Chimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer treatment by engineering patients' T cells to specifically target cancer cells. Traditional CAR-T cell manufacturing methods use viral transduction to integrate CAR genes into T cells, but this can cause severe side effects and immune reactions and is costly. To overcome these challenges, non-viral methods, such as plasmid DNA (pDNA) transfection, are being explored. Here, a high-throughput intracellular delivery platform that integrates microfluidic mechanoporation with lipid nanoparticle (LNP)-based delivery, LNP + Squeeze, is introduced. This system enhances pDNA transfection efficiency in T cells while maintaining cell viability compared to other non-viral transfection methods like electroporation. This platform successfully engineers CAR-T cells using primary human T cells with a high transfection efficiency and demonstrates potent cytotoxicity against melanoma cells. This approach offers a promising, cost-effective, and scalable alternative to viral methods, potentially improving the accessibility and efficacy of CAR-T cell therapies.

The efficient generation of monodispersed droplets holds great promise for micro‐/nanoparticle synthesis and biochemical analysis. However, it remains a challenge to achieve high‐throughput generation of monodispersed droplets in a portable and/or parallel manner in nonexpert biomedical laboratories. Herein, a versatile microchannel array (μCA) device is reported that is portable, multifaceted, reliable, and mass‐manufacturable, supporting the high‐throughput generation of monodispersed droplets in different ways. This device consists of a silicon‐based μCA chip based on the step emulsification principle, as well as two matching plastic containers, both of which can be mass‐manufactured by traditional microfabrication methods at low material costs. With the μCA device, aqueous solution can be dispersed into emulsion droplets by various modes, such as mechanical pump‐based large‐scale, handheld syringe‐based portable, and gas pump‐based highly parallel droplet generation. Furthermore, it is demonstrated that our cost‐effective device can be applied for digital polymerase chain reaction analysis, supporting the need for more accessible microfluidic systems. Thus, the present device is expected to have a significant impact on both benchside and bedside applications.

Organoids are becoming increasingly relevant in biology and medicine for their physiological complexity and accuracy in modeling human disease. To fully assess their biological profile while preserving their spatial information, spatiotemporal imaging tools are warranted. While previously developed imaging techniques, such as four-dimensional (4D) live imaging and light-sheet imaging have yielded important clinical insights, these technologies lack the combination of cyclic and multiplexed analysis. To address these challenges, bioorthogonal click chemistry is applied to display the first demonstration of multiplexed cyclic imaging of live and fixed patient-derived glioblastoma tumor organoids. This technology exploits bioorthogonal click chemistry to quench fluorescent signals from the surface and intracellular of labeled cells across multiple cycles, allowing for more accurate and efficient molecular profiling of their complex phenotypes. Herein, the versatility of this technology is demonstrated for the screening of glioblastoma markers in patient-derived human glioblastoma organoids while conserving their viability. It is anticipated that the findings and applications of this work can be broadly translated into investigating physiological developments in other organoid systems.

Extracellular vesicles (EVs) are promising for molecular diagnostics, but current analyses are limited by the rarity and compositional heterogeneity of EV protein expression. Therefore, single EV profiling methods require high sensitivity, multiplexing, and throughput to address these issues. Here a single EV analysis technique that utilizes squeezable methacrylated hyaluronic acid hydrogel microparticles (MHPs) is described as a scaffold to immobilize EVs and perform an integrated rolling circle amplification (RCA) assay for an ultra-sensitive and multiplex analysis of single EV proteins. EVs are prepared into MHPs in a high-throughput manner with droplet microfluidics and optimally labeled with antibody-oligonucleotide conjugates in MHPs without steric limitations. By designing MHPs with high compressibility, single EV protein signals are amplified as RCA products that can be aligned on the same plane by physically squeezing MHPs and visualized with low magnification. This method provides a simple and scalable single EV imaging analysis pipeline for identifying multiplex marker expression patterns from single EVs. For validation, the single EV heterogeneity of highly expressed cancer cell markers is profiled across different cancer cell lines. These findings exemplify squeezable MHPs as a robust platform with high sensitivity, multiplexing, and scalability for resolving single EV heterogeneity and advancing molecular assay technologies.

