates. We also demonstrated the potential application of the system for culturing mesenchymal stem cells on microcarriers. This miniature auto-perfusion system provides an excellent solution to maintain cell-favorable conditions and high cell concentration in small-scale cultures for research and clinical uses.

Correction: Huang et al. Deep-Learning Based Label-Free Classification of Activated and Inactivated Neutrophils for Rapid Immune State Monitoring. Sensors 2021, 21, 512.

The authors wish to make the following correction to their paper [...].

Deep-Learning Based Label-Free Classification of Activated and Inactivated Neutrophils for Rapid Immune State Monitoring.

The differential count of white blood cells (WBCs) is one widely used approach to assess the status of a patient's immune system. Currently, the main methods of differential WBC counting are manual counting and automatic instrument analysis with labeling preprocessing. But these two methods are complicated to operate and may interfere with the physiological states of cells. Therefore, we propose a deep learning-based method to perform label-free classification of three types of WBCs based on their morphologies to judge the activated or inactivated neutrophils. Over 90% accuracy was finally achieved by a pre-trained fine-tuning Resnet-50 network. This deep learning-based method for label-free WBC classification can tackle the problem of complex instrumental operation and interference of fluorescent labeling to the physiological states of the cells, which is promising for future point-of-care applications.

Continuous Online Protein Quality Monitoring during Perfusion Culture Production Using an Integrated Micro/Nanofluidic System.

We demonstrate a new micro/nanofluidic system for continuous and automatic monitoring of protein product size and quantity directly from the culture supernatant during a high-cell-concentration CHO cell perfusion culture. A microfluidic device enables clog-free cell retention for a bench-scale (350 mL) perfusion bioreactor that continuously produces the culture supernatant containing monoclonal antibodies (IgG1). A nanofluidic device directly monitors the protein size and quantity in the culture supernatant. The continuous-flow and fully automated operation of this nanofluidic protein analytics reduces design complexity and offers more detailed information on protein products than offline and batch-mode conventional analytics. Moreover, chemical and mechanical robustness of the nanofluidic device enables continuous monitoring for several days to a week. This continuous and online protein quality monitoring could be deployed at different steps and scales of biomanufacturing to improve product quality and manufacturing efficiency.

Massively Multiplexed Submicron Particle Patterning in Acoustically Driven Oscillating Nanocavities.

Nanoacoustic fields are a promising method for particle actuation at the nanoscale, though THz frequencies are typically required to create nanoscale wavelengths. In this work, the generation of robust nanoscale force gradients is demonstrated using MHz driving frequencies via acoustic-structure interactions. A structured elastic layer at the interface between a microfluidic channel and a traveling surface acoustic wave (SAW) device results in submicron acoustic traps, each of which can trap individual submicron particles. The acoustically driven deformation of nanocavities gives rise to time-averaged acoustic fields which direct suspended particles toward, and trap them within, the nanocavities. The use of SAWs permits massively multiplexed particle manipulation with deterministic patterning at the single-particle level. In this work, 300 nm diameter particles are acoustically trapped in 500 nm diameter cavities using traveling SAWs with wavelengths in the range of 20-80 µm with one particle per cavity. On-demand generation of nanoscale acoustic force gradients has wide applications in nanoparticle manipulation, including bioparticle enrichment and enhanced catalytic reactions for industrial applications.

Techno-economic analysis of multi-stage ion concentration polarization with recirculation for treatment of oil produced water.

The concept of recirculation of diluate/concentrate stream is implemented in multi-stage ion concentration polarization (ICP) desalination to deal with the issue of uncontrolled concentrate streams and deteriorated overall recovery rate to treat highly concentrated oil produce water from refineries. An improved empirical optimization model was established to calculate total energy consumption for operating cost and required membrane area for capital cost for a given set of operating parameters, feed salinity, salt removal ratio, and flow velocity. Using the empirical optimization model, a techno-economic analysis is performed to evaluate the feasibility of two-stage ICP system with recirculation loops. Brine of 160 g/kg is set as the system feed stream, whereas other operating conditions such as dilaute and concentrate streams are being controlled/fixed with 20 g/kg and ~250 g/kg respectively. Also, the system can be flexibly controlled to produce a specific concentration of product water and a recovery ratio with a corresponding water cost. With careful choices of recirculation rates, one can significantly increase the recovery ratio of two-stage ICP brine treatment process (from 25% to 39%) with only minor increase in overall cost (from $16.4-25.9/m3 to $20.6-22.54/m3), which is favourable for brine waste treatment application.

