Microfluidic Chamber Research Articles

P-050 Microfluidic chip-sorted spermatozoa selection yields embryo euploidy rate and reproductive outcomes similar to density-gradient centrifugation

Abstract Study question Does microfluidic chip-sorted spermatozoa selection (MSS) improve good quality blastocyst (GQB) and embryo euploidy rates compared to density gradient centrifugation (DGC)? Summary answer The use of MSS before intracytoplasmatic sperm injection (ICSI) does not improve good quality blastocist and euploidy rates comparing to DGC. What is known already The integrity of sperm DNA plays a crucial role in embryonic development. Conventional sperm selection methods, such as DGC, may potentially damage sperm DNA due to oxidative stress generated during centrifugation. One of the recent advances in sperm selection techniques is the microfluidic chamber, which, requiring less processing time and eliminating the need for centrifugation, allows for the selection of sperm with better mobility, morphology, and reduced DNA damage. Consequently, improved GQB and euploid embryo rates, as well as enhanced reproductive outcomes, are expected. Study design, size, duration Retrospective study of 1739 blastocysts from 404 couples undergoing 544 autologous cycles of ICSI/PGT-A (preimplantation genetic testing for aneuploidies) from January 2020 to December 2022. Those patients with sperm concentration <5 × 106/mL or motility <10%, altered karyotype, women aged > 43 years, uterine anomalies and thrombophilia were excluded. Besides comparing euploidy rates and blastocyst formation, reproductive outcomes were analyzed and compared. Participants/materials, setting, methods According to the sperm selection technique, two groups were established. One group underwent MSS (ZyMōt ICSI® or ZyMōt Multi®), resulting in 295 blastocysts from 96 cycles. It was compared with a control group (DGC) including 1444 blastocysts from 448 ICSI/PGT-A cycles. PGT-A was performed in day 5/6 using Next Generation Sequencing (NGS) technology. For a 95% confidence interval, statistical significance was considered at p < 0.05. Chi-square analysis was used for the statistical evaluation. Main results and the role of chance Both groups were comparable in terms of demographic data, including age, sperm analysis parameters, and cycle characteristics. There were no significant differences between the DGC and MSS groups in the women’s age (mean ± SD) (37.9 ± 3.9 vs. 38 ± 3.9 years, p = 0.99) or men’s age (38.4 ± 7.1 vs. 39.8 ± 5.9 years, p = 0.11). No significant differences were found between the DGC and MSS groups in terms of GQB rate (49.3% vs. 50.7%, p = 0.55), euploidy rate (35.8% vs. 32.2%, p = 0.23), fertilization rate (69.5% vs. 69.5%, p = 0.99), clinical pregnancy rate per transfer (63% vs. 76%, p = 0.13), clinical miscarriage (9% vs. 21%, p = 0.33), and live birth rate per transfer (44% vs. 37%, p = 0.42). The results also showed a higher number of euploid embryos per initiated cycle in the DGC group (1.154) compared to the MSS group (0.989). Despite this difference, the results were not statistically significant (p > 0.05). Limitations, reasons for caution The limitations of this study include the potential impact of selection bias, as it is a retrospective study in which groups were not randomly assigned. Additionally, the low number of blastocysts in the MSS group and the potential bias of DNA fragmentation, which was not evaluated in the study. Wider implications of the findings Both methods demonstrated similar effectiveness. While MSS could potentially be more effective in samples with high DNA fragmentation and they feature a user-friendly application, its major limitation lies in its challenging application with samples having a low concentration of motile sperm. Specific semen samples may need different sperm selection methods. Trial registration number Not Applicable

Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons.

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment-specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age-dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intramitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment-dependent manner during ageing.