Over the last few decades, the discipline of microfluidics has grown in importance in the medical, engineering, information technology, and energy fields. One of the more popular subdisciplines that have emerged in this area, though, is droplet microfluidics. Droplet microfluidics entails the creation of tens of thousands of monodisperse droplets for performing countless amount of reactions and high-throughput analyses. Because their parameters are easily adjustable, a plethora of droplets with different compositions and structures can be formed and investigated. In this chapter, we aim to provide a comprehensive overview of the fundamentals, methodologies, and applications behind droplet production and its significance in the microfluidic field. The highlighted material will provide a better understanding of droplet microfluidics' past, present, and future.

The use of mesenchymal stem cells (MSCs) in human and veterinary clinical applications has become a subject of increasing importance due to their roles in immunomodulation and regenerative processes. MSCs are especially relevant in equine medicine because they may have the ability to treat prevalent musculoskeletal disorders, among other conditions. However, recent evidence suggests that the components secreted by MSCs, particularly extracellular vesicles (EVs), are responsible for these properties. EVs contain proteins and nucleic acids, which possess an active role in intercellular communication and can be used as therapeutics. However, because the intersection of equine veterinary medicine with EVs remains a relatively new field, there is a demand to identify biomarkers that can discern and enrich for therapeutic EVs, progressing their clinical efficacy. In this study, we identified and characterized 84 miRNAs, between three equine donors involved in immunomodulation in cell and EV subjects. We discovered distinct groups of shared miRNAs, like miR-21-5p and miR-451a, that are abundant and enriched between the donors' EVs, respectively. By mapping and comparing the MSC-EV miRNA expression, we discovered many pathways that are involved in immunomodulation and tissue regenerative processes related to equine clinical applications. Therefore, the miRNAs highlighted in this article can be used as valuable biomarkers for screening MSC-derived EVs for potential equine therapy.

Extracellular vesicles (EVs) are lipid-bound nanometer-sized vesicles released by all cell types that contain molecular payload such as proteins and/or nucleic acids. EVs are a key facet of cell-to-cell communication and have the potential to be used in the diagnosis of numerous diseases, chief among them being cancer. However, most methods of EV analysis struggle to identify the rare, malformed proteins indicative of tumor cells as tumor EVs represent only a tiny fraction of the bulk EVs present in the bloodstream. Here, we present a method of single EV analysis, utilizing droplet microfluidics to encapsulate EVs, which are labeled with DNA barcodes linked to antibodies, in droplets with the DNA extension used to amplify the signals associated with each EV. The amplified DNA can then be sequenced to assess the protein content of individual EVs, enabling the detection of rare proteins and EV subpopulations within a bulk EV sample.

Extracellular vesicles (EVs) have emerged as a promising source of biomarkers for disease diagnosis. However, current diagnostic methods for EVs present formidable challenges, given the low expression levels of biomarkers carried by EV samples, as well as their complex physical and biological properties. Herein, a highly sensitive double digital assay is developed that allows for the absolute quantification of individual molecules from a single EV. Because the relative abundance of proteins is low for a single EV, tyramide signal amplification (TSA) is integrated to increase the fluorescent signal readout for evaluation. With the integrative microfluidic technology, the technology's ability to compartmentalize single EVs is successfully demonstrated, proving the technology's digital partitioning capacity. Then the device is applied to detect single PD‐L1 proteins from single EVs derived from a melanoma cell line and it is discovered that there are ≈2.7 molecules expressed per EV, demonstrating the applicability of the system for profiling important prognostic and diagnostic cancer biomarkers for therapy response, metastatic status, and tumor progression. The ability to accurately quantify protein molecules of rare abundance from individual EVs will shed light on the understanding of EV heterogeneity and discovery of EV subtypes as new biomarkers.

The ability to efficiently detect low-abundance protein biomarkers in tiny blood samples is a significant challenge in clinical and laboratory settings. Currently, high-sensitivity approaches require specialized instrumentation, involve multiple washing steps, and lack the ability to parallelize, preventing their widespread implementation. Herein, we developed a parallelized, wash-free, and ultrasensitive centrifugal droplet digital protein detection (CDPro) technology that achieves a femtomolar limit of detection (LoD) of target proteins with sub-microliters of plasma. The CDPro combines two techniques, namely a centrifugal microdroplet generation device and a digital immuno-PCR assay. Miniaturized centrifugal devices enable emulsification of hundreds of samples within 3 minutes using a common centrifuge. The bead-free digital immuno-PCR assay not only eliminates the need for multistep washing, but also possesses ultra-high detection sensitivity and accuracy. We characterized the performance of CDPro using recombinant interleukins (IL-3 and IL-6) as example targets and reported a LoD of 0.0128 pg mL-1. We also quantified IL-6 from 7 human clinical blood samples using the CDPro with only 0.5 μL plasma, which showed excellent agreement with an existing clinical protein diagnostic system with 25 μL plasma from those samples (R2 = 0.98).

