Machine Learning for Two-Phase Flow Separation in a Liquid-Liquid Interface Manipulation Separator.

Two-phase flow separation is a key step in various downstream purification processes. The use of a separator with controllable flow behavior is recommended to avoid contamination. In this study, a core-annular separator for biphasic flow separation with four different chemical polarities was developed, and two machine learning-based methods were proposed for answering two emergent questions to meet real industrial needs. (1) Could complete two-phase separation be achieved under these operating conditions? (2) Could the separation process be accelerated by determining the maximum input flow rate of the water? Process prediction for automation, machine learning-based classifiers, and multilayer perceptron were used to address these questions by predicting successful separation and the maximum input flow rates of unknown water-solvent systems with limited experimental data as training samples. The core-annular separator achieved complete two-phase water-solvent separation at a maximum total input flow rate of 4000 µL min-1. Moreover, the classification accuracy for complete separation reached 92.2%, and the multilayer perceptron network had the best performance for predicting the flow rate. This liquid-liquid interface manipulation separator and machine learning method could decrease the cost of relevant process development.

Development of Real-Time Transendothelial Electrical Resistance Monitoring for an In Vitro Blood-Brain Barrier System.

Three-dimensional (3D) cell cultures and organs-on-a-chip have been developed to construct microenvironments that resemble the environment within the human body and to provide a platform that enables clear observation and accurate assessments of cell behavior. However, direct observation of transendothelial electrical resistance (TEER) has been challenging. To improve the efficiency in monitoring the cell development in organs-on-a-chip, in this study, we designed and integrated commercially available TEER measurement electrodes into an in vitro blood-brain barrier (BBB)-on-chip system to quantify TEER variation. Moreover, a flowing culture medium was added to the monolayered cells to simulate the promotion of continuous shear stress on cerebrovascular cells. Compared with static 3D cell culture, the proposed BBB-on-chip integrated with electrodes could measure TEER in a real-time manner over a long period. It also allowed cell growth angle measurement, providing instant reports of cell growth information online. Overall, the results demonstrated that the developed system can aid in the quantification of the continuous cell-pattern variations for future studies in drug testing.

Bioinspired Durable Superhydrophobic Surface from a Hierarchically Wrinkled Nanoporous Polymer.

Inspired by complex multifunctional leaves, in this study, we created robust hierarchically wrinkled nanoporous polytetrafluoroethene (PTFE) surfaces that exhibit superhydrophobic properties by combination of PTFE micellization and spontaneous surface wrinkling on a commercially available thermoretractable polystyrene (PS) sheet. A PTFE dispersion was coated onto the PS sheet, followed by thermal treatment to remove the surfactants surrounding the PTFE particles, and surface wrinkling was induced through a dynamic thermal contraction process. Thermally induced contraction from the PS sheet provided the driving force for developing and stabilizing micrometer-sized wrinkle formation, whereas the nanometer-sized PTFE particle aggregation formed a rigid nanoporous film, providing its intrinsic hydrophobic character. By combining the hierarchical interfacial structure and chemical composition, hierarchically wrinkled nanoporous PTFE surfaces were fabricated, which exhibited extremely high water repellence (water contact angle of ∼167°) and a water rolling-off angle lower than 5°. The wrinkled patterns could intimately bind the nanoporous PTFE layer through enhanced adhesion from their curved surface and viscous liquid surfactants, making these surfaces mechanically robust and offering potentially extendable alternatives with self-cleaning, antifouling, and drag-reducing properties.

Preparation of neuronal co-cultures with single cell precision.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (<10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (>85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Whole cell quenched flow analysis.

This paper describes a microfluidic quenched flow platform for the investigation of ligand-mediated cell surface processes with unprecedented temporal resolution. A roll-slip behavior caused by cell-wall-fluid coupling was documented and acts to minimize the compression and shear stresses experienced by the cell. This feature enables high-velocity (100-400 mm/s) operation without impacting the integrity of the cell membrane. In addition, rotation generates localized convection paths. This cell-driven micromixing effect causes the cell to become rapidly enveloped with ligands to saturate the surface receptors. High-speed imaging of the transport of a Janus particle and fictitious domain numerical simulations were used to predict millisecond-scale biochemical switching times. Dispersion in the incubation channel was characterized by microparticle image velocimetry and minimized by using a horizontal Hele-Shaw velocity profile in combination with vertical hydrodynamic focusing to achieve highly reproducible incubation times (CV = 3.6%). Microfluidic quenched flow was used to investigate the pY1131 autophosphorylation transition in the type I insulin-like growth factor receptor (IGF-1R). This predimerized receptor undergoes autophosphorylation within 100 ms of stimulation. Beyond this demonstration, the extreme temporal resolution can be used to gain new insights into the mechanisms underpinning a tremendous variety of important cell surface events.

Microfluidic construction of minimalistic neuronal co-cultures.

In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10-100-fold less than existing systems). The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency Epatt was >75% during lengthy in chip culture, with ∼85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health.

Ultrafast cell switching for recording cell surface transitions: new insights into epidermal growth factor receptor signalling.

A pinched-flow deflection technology was developed for rapid single cell switching between biochemical microenvironments. Millisecond switching was used to stimulate and preserve epidermal growth factor receptor (EGFR) autophosphorylation transitions. Intramolecular phosphorylation initiates signal transduction, is silenced by phosphatase activity until EGFR dimerization enables intermolecular phosphorylation to initiate downstream signalling.

Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits.

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research.

A microfluidic array with cellular valving for single cell co-culture.

We present a highly parallel microfluidic approach for contacting single cell pairs. The approach combines a differential fluidic resistance trapping method with a novel cellular valving principle for homotypic and heterotypic single cell co-culturing. Differential fluidic resistance was used for sequential single cell arraying, with the adhesion and flattening of viable cells within the microstructured environment acting to produce valves in the open state. Reversal of the flow was used for the sequential single cell arraying of the second cell type. Plasma stencilling, along the linear path of least resistance, was required to confine the cells within the trap regions. Prime flow conditions with minimal shear stress were identified for highly efficient cell arraying (∼99%) and long term cell culture. Larger trap dimensions enabled the highest levels of cell pairing (∼70%). The single cell co-cultures were in close proximity for the formation of connexon structures and the study of contact modes of communication. The research further highlights the possibility of using the natural behaviour of cells as the working principle behind responsive microfluidic elements.