Stable, Conductive, Adhesive Polymer Patterning Inside a Microfluidic Chamber for Endothelial Cell Alignment

AbstractEndothelial cells (ECs) line the inner walls of blood vessels, respond to shear stress by elongating in the direction of flow. Engineering aligned ECs in vitro is essential for modeling human vascular diseases and for drug testing. Current microfluidic approaches mainly rely on unidirectional laminar flow, uniform coating of surfaces to improve cellular adhesion or alteration of the surface topography. Challenges persist due to shear stress‐induced changes in cellular behavior, especially in complex multicellular environments and the time needed for the cells to align and polarize inside the microfluidic conduits. Generally, protein coating processes and physical treatments are also not compatible with the steps required for the assembly of microfluidic devices. This approach employs aerosol jet printing (AJP) to precisely pattern poly(3,4‐ethylenedioxythiophene) polystyrene sulphonate (PEDOT:PSS) within microfluidic chambers in a single step. It is shown that the PEDOT:PSS is biocompatible and facilitates EC adhesion, patterning, elongation, and alignment. Under capillary flow, the cells retain their pattern‐induced morphology over 7 d, confirming the efficacy of the approach in promoting cellular organization, eliminating the need for external pumps. Furthermore, it is demonstrated that the PEDOT:PSS pattern retains structural integrity and electrical stability following oxygen plasma treatment, required for assembling of fully enclosed microfluidic devices.

Environmentally friendly calcium carbonate-polydopamine microcapsules with superior mechanical, barrier, and adhesive properties

There is a rising need to deliver perfume molecules to fabric surfaces during washing cycles to enhance consumers' perception. With upcoming regulations phasing out intentionally produced microplastics by 2027, the focus is on sustainable alternatives to conventional non-biodegradable synthetic microcapsules (e.g. melamine-formaldehyde). Calcium carbonate (CaCO3) has shown promise to form the microcapsule shells due to its environmental benignity, inexpensiveness, and potential for liquid formulations, particularly detergents. Notwithstanding, its inherent porosity undermines the performance of the ensuing microcapsules. Bio-inspired by the adhesive properties of mussel proteins, dopamine is proposed for forming a protective organic coating on CaCO3-based microcapsules. This research aims towards developing primary microcapsules with CaCO3 shells encapsulating hexylsalicylate (HS) as a perfume oil, applying a polydopamine (PDA) coating via oxidative auto-polymerisation of dopamine-hydrochloride (pH 8.5), and conducting a comprehensive analysis of morphological, mechanical, barrier, and adhesive properties through advanced techniques, namely fluorescent-sensing/scanning electron microscopy (SEM), micromanipulation, UV–Vis spectrometry, and a microfluidic chamber fitted with polyethylene-terephthalate (PET) fabrics. The obtained microcapsules (D[3, 2]=31.2 ± 0.4 μm) exhibited a spherical core-shell structure with a smooth PDA-coated surface. Mechanical assessments reveal remarkable rupture stress (2.2 ± 0.3 MPa) comparable to that of commercial microcapsules. After one month in water, ∼40% of HS was released from PDA-coated microcapsules, while the primary ones released the entire amount within 4 h. When mimicking washing conditions (pH 9), the PDA-coated microcapsules demonstrated improved retention (∼60%) on the PET substrate at hydrodynamic shear stress of ∼1 Pa, whereas that of the primary microcapsules was below 10%. Overall, this study suggests the successful fabrication of eco-friendly microcapsules featuring a hybrid inorganic-organic shell, with enhanced mechanical strength, reduced leakage, and improved adhesion, showcasing their potential in applications within the fast-moving consumer goods industry.

Detection of α-Galactosidase A Reaction in Samples Extracted from Dried Blood Spots Using Ion-Sensitive Field Effect Transistors.

Fabry disease is a lysosomal storage disorder caused by a significant decrease in the activity or absence of the enzyme α-galactosidase A. The diagnostics of Fabry disease during newborn screening are reasonable, due to the availability of enzyme replacement therapy. This paper presents an electrochemical method using complementary metal-oxide semiconductor (CMOS)-compatible ion-sensitive field effect transistors (ISFETs) with hafnium oxide-sensitive surfaces for the detection of α-galactosidase A activity in dried blood spot extracts. The capability of ISFETs to detect the reaction catalyzed by α-galactosidase A was demonstrated. The buffer composition was optimized to provide suitable conditions for both enzyme and ISFET performance. The use of ISFET structures as sensor elements allowed for the label-free detection of enzymatic reactions with melibiose, a natural substrate of α-galactosidase A, instead of a synthetic fluorogenic one. ISFET chips were packaged with printed circuit boards and microfluidic reaction chambers to enable long-term signal measurement using a custom device. The packaged sensors were demonstrated to discriminate between normal and inhibited GLA activity in dried blood spots extracts. The described method offers a promising solution for increasing the widespread distribution of newborn screening of Fabry disease.