One-Step Nucleic Acid Purification and Noise-Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow-Abundance Nucleic Acid Detection.

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point-of-care diagnostics. Currently, nucleic acid (NA) purification remains time-consuming and labor-intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one-step, liquid-phase NA purification that is simpler and faster than conventional solid-phase extraction. By further re-concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non-specific amplification caused by non-optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non-optimal PCR designs, which is 10- and 1000-fold fewer than those of the standard bench-top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point-of-care diagnosis.

Low-dose anti-inflammatory combinatorial therapy reduced cancer stem cell formation in patient-derived preclinical models for tumour relapse prevention.

BACKGROUND: Emergence of drug-resistant cancer phenotypes is a challenge for anti-cancer therapy. Cancer stem cells are identified as one of the ways by which chemoresistance develops. METHOD: We investigated the anti-inflammatory combinatorial treatment (DA) of doxorubicin and aspirin using a preclinical microfluidic model on cancer cell lines and patient-derived circulating tumour cell clusters. The model had been previously demonstrated to predict patient overall prognosis. RESULTS: We demonstrated that low-dose aspirin with a sub-optimal dose of doxorubicin for 72 h could generate higher killing efficacy and enhanced apoptosis. Seven days of DA treatment significantly reduced the proportion of cancer stem cells and colony-forming ability. DA treatment delayed the inhibition of interleukin-6 secretion, which is mediated by both COX-dependent and independent pathways. The response of patients varied due to clinical heterogeneity, with 62.5% and 64.7% of samples demonstrating higher killing efficacy or reduction in cancer stem cell (CSC) proportions after DA treatment, respectively. These results highlight the importance of using patient-derived models for drug discovery. CONCLUSIONS: This preclinical proof of concept seeks to reduce the onset of CSCs generated post treatment by stressful stimuli. Our study will promote a better understanding of anti-inflammatory treatments for cancer and reduce the risk of relapse in patients.

Femtomolar Detection of Lipopolysaccharide in Injectables and Serum Samples Using Aptamer-Coupled Reduced Graphene Oxide in a Continuous Injection-Electrostacking Biochip.

A method for microfluidic sample preconcentration to detect femtomolar level of lipopolysaccharide (LPS) is introduced, enabled by 6-carboxyfluorescein (6-FAM) labeled aptamer-LPS binding along with reduced graphene oxide (rGO). The free FAM-aptamers can be adsorbed onto the surface of rGO, resulting in fluorescence quenching of background signals. Conversely, the aptamer-LPS complex cannot be adsorbed by rGO, so the fluorescence is maintained and detected. When an electric field is applied across the microchannel with Nafion membrane in the chip, only the fluorescence of aptamer-LPS complex can be detected and stacked by continuous injection-electrostacking (CI-ES). The method shows a high selectivity (in the presence of pyrophosphate, FAD+, NAD+, AMP, ADP, ATP, phosphatidylcholine, LTA, and ß-d-glucans which respond positively to LAL) to LPS and an extreme sensitivity with the limit of detection (LOD) at 7.9 fM (7.9 × 10-4 EU/mL) and 8.3 fM (8.3 × 10-4 EU/mL) for water sample and serum sample, respectively. As a practical application, this method can detect LPS in injections and serum samples of human and sepsis model mouse and quickly distinguish Gram-negative bacteria Escherichia coli ( E. coli) from Gram-positive bacteria Staphylococcus aureus ( S. aureus) and fungus Candida albicans ( C. albicans). More importantly, by changing the aptamers based on different targets, we can detect different analytes. Therefore, aptamer-coupled rGO in a CI-ES biochip is a universal, sensitive, and specific method. For TOC only.