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1. Electronic profiling of membrane antigen expression via immunomagnetic cell manipulation

   https://doi.org/10.1039/c9lc00297a  

   Civelekoglu, Ozgun; Wang, Ningquan; Boya, Mert; Ozkaya-Ahmadov, Tevhide; Liu, Ruxiu; Sarioglu, A. Fatih (January 2019, Lab on a Chip)

   Membrane antigens control cell function by regulating biochemical interactions and hence are routinely used as diagnostic and prognostic targets in biomedicine. Fluorescent labeling and subsequent optical interrogation of cell membrane antigens, while highly effective, limit expression profiling to centralized facilities that can afford and operate complex instrumentation. Here, we introduce a cytometry technique that computes surface expression of immunomagnetically labeled cells by electrically tracking their trajectory under a magnetic field gradient on a microfluidic chip with a throughput of >500 cells per min. In addition to enabling the creation of a frugal cytometry platform, this immunomagnetic cell manipulation-based measurement approach allows direct expression profiling of target subpopulations from non-purified samples. We applied our technology to measure epithelial cell adhesion molecule expression on human breast cancer cells. Once calibrated, surface expression and size measurements match remarkably well with fluorescence-based measurements from a commercial flow cytometer. Quantitative measurements of biochemical and biophysical cell characteristics with a disposable cytometer have the potential to impact point of care testing of clinical samples particularly in resource limited settings. 

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2. WASp Is Crucial for the Unique Architecture of the Immunological Synapse in Germinal Center B-Cells

   https://doi.org/10.3389/fcell.2021.646077  

   Li, Yanan; Bhanja, Anshuman; Upadhyaya, Arpita; Zhao, Xiaodong; Song, Wenxia (June 2021, Frontiers in Cell and Developmental Biology)

   null (Ed.)

   B-cells undergo somatic hypermutation and affinity maturation in germinal centers. Somatic hypermutated germinal center B-cells (GCBs) compete to engage with and capture antigens on follicular dendritic cells. Recent studies show that when encountering membrane antigens, GCBs generate actin-rich pod-like structures with B-cell receptor (BCR) microclusters to facilitate affinity discrimination. While deficiencies in actin regulators, including the Wiskott-Aldrich syndrome protein (WASp), cause B-cell affinity maturation defects, the mechanism by which actin regulates BCR signaling in GBCs is not fully understood. Using WASp knockout (WKO) mice that express Lifeact-GFP and live-cell total internal reflection fluorescence imaging, this study examined the role of WASp-mediated branched actin polymerization in the GCB immunological synapse. After rapid spreading on antigen-coated planar lipid bilayers, GCBs formed microclusters of phosphorylated BCRs and proximal signaling molecules at the center and the outer edge of the contact zone. The centralized signaling clusters localized at actin-rich GCB membrane protrusions. WKO reduced the centralized micro-signaling clusters by decreasing the number and stability of F-actin foci supporting GCB membrane protrusions. The actin structures that support the spreading membrane also appeared less frequently and regularly in WKO than in WT GCBs, which led to reductions in both the level and rate of GCB spreading and antigen gathering. Our results reveal essential roles for WASp in the generation and maintenance of unique structures for GCB immunological synapses. 

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3. Recent advances of droplet-based microfluidics for engineering artificial cells

   https://doi.org/10.1016/j.slast.2023.05.002  

   Fasciano, Samantha; Wang, Shue (May 2023, SLAS Technology)

   The goal of engineering artificial cells is to build a living cell with the least amount of parts and complexity. Artificial cells hold great potential for several applications, including membrane protein interactions, gene expression, biomaterials, and drug development. It is critical to generate robust, stable artificial cells using high throughput, easy-to-control, and flexible techniques. Recently, droplet-based microfluidic techniques have shown great potential for the synthesis of vesicles and artificial cells. Here, we summarized the recent advances in droplet-based microfluidic techniques for the fabrication of vesicles and artificial cells. We first reviewed the different types of droplet-based microfluidic devices, including flow-focusing, T-junction, and coflowing. Next, we discussed the formation of multi-compartmental vesicles and artificial cells based on droplet-based microfluidics. The applications of artificial cells for studying gene expression dynamics, artificial cell-cell communications, and mechanobiology are highlighted and discussed. Finally, the current challenges and future outlook of droplet-based microfluidic methods for engineering artificial cells are discussed. This review will provide insights into scientific research in synthetic biology, microfluidic devices, membrane interactions, and mechanobiology. 

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4. Hydrogel-based microfluidic device with multiplexed 3D in vitro cell culture

   https://doi.org/10.1038/s41598-022-22439-y  

   Clancy, Allison; Chen, Dayi; Bruns, Joseph; Nadella, Jahnavi; Stealey, Samuel; Zhang, Yanjia; Timperman, Aaron; Zustiak, Silviya P. (December 2022, Scientific Reports)

   Abstract Microfluidic devices that combine an extracellular matrix environment, cells, and physiologically relevant perfusion, are advantageous as cell culture platforms. We developed a hydrogel-based, microfluidic cell culture platform by loading polyethylene glycol (PEG) hydrogel-encapsulated U87 glioblastoma cells into membrane-capped wells in polydimethyl siloxane (PDMS). The multilayer microfluidic cell culture system combines previously reported design features in a configuration that loads and biomimetically perfuses a 2D array of cell culture chambers. One dimension of the array is fed by a microfluidic concentration gradient generator (MCGG) while the orthogonal dimension provides loading channels that fill rows of cell culture chambers in a separate layer. In contrast to typical tree-like MCGG mixers, a fractional serial dilution of 1, ½, ¼, and 0 of the initial solute concentration is achieved by tailoring the input microchannel widths. Hydrogels are efficiently and reproducibly loaded in all wells and cells are evenly distributed throughout the hydrogel, maintaining > 90% viability for up to 4 days. In a drug screening assay, diffusion of temozolomide and carmustine to hydrogel-encapsulated U87 cells from the perfusion solution is measured, and dose–response curves are generated, demonstrating utility as an in vitro mimic of the glioblastoma microenvironment. 

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5. Functional Characterization of Hexacorallia Phagocytic Cells

   https://doi.org/10.3389/fimmu.2021.662803  

   Snyder, Grace A.; Eliachar, Shir; Connelly, Michael T.; Talice, Shani; Hadad, Uzi; Gershoni-Yahalom, Orly; Browne, William E.; Palmer, Caroline V.; Rosental, Benyamin; Traylor-Knowles, Nikki (July 2021, Frontiers in Immunology)

   Phagocytosis is the cellular defense mechanism used to eliminate antigens derived from dysregulated or damaged cells, and microbial pathogens. Phagocytosis is therefore a pillar of innate immunity, whereby foreign particles are engulfed and degraded in lysolitic vesicles. In hexacorallians, phagocytic mechanisms are poorly understood, though putative anthozoan phagocytic cells (amoebocytes) have been identified histologically. We identify and characterize phagocytes from the coral Pocillopora damicornis and the sea anemone Nematostella vectensis . Using fluorescence-activated cell sorting and microscopy, we show that distinct populations of phagocytic cells engulf bacteria, fungal antigens, and beads. In addition to pathogenic antigens, we show that phagocytic cells engulf self, damaged cells. We show that target antigens localize to low pH phagolysosomes, and that degradation is occurring within them. Inhibiting actin filament rearrangement interferes with efficient particle phagocytosis but does not affect small molecule pinocytosis. We also demonstrate that cellular markers for lysolitic vesicles and reactive oxygen species (ROS) correlate with hexacorallian phagocytes. These results establish a foundation for improving our understanding of hexacorallian immune cell biology. 

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