Real-time imaging of axonal membrane protein life cycles.

The construction of neuronal membranes is a dynamic process involving the biogenesis, vesicular packaging, transport, insertion and recycling of membrane proteins. Optical imaging is well suited for the study of protein spatial organization and transport. However, various shortcomings of existing imaging techniques have prevented the study of specific types of proteins and cellular processes. Here we describe strategies for protein tagging and labeling, cell culture and microscopy that enable the real-time imaging of axonal membrane protein trafficking and subcellular distribution as they progress through some stages of their life cycle. First, we describe a process for engineering membrane proteins with extracellular self-labeling tags (either HaloTag or SNAPTag), which can be labeled with fluorescent ligands of various colors and cell permeability, providing flexibility for investigating the trafficking and spatiotemporal regulation of multiple membrane proteins in neuronal compartments. Next, we detail the dissection, transfection and culture of dorsal root ganglion sensory neurons in microfluidic chambers, which physically compartmentalizes cell bodies and distal axons. Finally, we describe four labeling and imaging procedures that utilize these enzymatically tagged proteins, flexible fluorescent labels and compartmentalized neuronal cultures to study axonal membrane protein anterograde and retrograde transport, the cotransport of multiple proteins, protein subcellular localization, exocytosis and endocytosis. Additionally, we generated open-source software for analyzing the imaging data in a high throughput manner. The experimental and analysis workflows provide an approach for studying the dynamics of neuronal membrane protein homeostasis, addressing longstanding challenges in this area. The protocol requires 5-7 days and expertise in cell culture and microscopy.

Enabling accessibility to the CellScape spatial biology workflow for researchers using standard histology protocols and pre-slided specimens.

e15185 Background: Solid tumor specimen collection during clinical trials often follows a standard histology protocol, ending in the generation of FFPE sections mounted onto 1 mm histology slides. Though a common sample format for researchers developing tissue-based biomarkers, slides present a data quality challenge for multiplexed spatial biology workflows. Here we present data describing an innovative flow cell design fully compatible with pre-slided tissue specimens. We collected spatial biology data using this slide-compatible flow cell and compared it to the gold standard direct mounting on coverglass. Several rounds of marker staining and imaging demonstrate equivalency in method outcomes including image resolution, signal to noise, and re-interrogation capacity. Methods: FFPE biopsy tissue specimens (tumor punch biopsies and a tonsil section) were mounted on standard histology slides. These specimens were incorporated into flow cells using a new glass coverslip and porting device, CellScape™ Slide, that pairs with a standard histology slide to form a microfluidic chamber. After mounting, each sample was probed for consensus CD markers (CD45, CD3, CD4,CD8,CD20, etc.) through several rounds of iterative immunofluorescence staining using CellScape™ Precise Spatial Multiplexing. The resulting multi-layered whole slide image dataset was then interrogated for spatial resolution and signal-to-noise using digital image analysis. Results: Spatial resolution and signal-to-noise ratio (SNR) were assessed through comparison to data collected from similar samples using the existing CellScape microfluidic chip. The lowest intensity signal collected with at least a 30:1 SNR (acceptable for image analysis) was equivalent or lower in the CellScape Slide preparation (equivalent sensitivity). The highest intensity signal collected was equivalent between the two flow cell designs. Comparing stain quality (specificity, sensitivity) among markers stained through multiple rounds of iterative staining, imaging, and signal remove via photoinactivation showed that the CellScape Slide flow cell is on par with or better than the existing CellScape chip. Further, the usable clear window of the CellScape Slide flow chamber, measuring 16 mm x 44 mm, was found to be greater than twice that of the existing chip. Literature review determined this to also be the largest imaging window available among commercially available spatial biology platforms. Conclusions: Assessment of the CellScape Slide flow cell, innovative in providing access to the CellScape spatial biology workflow for pre-mounted FFPE tissue, determined that the optical performance of the new flow cell is at least on par with the existing flow cell. Further, the usable window is over twice the area, and the new flow cell is fully capable of supporting re-interrogation of the sample.

