Live Organoid Cyclic Imaging.

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.

Double Digital Assay for Single Extracellular Vesicle and Single Molecule Detection.

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.

Equine mesenchymal stem cell derived extracellular vesicle immunopathology biomarker discovery.

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.

Transport of Maternally Administered Pharmaceutical Agents Across the Placental Barrier In Vitro.

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 6ß-naltrexol, respectively. In acellular model, fetal channel showed about 10.5 and 10.3% of the maternal initial concentration for naltrexone and 6ß-naltrexol, respectively. Gene expressions of epithelial cells after direct administration of naltrexone and 6ß-naltrexol to the maternal channel and endothelial cells after exposure due to transport through placental barrier are also reported.

Manufacturing of poly(ethylene glycol diacrylate)-based hollow microvessels using microfluidics.

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.

Construction of coarse-grained order parameters in nonequilibrium systems.

We develop a renormalization-group (RG) procedure that includes important system-specific features. The key ingredient is to systematize the coarse-graining procedure that generates the RG flow. The coarse-graining technology comes from the control and operator theoretic model reduction. The resulting "generalized" RG is a consistent generalization of the Wilsonian RG. We apply the procedure to a deterministic nonlinear wave equation (NLWE) with probabilistic initial conditions. We derive the form of the projection operator from the dynamics of the NLWE and then use it to generate the RG flow for the distribution of initial conditions. The probability density of the initial conditions is chosen to be a Boltzmann weight that is quartic in the field variables. In our calculation, we find that in contrast to conventional implementations of the RG, naïve power counting breaks down. We also show that the resulting RG equations are different from those derived from the conventional RG.

Drug transport across the human placenta: review of placenta-on-a-chip and previous approaches.

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.