BACKGROUND:Vascular activation is characterized by increased proinflammatory, pro thrombotic, and proadhesive signaling. Several chronic and acute conditions, including Bcr-abl-negative myeloproliferative neoplasms (MPNs), graft-vs-host disease, and COVID-19 have been noted to have increased activation of the janus kinase (JAK)-signal transducer and downstream activator of transcription (STAT) pathways. Two notable inhibitors of the JAK-STAT pathway are ruxolitinib (JAK1/2 inhibitor) and fedratinib (JAK2 inhibitor), which are currently used to treat MPN patients. However, in some conditions, it has been noted that JAK inhibitors can increase the risk of thromboembolic complications. OBJECTIVES:We sought to define the anti-inflammatory and antithrombotic effects of JAK-STAT inhibitors in vascular endothelial cells. METHODS:We assessed endothelial activation in the presence or absence of ruxolitinib or fedratinib by using immunoblots, immunofluorescence, qRT-PCR, and function coagulation assays. Finally, we used endothelialized microfluidics perfused with blood from normal and JAK2V617F+ individuals to evaluate whether ruxolitinib and fedratinib changed cell adhesion. RESULTS:We found that both ruxolitinib and fedratinib reduced endothelial cell phospho-STAT1 and STAT3 signaling and attenuated nuclear phospho-NK-κB and phospho-c-Jun localization. JAK-STAT inhibition also limited secretion of proadhesive and procoagulant P-selectin and von Willebrand factor and proinflammatory IL-6. Likewise, we found that JAK-STAT inhibition reduced endothelial tissue factor and urokinase plasminogen activator expression and activity. CONCLUSIONS:By using endothelialized microfluidics perfused with whole blood samples, we demonstrated that endothelial treatment with JAK-STAT inhibitors prevented rolling of both healthy control and JAK2V617F MPN leukocytes. Together, these findings demonstrate that JAK-STAT inhibitors reduce the upregulation of critical prothrombotic pathways and prevent increased leukocyte-endothelial adhesion.

For microfluidic device fabrication in the research, industry, and commercial areas, the curing and transfer of patterns on photoresist relies on ultraviolet (UV) light. Often, this step is performed by commercial mask aligner or UV lamp exposure systems; however, these machines are often expensive, large, and inaccessible. To find an alternative solution, we present an inexpensive, customizable, and lightweight UV exposure system that is user-friendly and readily available for a homemade cleanroom. We fabricated a portable UV exposure system that costs under $200. The wafer holder's adjustable height enabled for the selection of the appropriate curing distance, demonstrating our system's ability to be easily tailored for different applications. The high light uniformity across a 4" diameter wafer holder (light intensity error ~2.9%) was achieved by adding a light diffusing film to the apparatus. These values are comparable to the light uniformity across a 5" diameter wafer holder from a commercial mask aligner (ABM 3000HR Mask Aligner), that has a light intensity error of ~4.0%. We demonstrated the ability to perform photolithography with high quality by fabricating microfluidic devices and generating uniform microdroplets. We achieved comparable quality to the wafer patterns, microfluidic devices, and droplets made from the ABM 3000HR Mask Aligner.

To understand the transport of pharmaceutical agents and their effects on developing fetus, we have created a placental microsystem that mimics structural phenotypes and physiological characteristic of a placental barrier. We have shown the formation of a continuous network of epithelial adherens junctions and endothelial cell-cell junctions confirming the integrity of the placental barrier. More importantly, the formation of elongated microvilli under dynamic flow condition is demonstrated. Fluid shear stress acts as a mechanical cue triggering the microvilli formation. Pharmaceutical agents were administered to the maternal channel, and the concentration of pharmaceutical agents in fetal channel for coculture and control models were evaluated. In fetal channel, the coculture model exhibited about 2.5 and 2.2% of the maternal initial concentration for naltrexone and 6β-naltrexol, respectively. In acellular model, fetal channel showed about 10.5 and 10.3% of the maternal initial concentration for naltrexone and 6β-naltrexol, respectively. Gene expressions of epithelial cells after direct administration of naltrexone and 6β-naltrexol to the maternal channel and endothelial cells after exposure due to transport through placental barrier are also reported.