Carbon Dioxide Concentration Alters the Dynamics of Oxygen-Mediated Capillary Blood Flow Response s in Skeletal Muscle

Objective: We sought to quantify how different muscle carbon dioxide concentrations ([CO2]) alter the dynamics of oxygen-mediated blood flow responses in skeletal muscle capillaries. Methods: 6 male Sprague Dawley rats (164-215g) were anaesthetized, instrumented for systemic monitoring, and mechanically ventilated. The extensor digitorum longus muscle was isolated via blunt dissection, externalized, and reflected over a microfluidic gas exchange chamber mounted in the stage of an inverted microscope. Intravital video of capillary blood flow was recorded during 4-minute O2 challenges consisting of a 1-minute baseline at 7% oxygen concentration ([O2]), followed by 3 minutes at 2% [O2]. Before recordings, the muscle was equilibrated to constant background [CO2] of either 2%, 5%, or 8% for 5 minutes. Recordings were analyzed offline using custom MATLAB software. The time transients () of capillary hemodynamic responses were determined using a least-squared regression fit (LSRF) to single- and double-exponential models. All animal protocols were approved by Memorial University’s Animal Care Committee. Results: Baseline red blood cell (RBC) velocity was significantly different at 5% [CO2] (213.2±179.4 μm/s) compared to 2% [CO2] (177.7±162.6 μm/s, p=0.0016), and 8% [CO2] (250.3±193.6 μm/s, p<0.0001). RBC oxygen saturations (SO2) were significantly different beyond 3 s post-baseline in all [CO2] conditions ( p<0.02, =3.2-4.3s). Minimum RBC SO2 was 52%, 46%, and 38% below baseline at 12s, 14s, and 12s for 2%, 5%, and 8% [CO2] conditions, respectively. RBC velocity was significantly increased during 2% [CO2] by 3 s post-baseline (177.7±162.6 vs 188.7±168.6 μm/s, p<0.0001), during 5% [CO2] by 6 s post-baseline (213.2±179.4 vs 224.2±189.9 μm/s, p=0.0190), and during 8% [CO2] by 12 s post-baseline (250.3±193.6 vs 267.0±199.9 μm/s, p=0.0062). Time transients of the fast component of the oxygen-mediated velocity response, as determined by LSRF, were 1.9s for 2% [CO2], 4.8s for 5% [CO2], and 9.8s for 8% [CO2]. Velocity was increased at the end of the O2 challenge by 39%, 33%, and 36% during 2%, 5%, and 8% [CO2] conditions, respectively. Baseline RBC supply rate (SR) was significantly different at 5% [CO2] (13.5±18.9 cells/s) compared to 2% [CO2] (9.5±15.3 cells/s, p=0.0090), and 8% [CO2] (19.0±23.6 cells/s, p<0.0001). At 2% [CO2], SR increased significantly by 9 s post-baseline (9.5±15.2 vs 10.9±16.9 cells/s, p=0.0001), by 10 s post-baseline for 5% [CO2] (13.5±18.9 vs 14.9±20.6 cells/s, p=0.0099), and by 13 s post-baseline during 8% [CO2] (19.0±23.6 vs 21.1±25.2 cells/s, p=0.0204). The fast component of the oxygen-mediated SR response had a of 2.7s for 2% [CO2], 4.5s for 5% [CO2], and 6.9s for 8% [CO2] as determined by LSRF. Overall SR was increased by 74%, 51%, and 39% at the end of the O2 challenge during 2%, 5%, and 8% [CO2], respectively. As well, capillary hematocrits were significantly different during baseline at 5% [CO2] (21.4±16.1%) compared to 2% [CO2] (18.2±22.2%, p=0.0113), and 8% [CO2] (27.2±21.4%, p<0.0001). Conclusion: We have quantified the fast component of oxygen-mediated capillary hemodynamic responses to an acute decrease in skeletal muscle [O2]. The time transients of O2-mediated responses vary with tissue [CO2] such that increasing CO2 results in slower response dynamics. As well, increasing background [CO2] provokes higher capillary RBC velocity, SR, and hematocrit both at baseline and during low O2 challenges as expected. This project was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant awarded to GMF. Student support was provided by Memorial University's School of Graduate Studies and Faculty of Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanical Characterization of the Erythrocyte Membrane Using a Capacitor-Based Technique.

