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Multi-compartment microfluidic devices have become valuable tools for experimental neuroscientists, improving the organization of neurons and access to their distinct subcellular microenvironments for measurements and manipulations. While murine neurons are extensively used within these devices, there is a growing need to culture and maintain human neurons differentiated from stem cells within multi-compartment devices. Human neuron cultures have different metabolic demands and require longer culture times to achieve synaptic maturation. We tested different channel heights (100 μm, 400 μm, and open) to determine whether greater exposure to media for nutrient exchange might improve long-term growth of NIH-approved H9 embryonic stem cells differentiated into glutamatergic neurons. Our data showed an opposite result with both closed channel configurations having greater synaptic maturation compared to the open compartment configuration. These data suggest that restricted microenvironments surrounding neurons improve growth and maturation of neurons. We next tested whether covalently bound poly-D-lysine (PDL) might improve growth and maturation of these neurons as somata tend to cluster together on PDL adsorbed surfaces after long culture periods (>30 days). We found that covalently bound PDL greatly improved the differentiation and maturation of stem cell-derived neurons within the devices. Lastly, experimental paradigms using the multi-compartment platform show that axons of human stem cell derived neurons intrinsically regenerate in the absence of inhibitory cues and that isolated axons form presynaptic terminals when presented with synaptic targets.

Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods.

As a subspecies of extracellular vesicles (EVs), exosomes have provided promising results in diagnostic and theranostic applications in recent years. The nanometer-sized exosomes can be extracted by liquid biopsy from almost all body fluids, making them especially suitable for mainly non-invasive point-of-care (POC) applications. To achieve this, exosomes must first be separated from the respective biofluid. Impurities with similar properties, heterogeneity of exosome characteristics, and time-related biofouling complicate the separation. This practical review presents the state-of-the-art methods available for the separation of exosomes. Furthermore, it is shown how new separation methods can be developed. A particular focus lies on the fabrication and design of microfluidic devices using highly selective affinity separation. Due to their compactness, quick analysis time and portable form factor, these microfluidic devices are particularly suitable to deliver fast and reliable results for POC applications. For these devices, new manufacturing methods (e.g., laminating, replica molding and 3D printing) that use low-cost materials and do not require clean rooms are presented. Additionally, special flow routes and patterns that increase contact surfaces, as well as residence time, and thus improve affinity purification are displayed. Finally, various analyses are shown that can be used to evaluate the separation results of a newly developed device. Overall, this review paper provides a toolbox for developing new microfluidic affinity devices for exosome separation.

Optical biosensors have a significant impact on various aspects of our lives. In many applications of optical biosensors, fluidic chambers play a crucial role in facilitating controlled fluid delivery. It is essential to achieve complete liquid replacement in order to obtain accurate and reliable results. However, the configurations of fluidic chambers vary across different optical biosensors, resulting in diverse fluidic volumes and flow rates, and there are no standardized guidelines for liquid replacement. In this paper, we utilize COMSOL Multiphysics, a finite element analysis software, to investigate the optimal fluid volume required for two types of fluidic chambers in the context of the oblique–incidence reflectivity difference (OI-RD) biosensor. We found that the depth of the fluidic chamber is the most crucial factor influencing the required liquid volume, with the volume being a quadratic function of the depth. Additionally, the required fluid volume is also influenced by the positions on the substrate surface bearing samples, while the flow rate has no impact on the fluid volume.

The margination dynamics of microparticles with different shapes has been analyzed within a laminar flow mimicking the hydrodynamic conditions in the microcirculation. Silica spherical particles, quasi-hemispherical and discoidal silicon particles have been perfused in a parallel plate flow chamber. The effect of the shape and density on their margination propensity has been investigated at different physiologically relevant shear rates S. Simple scaling laws have been derived showing that the number n of marginating particles scales as S - 0.63 for the spheres; S - 0.85 for discoidal and S - 1 for quasi-hemispherical particles, regardless of their density and size. Within the range considered for the shear rate, discoidal particles marginate in a larger number compared to quasi-hemispherical and spherical particles. These results may be of interest in drug delivery and bio-imaging applications, where particles are expected to drift towards and interact with the walls of the blood vessels.

ObjectiveTo test a novel method to select spermatozoa with high chromatin integrity.DesignSpecimens with high sperm chromatin fragmentation (SCF) were selected by density gradient selection (DGS) and microfluidic sperm sorting (MSS).SettingAcademic medical center.Patient(s)Ejaculates from consenting men were processed by DGS/MSS. Couples underwent ICSI cycles with spermatozoa processed by DGS/MSS. Clinical outcomes were evaluated after embryo transfer.Intervention(s)SCF was measured by TUNEL. ICSI with spermatozoa selected by DGS and MSS was performed.Main outcome measure(s)Fertilization, embryo implantation, and pregnancy outcomes were compared between DGS and MSS.Result(s)A total of 23 men had an average SCF of 20.7 ± 10%. After DGS and MSS, the SCF was 12.5 ± 5% and 1.8 ± 1%, respectively. In couples who underwent ICSI, the average SCF was 28.8 ± 9%, which fell to 21.0 ± 9% after DGS and 1.3 ± 0.7% after MSS. Four couples underwent 11 ICSI cycles with DGS and achieved one (25%) pregnancy that resulted in pregnancy loss. In four subsequent ICSI cycles with MSS, an ongoing clinical pregnancy rate of 50% was achieved. Five additional couples underwent 12 cycles of ICSI with DGS. After preimplantation genetic testing for aneuploidy, 30.3% of the embryos were euploid. One pregnancy was achieved, resulting in pregnancy loss. With MSS, 31.5% of the embryos were euploid and 4 couples obtained a pregnancy. Finally, sixteen couples underwent 20 ICSI cycles solely with MSS at our center. Of these couples, 8 had failed 13 ICSI cycles with DGS elsewhere. These couples achieved an overall implantation of 34.5% (10/29) and a pregnancy rate of 58.8% (10/17).Conclusion(s)Microfluidic selection yielded spermatozoa with optimal genomic integrity and improved chances of obtaining a euploid conceptus.

