Extreme wettability empowered under oil open-channel microfluidics bridges the gap between open- and closed-channel microfluidics.

Tape-based razor-printing is a flexible and affordable ultra-rapid prototyping approach for microscale device fabrication. However, integration of this prototyping approach into cell-based assay development has been limited to proof of principle demonstrations. This is in large part due to lack of an established or well-characterized option for biocompatible adhesive tape. Without such an option, integration of these areas will remain unexplored. Therefore, to address this critical hurdle, we characterized microscale devices made using a potentially biocompatible double-sided adhesive, ARCare 90106. We validated tape-based device performance against 96-well plates and PDMS microdevices with respect to cell viability, hydrophobic small molecule sequestration, the potential for leaching compounds, use in fluorescence microscopy, and outgassing (bubble formation). Results supported the tape as a promising tool for future cell-based assay development. Therefore, we subsequently demonstrated specific strengths enabled by the ultra-rapid (<1 h per prototype) and affordable (∼$1200 cutting plotter, <$0.05 per prototype) approach. Specifically, data demonstrate the ability to integrate disparate materials for advanced sticker-device functionality such as bonding of polystyrene devices to glass substrates for microscopy applications, inclusion of membranes, and incorporation of different electrospun biomaterials into a single device. Likewise, the approach allowed rapid adoption by uninitiated users. Overall, this study provides a necessary and unique contribution to the largely separate fields of tape-based razor-printing and cell-based microscale assay development by addressing a critical barrier to widespread integration and adoption while also demonstrating the potential for new and future applications.

Antimicrobial susceptibility testing (AST) remains the cornerstone of effective antimicrobial selection and optimization in patients. Despite recent advances in rapid pathogen identification and resistance marker detection with molecular diagnostics (e.g., qPCR, MALDI-TOF MS), phenotypic (i.e., microbial culture-based) AST methods – the gold standard in hospitals/clinics – remain relatively unchanged over the last few decades. Microfluidics-based phenotypic AST has been growing fast in recent years, aiming for rapid (i.e., turnaround time <8 h), high-throughput, and automated species identification, resistance detection, and antibiotics screening. In this pilot study, we describe the application of a multi-liquid-phase open microfluidic system, named under-oil open microfluidic systems (UOMS), to achieve a rapid phenotypic AST. UOMS provides an open microfluidics-based solution for rapid phenotypic AST (UOMS-AST) by implementing and recording a pathogen's antimicrobial activity in micro-volume testing units under an oil overlay. UOMS-AST allows free physical access (e.g., by standard pipetting) to the system and label-free, single-cell resolution optical access. UOMS-AST can accurately and rapidly determine antimicrobial activities [including susceptibility/resistance breakpoint and minimum inhibitory concentration (MIC)] from nominal sample/bacterial cells in a system aligned with clinical laboratory standards where open systems and optical microscopy are predominantly adopted. Further, we combine UOMS-AST with a cloud lab data analytic technique for real-time image analysis and report generation to provide a rapid (<4 h) sample-to-report turnaround time, shedding light on its utility as a versatile (e.g., low-resource setting and manual laboratory operation, or high-throughput automated system) phenotypic AST platform for hospital/clinic use.

Optimal CD8 T cell immunity is orchestrated by signaling events initiated by TCR recognition of peptide Ag in concert with signals from molecules such as CD28 and 4-1BB. The molecular mechanisms underlying the temporal and spatial signaling dynamics in CD8 T cells remain incompletely understood. In this study, we show that stimulation of naive CD8 T cells with agonistic CD3 and CD28 Abs, mimicking TCR and costimulatory signals, coordinately induces 4-1BB and cRel to enable elevated cytosolic cRel:IκBα complex formation and subsequent 4-1BB–induced IκBα degradation, sustained cRel activation, heightened IL-2 production and T cell expansion. NfkbiaNES/NES CD8 T cells harboring a mutated IκBα nuclear export sequence abnormally accumulate inactive cRel:IκBα complexes in the nucleus following stimulation with agonistic anti-CD3 and anti-CD28 Abs, rendering them resistant to 4-1BB induced signaling and a disrupted chain of events necessary for efficient T cell expansion. Consequently, CD8 T cells in NfkbiaNES/NES mice poorly expand during viral infection, and this can be overcome by exogenous IL-2 administration. Consistent with cell-based data, adoptive transfer experiments demonstrated that the antiviral CD8 T cell defect in NfkbiaNES/NES mice was cell intrinsic. Thus, these results reveal that IκBα, via its unique nuclear export function, enables, rather than inhibits 4-1BB–induced cRel activation and IL-2 production to facilitate optimal CD8 T cell immunity.

Concentration gradients of biochemical stimuli such as morphogens play a critical role in directing cell fate patterning across species and throughout development but are not commonly recapitulated in vitro. While in vitro biomolecule gradients have been generated using customized microfluidic platforms, broad implementation has been limited because these platforms introduce new variables to cell culture such as externally driven flow, culture in a specialized matrix, or extended time for in situ long range diffusion. Here we introduce a method that enables preforming and then transferring user-controlled gradients to cells in standard “open” cultures. Our gradient patterning devices are modular and decoupled from the culture substrate. We find that gradient generation and transfer are predictable by finite element modeling and that device and loading parameters can be used to tune the stimulus pattern. Furthermore, we demonstrate use of these devices to spatially define morphogen signal gradients and direct peri-gastrulation fate stratification of human pluripotent stem cells. This method for extrinsic application of biochemical signal gradients can thus be used to spatially influence cellular fate decisions in a user-controlled manner.