Pathological processes often change the mechanical properties of cells. Increased rigidity could be a marker of cellular malfunction. Erythrocytes are a type of cell that deforms to squeeze through tiny capillaries; changes in their rigidity can dramatically affect their functionality. Furthermore, differences in the homeostatic elasticity of the cell can be used as a tool for diagnosis and even for choosing the adequate treatment for some illnesses. More accurate types of equipment needed to study biomechanical phenomena at the single-cell level are very costly and thus out of reach for many laboratories around the world. This study presents a simple and low-cost technique to study the rigidity of red blood cells (RBCs) through the application of electric fields in a hand-made microfluidic chamber that uses a capacitor principle. As RBCs are deformed with the application of voltage, cells are observed under a light microscope. From mechanical force vs. deformation data, the elastic constant of the cells is determined. The results obtained with the capacitor-based method were compared with those obtained using optical tweezers, finding good agreement. In addition, P. falciparum-infected erythrocytes were tested with the electric field applicator. Our technique provides a simple means of testing the mechanical properties of individual cells.

A Microfluidic Strategy to Capture Antigen‐Specific High‐Affinity B Cells

Assessing B cell affinity to pathogen‐specific antigens prior to or following exposure could facilitate the assessment of immune status. Current standard tools to assess antigen‐specific B cell responses focus on equilibrium binding of the secreted antibody in serum. These methods are costly, time‐consuming, and assess antibody affinity under zero force. Recent findings indicate that force may influence BCR‐antigen binding interactions and thus immune status. Herein, a simple laminar flow microfluidic chamber in which the antigen (hemagglutinin of influenza A) is bound to the chamber surface to assess antigen‐specific BCR binding affinity of five hemagglutinin‐specific hybridomas from 65 to 650 pN force range is designed. The results demonstrate that both increasing shear force and bound lifetime can be used to enrich antigen‐specific high‐affinity B cells. The affinity of the membrane‐bound BCR in the flow chamber correlates well with the affinity of the matched antibodies measured in solution. These findings demonstrate that a microfluidic strategy can rapidly assess BCR‐antigen‐binding properties and identify antigen‐specific high‐affinity B cells. This strategy has the potential to both assess functional immune status from peripheral B cells and be a cost‐effective way of identifying individual B cells as antibody sources for a range of clinical applications.

Shear-Induced Nonequilibrium Patterns in Lipid Bilayer Membranes Exhibiting Phase Separation.

The nonequilibrium dynamics of a fluid lipid membrane under external stimuli is an important issue that spans disciplines such as soft matter, biophysical chemistry, and interface science. This study investigated the dynamic response of lipid vesicles with order-disorder phase separation, which mimics a plasma membrane heterogeneity, to shear flow. Lipid vesicles were immobilized in a microfluidic chamber, and shear-induced nonequilibrium patterns on the membrane surface were observed by an optical microscope. We found that phase-separated membranes exhibit a dissipative structure of stripe patterns along the vortex flow on the membrane surface, and the number of stripes increased with the flow rate. At a high flow rate, the membrane exhibited a stripe-to-wave transition, where striped domains often migrated and the replacement of two different phases happened at vortex centers with time. We obtained a dynamic phase diagram of the shear-induced wave pattern by changing the flow rate, membrane components, and temperature. These findings could provide insight into the dissipative structures of lipid membranes out of equilibrium and flow-mediated mechanotransduction of biological membranes.

Mathematical modeling of transdermal delivery of topical drug formulations in a dynamic microfluidic diffusion chamber in health and disease.

Mathematical models of epidermal and dermal transport are essential for optimization and development of products for percutaneous delivery both for local and systemic indication and for evaluation of dermal exposure to chemicals for assessing their toxicity. These models often help directly by providing information on the rate of drug penetration through the skin and thus on the dermal or systemic concentration of drugs which is the base of their pharmacological effect. The simulations are also helpful in analyzing experimental data, reducing the number of experiments and translating the in vitro investigations to an in-vivo setting. In this study skin penetration of topically administered caffeine cream was investigated in a skin-on-a-chip microfluidic diffusion chamber at room temperature and at 32°C. Also the transdermal penetration of caffeine in healthy and diseased conditions was compared in mouse skins from intact, psoriatic and allergic animals. In the last experimental setup dexamethasone, indomethacin, piroxicam and diclofenac were examined as a cream formulation for absorption across the dermal barrier. All the measured data were used for making mathematical simulation in a three-compartmental model. The calculated and measured results showed a good match, which findings indicate that our mathematical model might be applied for prediction of drug delivery through the skin under different circumstances and for various drugs in the novel, miniaturized diffusion chamber.