The outcome of vascular-targeted therapies is generally determined by how efficiently vascular-targeted carriers localize and adhere to the endothelial wall at the targeted site. This study investigates the impact of leukocytes, platelets and red blood cells on the margination of vascular-targeted polymeric nanospheres and microspheres under various physiological blood flow conditions. We report that red blood cells either promote or hinder particle adhesion to an endothelial wall in a parallel plate flow chamber depending on the blood flow pattern, hematocrit, and particle size. Leukocytes prevent microspheres – but not nanospheres – from adhering in laminar and pulsatile flows via (1) competition for the available binding space and (2) physical removal of previously bound spheres. In recirculating blood flow, the negative effect of leukocytes on particle adhesion is minimal for large microspheres in the disturbed flow region beyond the flow reattachment. Resting platelets were found to have no effect on particle binding likely due to their dimensions and minimal interaction with the endothelial wall. Overall, the findings of the present work would be critical for designing effective vascular-targeted carriers for imaging and drug delivery applications in several human diseases.

The simultaneous determination of adhesion and deformability parameters of erythrocytes was carried out through a microfluidic device, which uses an inverted optical microscope with new image acquisition and analysis technologies. Also, an update of the models describing erythrocyte adhesion and deformation was proposed. Measurements were carried out with red blood cells suspended in saline solution with human serum albumin at different concentrations. Erythrocytes adhered to a glass surface were subjected to different low shear stress (from 0.04 to 0.25 Pa), causing cellular deformation and dissociation. The maximum value obtained of the erythrocyte deformability index was 0.3, and that of the adhesion energy per unit area was 1.1 × 10−6 Pa m, both according to previous works. The obtained images of RBCs adhered to glass reveal that the adhesion is stronger in a single point of the cell, suggesting a ligand migration that concentrates the adhesion in a “spike-like tip” in the cell. Moreover, adhesion energy results indicate that the energy required to separate erythrocytes in media with a lower albumin concentration is greater. Both results could be explained by the mobility of membrane receptors.

Organ-on-a-Chip (OOC) platforms are microfluidic systems that recreate key features of human organ physiology in vitro via controlled perfusion. Fluid mechanical stimuli strongly influence cell morphology and function, making this important for cardiovascular OOC applications exposed to pulsatile blood flow. However, many existing OOC devices employ relatively simple chamber geometries and steady inflow assumptions, which may cause non-uniform shear exposure to cells, create stagnant regions with prolonged residence time, and overlook the specific effects of pulsatile perfusion. Here, we used computational fluid dynamics (CFD) to investigate how chamber geometry and inflow conditions shape the near-wall flow environment on a cell culture surface at a matched cycle-averaged volumetric flow rate. Numerical results demonstrated that pillarized chambers markedly reduced relative residence time (RRT) versus the flat chamber, and the small pillar configuration produced the most uniform time-averaged wall shear stress (TAWSS) distribution among the tested designs. Phase-resolved analysis further showed that wall shear stress varies with waveform phase, indicating that steady inflow may not capture features of pulsatile perfusion. These findings provide practical guidance for pillar geometries and perfusion conditions to create more controlled and physiologically relevant microenvironments in OOC platforms, thus improving the reliability of cell experimental readouts.

Chorea acanthocytosis (ChAc), an ultra-rare devastating neurodegenerative disease, is caused by mutations in the VPS13A gene, which encodes for the protein chorein. Affected patients suffer from chorea, orofacial dyskinesia, epilepsy, parkinsonism as well as peripheral neuropathy. Although medium spinal neurons of the striatum are mainly affected, other regions are impaired as well over the course of the disease. Animal studies as well as studies on human erythrocytes suggest Lyn-kinase inhibition as valuable novel opportunity to treat ChAc. In order to investigate the peripheral neuropathy aspect, we analyzed induced pluripotent stem cell derived midbrain/hindbrain cell cultures from ChAc patients in vitro. We observed dendritic microtubule fragmentation. Furthermore, by using in vitro live cell imaging, we found a reduction in the number of lysosomes and mitochondria, shortened mitochondria, an increase in retrograde transport and hyperpolarization as measured with the fluorescent probe JC-1. Deep phenotyping pointed towards a proximal axonal deterioration as the primary axonal disease phenotype. Interestingly, pharmacological interventions, which proved to be successful in different models of ChAc, were ineffective in treating the observed axonal phenotypes. Our data suggests that treatment of this multifaceted disease might be cell type and/or neuronal subtype specific, and thus necessitates precision medicine in this ultra-rare disease.