Opioid use disorder (OUD) has become a growing concern in the U.S. and has been a dominant presence among pregnant women, resulting in an unprecedented amount of prescription medications, particularly naltrexone (NTX), prescribed for pregnant women. Because of unknown potential harm that NTX can impose on the fetus and its premature brain, the needs for safety and regulation of NTX are still undetermined. To address this issue, a microfluidic device is fabricated to mimic structural phenotypes and physiological characteristic of an in vivo placental barrier to evaluate near-transport simulations of NTX and its primary metabolite, 6β-naltrexol, across the placental barrier. Following transport analysis, cell layers are evaluated for possible gene-expressions released by an in vivo human placenta during NTX and 6β-naltrexol placental exposure. When a 100 ng/mL dose of NTX and 6β-naltrexol (1:1) is administered to the maternal channel, the mean fetal concentration for co-culture models exhibited ~2.5 % of NTX and ~2.2% of 6β-naltrexol of the initial maternal concentration. To prototype and simulate fetal-brain exposure, perfusate from a fetal channel is directed to cultured N27 cells that are then evaluated for gene-expression.### Competing Interest StatementThe authors have declared no competing interest.

Introduction The JAK2V617F+ mutation occurs in up to 95% of patients with polycythemia vera (PV) and increases the risk of thrombosis 6-fold. Recent studies demonstrate that JAK2V617F+ endothelial cells express pro-adhesive proteins, suggesting that the endothelium may contribute to increased thrombosis.1-3 The targeted JAK1/JAK2 inhibitor ruxolitinib is an approved second-line therapy for PV patients and is effective in alleviating constitutional symptoms, lowering hematocrit, and reducing cell number. However, there is limited data regarding efficacy of ruxolitinib in reducing thrombosis. Although recent work has demonstrated ruxolitinib reduces neutrophil extracellular trap formation,4 the vascular effects of ruxolitinib are unknown. Therefore, we hypothesize that ruxolitinib reduces endothelial cell pro-adhesive activation leading to decreased rolling and adhesion of JAK2V617F+ samples. Methods To mimic JAK2V617F activation, primary human umbilical vein endothelial cells (HUVEC, passage 1-5) were treated with TNF-α (10 ng/mL) +/- ruxolitinib (400 nM-4 µM, Selleckchem) and characterized 4h later. For confocal microscopy, cells were fixed with paraformaldehyde, permeabilized and stained for either VWF, VCAM-1, or P-selectin along with 2-(4-amidinophenyl)-1H-indole-carboxamide (DAPI, Thermo Fisher). Images were captured on Olympus FluoView FV1000 IX2 Inverted Confocal microscope. Secretion of VWF, VCAM-1 and P-selectin into conditioned media was assessed with ELISA (Molecular Innovations; BioLegend). Citrated normal and JAK2V617F+whole blood was obtained per IRB and labeled with calcein AM (ThermoFisher). To quantify leukocyte and platelet velocity and adhesion, an endothelialized poly-di-methyl-siloxane (PDMS) microchannel was prepared and treated with TNF-α (10 ng/mL) +/- ruxolitinib (400 nM-4 µM). Samples were perfused through the microfluidic at a shear stress of 0.35 dynes/cm2, and 10 s images were captured using a fluorescence microscope (Olympus). Cell velocity and adhesion were quantified using FIJI (NIH). Results Compared to TNF-α treated HUVEC alone, the combination of ruxolitinib + TNF-α reduced expression of VWF, VCAM-1 and P-selectin using both immunofluorescence and ELISA. In endothelialized microfluidic devices treated with TNF-α alone, both normal (n=3) and JAK2V617F+ (n=9) leukocyte and platelet velocity was significantly decreased and cell adhesion increased. This result was independent of hematocrit and platelet levels. In normal controls (n=2), TNF-α+ruxolitinib lead to a trend toward normal leukocyte and platelet velocity with decreased cell adhesion. Lastly, in a small subset of JAK2V617F+ patients (n=4), addition of ruxolitinib increased cell velocity. Conclusions In conclusion, in TNF-α-activated endothelial cells, treatment with ruxolitinib decreases pro-adhesive VWF, VCAM-1, and P-selectin expression. Using normal controls and JAK2V617F+ MPN blood samples, our TNF-α-activated endothelialized microfluidics model demonstrates significant reduction in leukocyte and platelet velocity and increased cell adhesion. In normal and JAK2V617F+ MPN whole blood, treatment of TNF-α-stimulated endothelium with ruxolitinib improves cell velocity. Further evaluation using JAK2V617F+ endothelial cells is planned. Collectively, these results suggest that ruxolitinib may reduce JAK2V617F+ thrombotic risk through reduction of pro-adhesive endothelial activation. Disclosures Vercellotti: CSL Behring: Research Funding.