Integration of wide-field imaging system with droplet microfluidics for monitoring living bacteria

Droplet microfluidics has been widely used to analyze chemicals and biological reactions at the single-molecule level. Microscopic systems are commonly used for imaging and analyzing droplets using high-magnification objective lenses. However, these systems are expensive, complex, and large, limiting the high-throughput characterization of droplets in reactions targeting disease-related biomarkers, including nucleic acids, proteins, and pathogens. Another concern is the time gap between droplet analysis within a microfluidic channel and imaging chamber, which can cause discrepancies in reaction time between droplets. To address this issue, we introduce the droplet analysis system for vast imaging and statistical tool (DropVIST)—a wide-field imaging system integrated with droplet microfluidics for rapid and accurate droplet analysis. DropVIST can detect eight differently colored droplets simultaneously using a wide-field imaging system and dFinder software, which can quantify the reacted droplets. The smallest droplet diameter for detection was 30 μm, and the maximum detection area was 201.84 cm2, suggesting that the system can theoretically analyze 3103,377 droplets in real-time. We validated the monitoring performance of DropVIST using clinical isolates of five types of living pathogenic bacteria and compared this with conventional approaches, including the microbroth dilution method and colony-forming unit assay. This platform provides a rapid, simple, and accurate tool for monitoring living bacteria at the single-cell level.

Characterizing the Janus colloidal particles in AC electric field and a step towards label-free cargo manipulation

In recent years, researchers have been exploring Janus particles, a unique type of colloidal system, which has gained enormous attention across diverse fields, from soft matter physics to biology. These particles, possessing an asymmetric surface, can be manipulated in the fluid using external energy sources. This versatility makes them suitable for mimicking biological systems and applications like cargo transportation, drug delivery, biosensing, and environmental cleanup. The study focuses on the response of metal-dielectric (Ti-PMMA) Janus spheres in the AC field, created through the Physical Vapor Deposition (PVD) technique. When subjected to a uniform AC electric field, these particles exhibit different dynamic behaviors at various frequency ranges. The particles show diverse responses from induced-charge electrophoresis (ICEP: 500 Hz-80 kHz) to reversed motion (r: ICEP:100 kHz-1 MHz) and linear chain formation (1 MHz). Additionally, they randomly cluster near the lower characteristic frequency (800 Hz) of the ICEP, whereas they exhibit the 3D motion at frequencies (100 Hz) below this lower characteristic value. The mechanisms behind these dynamic phenomena could be explained by considering the concept of electric double layer (EDL), self-dielectrophoresis (sDEP), dipolar interactions, hydrodynamic interactions, and electrothermal effects. These active Janus Colloids are further leveraged to manipulate 1 µm diameter PMMA passive microparticles and E. coli bacteria at 1 kHz, showcasing their potential applications in cargo delivery under various microfluidic research areas. The manipulation of payloads could be explained based on dielectrophoretic trapping due to locally generated field gradients around the surface of the Janus sphere. Therefore, the present work offers an unprecedented opportunity to study such out-of-equilibrium complex active matter systems and create novel functional materials. Moreover, it could also be extended as a fuel-free microrobotics system for various biomedical applications such as sensing and on-demand drug or cargo delivery inside the microfluidics chamber.

Abstract 7498: Acoustophoresis enriches tumor cell clusters from patients with metastatic prostate cancer

Abstract Purpose: A subset of aggressive prostate cancers (PCa) progress to invade surrounding tissue to form distant metastases by shed of circulating tumor cells (CTCs). However, due to the scarcity of CTCs among the millions of nucleated blood cells, there is an unmet need to improve CTC isolation assays. Our technique Acoustophoresis uses standing ultrasound waves in a flow-through microfluidic chamber to discriminate cells based on their size, density, and compressibility. Method: We analyzed the effectiveness of acoustophoretic circulating tumor cell (CTC) isolation from 6 mL PFA fixed red blood cell lysed blood from 12 metastatic prostate cancer patients and 20 healthy control subjects and compared it to the golden standard CellSearch. For acoustic cell separation, cells were diluted in 12 mL PBS based buffer and separated by acoustophoresis and analyzed by imaging flow cytometry using a standardized antibody panel (EpCAM, cytokeratin, CD45, CD66b, DAPI). Generally, CTCs, and clusters migrate faster than white blood cells (WBCs) when exposed to ultrasound in the microchannel during acoustophoresis and can therefore be separated from nucleated blood cells. Major findings: Our pilot study shows that acoustophoresis finds both singlets and clusters of CTCs in patients as well as aggregates of CTCs and WBCs. In total, 67 cancer cell containing cell clusters were identified. Most identified cell clusters were small, containing less than 4 cells. In addition, acoustophoresis is a label-free separation technique, and could therefore identify additional subclasses of CTCs among the detected CTCs with e.g., EpCAM low/- expression. Further, we provide extensive data for the effect of storage time on acoustophoretic separation performance for fixed prostate cancer cell line cells and WBCs. Demonstrating that sample processing can be done up to 72 hours after blood sampling. We believe this is to date the most comprehensive study of acoustic separation of CTCs in patients and control subjects. Conclusion: Label-free acoustophoresis is a potential method for future non-invasive molecular interrogation of metastatic cancers. We believe Acoustophoresis is a promising technology for CTC enrichment with the possibility to detect new subclasses of CTCs as well as cell clusters not detectable with CellSearch. CTC-clusters has previously been proposed as a promising and potent predictor to monitor progressive disease and is associated with earlier onset of metastatic disease and poor prognosis, which makes acoustophoresis ability to identify clusters of high interest. Citation Format: Cecilia Magnusson, Per Augustsson, Andreas Lenshof, Andreas Josefsson, Anders Bjartell, Yvonne Ceder, Hans Lilja, Thomas Laurell. Acoustophoresis enriches tumor cell clusters from patients with metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7498.

Abstract 5354: High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach

Abstract The immune checkpoint involving PD-1 and PD-L1 generates suppressive signals in T cells, which help to prevent autoimmunity by inducing a state of immune exhaustion. In the context of the tumor microenvironment (TME), however, cancer cells can manipulate these pathways to camouflage themselves from an immune attack. Blocking these checkpoints has thus emerged as a key immunotherapeutic tactic. Nonetheless, the success rate of immune checkpoint inhibitors (ICIs) has been mixed, even for patients who test positive for relevant diagnostic biomarkers. Patient stratification currently depends on immunohistochemical staining for checkpoint proteins like PD-L1, but these tests do not provide adequate stratification. A more comprehensive stratification, one that includes immune profiling of the TME plus an evaluation of immune checkpoint interactions, might provide better patient stratification and ICI responsiveness. Here we describe mIF that is augmented with a PD-1 and PD-L1 protein-protein interaction assay. Human formalin-fixed, paraffin-embedded (FFPE) tissue sections were subjected to standard histological processing and then incubated with primary antibodies against PD1 and PD-L1. Sections were then treated with oligonucleotide-modified secondary probes suitable for in situ Proximity Ligation Assay (isPLA), enabling the detection of PD1 and PD-L1 interactions. The PLA protocol utilized a fluorescent probe complementary to amplified nucleic acid to enhance the visibility of the protein interactions. The sample was then mounted in a closed microfluidic chamber and imaged on the CellScape™ platform for Precise Spatial Multiplexing. After imaging of the isPLA signal, the CellScape cyclic multiplex mIF staining approach was used to iteratively stain immune and structural markers on the tissue sample utilizing the VistaPlex™ Spatial Immune Profiling Assay Kit. This proof-of-concept study demonstrates that high-plex mIF can be augmented with an isPLA on human FFPE samples. The combined application of both methods allows visualization of PD1/PD-L1 protein-protein interactions and integrates this interaction within the spatial context of the surrounding cell populations. This approach allows for a more comprehensive insight into the interplay of different immune cell and non-immune cell populations during checkpoint activation processes in normal and neoplastic tissues. Citation Format: Arne Christians, Jannik Boog, Charles E. Jackson, Matthew H. Ingalls, J Spencer Schwarz, Sara Bodbin, Agata Zieba Wicher, Subham Basu, Oliver Braubach. High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5354.

Trapping and scattering of a multiflagellated bacterium by a hard surface.