The microvasculature is a vital organ that distributes nutrients within tissues, and collects waste products from them, and which defines the environmental conditions in both normal and disease situations. Here, a microfluidic chip was developed for the fabrication of poly(ethylene glycol diacrylate) (PEGDA)-based hollow self-standing microvessels having inner dimensions ranging from 15 μm to 73 μm and displaying biocompatibility/cytocompatibility. Macromer solutions were hydrodynamically focused into a single microchannel to form a concentric flow regime, and were subsequently solidified through photopolymerization. This approach uniquely allowed the fabrication of hollow microvessels having a defined structure and integrity suitable for cell culturing.

In the past few decades, the placenta became a very controversial topic that has had many researchers and pharmacists discussing the significance of the effects of pharmaceutical drug intake and how it is a possible leading cause towards birth defects. The creation of an in vitro microengineered model of the placenta can be used to replicate the interactions between the mother and fetus, specifically pharmaceutical drug intake reactions. As the field of nanotechnology significantly continues growing, nanotechnology will become more apparent in the study of medicine and other scientific disciplines, specifically microengineering applications. This review is based on past and current research that compares the feasibility and testing of the placenta-on-a-chip microengineered model to the previous and underdeveloped in vivo and ex vivo approaches. The testing of the practicality and effectiveness of the in vitro , in vivo and ex vivo models requires the experimentation of prominent pharmaceutical drugs that most mothers consume during pregnancy. In this case, these drugs need to be studied and tested more often. However, there are challenges associated with the in vitro , in vivo and ex vivo processes when developing a practical placental model, which are discussed in further detail.

Sister chromatid cohesion is essential for tension-sensing mechanisms that monitor bipolar attachment of replicated chromatids in metaphase. Cohesion is mediated by the association of cohesins along the length of sister chromatid arms. In contrast, centromeric cohesin generates intrastrand cohesion and sister centromeres, while highly cohesin enriched, are separated by >800 nm at metaphase in yeast. Removal of cohesin is necessary for sister chromatid separation during anaphase, and this is regulated by evolutionarily conserved polo-like kinase (Cdc5 in yeast, Plk1 in humans). Here we address how high levels of cohesins at centromeric chromatin are removed. Cdc5 associates with centromeric chromatin and cohesin-associated regions. Maximum enrichment of Cdc5 in centromeric chromatin occurs during the metaphase-to-anaphase transition and coincides with the removal of chromosome-associated cohesin. Cdc5 interacts with cohesin in vivo, and cohesin is required for association of Cdc5 at centromeric chromatin. Cohesin removal from centromeric chromatin requires Cdc5 but removal at distal chromosomal arm sites does not. Our results define a novel role for Cdc5 in regulating removal of centromeric cohesins and faithful chromosome segregation.

Cellular signal transduction pathways modify gene expression programs in response to changes in the environment, but the mechanisms by which these pathways regulate populations of genes under their control are not entirely understood. We present evidence that most mitogen-activated protein kinases and protein kinase A subunits become physically associated with the genes that they regulate in the yeast ( Saccharomyces cerevisiae) genome. The ability to detect this interaction of signaling kinases with target genes can be used to more precisely and comprehensively map the regulatory circuitry that eukaryotic cells use to respond to their environment.