Thiovulum majus, which is one of the fastest known bacteria, swims using hundreds of flagella. Unlike typical pusher cells, which swim in circular paths over hard surfaces, T. majus localize near hard boundaries by turning their flagella to exert a net force normal to the surface. To probe the torques that stabilize this hydrodynamically bound state, the trajectories of several thousand collisions between a T. majus cell and a wall of a quasi-two-dimensional microfluidic chamber are analyzed. Measuring the fraction of cells escaping the wall either to the left or to the right of the point of contact-and how this probability varies with incident angle and time spent in contact with the surface-maps the scattering dynamics onto a first passage problem. These measurements are compared to the prediction of a Fokker-Planck equationto fit the angular velocity of a cell in contact with a hard surface. This analysis reveals a bound state with a narrow basin of attraction in which cells orient their flagella normal to the surface. The escape angle predicted by matching these near field dynamics with the far-field hydrodynamics is consistent with observation. We discuss the significance of these results for the ecology of T. majus and their self-organization into active chiral crystals.

Amplifying Sensing Capabilities: Combining Plasmonic Resonances and Fresnel Reflections through Multivariate Analysis.

Multivariate analysis applied in biosensing greatly improves analytical performance by extracting relevant information or bypassing confounding factors such as nonlinear responses or experimental errors and noise. Plasmonic sensors based on various light coupling mechanisms have shown impressive performance in biosensing by detecting dielectric changes with high sensitivity. In this study, gold nanodiscs are used as metasurface in a Kretschmann setup, and a variety of features from the reflectance curve are used by machine learning to improve sensing performance. The nanostructures of the metasurface generate new plasmonic features, apart from the typical resonance that occurs in the classical Kretschmann mode of a gold thin film, related to the evanescent field beyond total internal reflection. When the engineered metasurface is integrated into a microfluidic chamber, the device provides additional spectral features generated by Fresnel reflections at all dielectric interfaces. The increased number of features results in greatly improved detection. Here, multivariate analysis enhances analytical sensitivity and sensor resolution by 200% and more than 20%, respectively, and reduces prediction errors by almost 40% compared to a standard plasmonic sensor. The combination of plasmonic metasurfaces and Fresnel reflections thus offers the possibility of improving sensing capabilities even in commonly available setups.

Multiplexed Single‐Cell Rheology Probing Using Surface Acoustic Waves

Cellular rheological properties affect cell function and are reflective of cell status. It is challenging to perform multiplexed single‐cell rheology probing with high controllability, particularly for adherent cells. A surface acoustic wave (SAW)‐based method is presented for this purpose. The method integrates the potent micromanipulation ability of acoustic waves in a microfluidic chamber with the ability of cell‐anchored microbeads to concentrate the acoustic energy to deform the cell. Two strategies are developed for placing a targeted microbead at a desired position on the cell membrane. The power‐law rheological dynamics with plastic components are applied to fit the creep (during the mechanical loading) and relaxation (after force removal) responses of the cell. With more than 400 measurements of adherent cells and each with detailed dynamics, a full range of viscoelastic behaviors of cells far beyond the typical rheology of previously reported adherent cells and unexpected negative plastic compliance is observed. The developed method supports in‐depth investigations of biomechanics at the cellular and subcellular levels, with considerable potential for extension to mechanical force‐based cell function regulation.

Multiple Labeling of Compartmentalized Cortical Neurons in Microfluidic Chambers.

Neurons are complex cells with two distinct compartments: the somatodendritic and the axonal domains. Because of their polarized morphology, it is challenging to study the differential cellular and molecular mechanisms that occur in axons and impact the soma and dendrites using conventional in vitro culture systems. Compartmentalized cultures offer a solution by physically and chemically separating the axonal from the somatodendritic domain of neurons. The microfluidic chamber model presented in this work is valuable for studying these mechanisms in primary cortical cultures derived from rat and mouse. In addition, this chamber model is compatible with various microscopy methods, such as phase contrast, and fluorescence imaging of living and fixed cells. Key features • Preparation and attachment of PDMS microfluidic chambers to glass coverslips. • Primary culture of cortical neurons and plating cortical neurons in microfluidic chamber. • Confirmation of compartmentalization using the retrograde transport of the fluorescently labeled form of cholera toxin subunit B (f-Ctb). • Immunofluorescence and multilabeling of compartmentalized cortical neurons. • Retrograde transport of fluorescently labeled BDNF.