Gina Layedra

Chagas disease remains a major neglected tropical disease, highlighting the need for new anti-Trypanosoma cruzi compounds and improved approaches for their phenotypic evaluation. Here, we extended a previously developed Lab-on-a-Chip (LOC) platform integrating a serial dilution generator and automated label-free motility analysis (TR_KF) to the direct screening of extracellular T. cruzi trypomastigotes. The platform was validated in two steps using benznidazole as the reference drug. First, automated motility quantification in conventional 96-well plates was compared with manual Neubauer counting, yielding comparable concentration–response curves and IC₅₀ values. Second, the complete LOC–TR_KF platform was compared with the conventional assay, producing an IC₅₀ that was not significantly different from the reference method. The validated platform was subsequently applied to screen ten candidate samples, identifying combinatorial libraries 2161-X and 2160-X as the most active against trypomastigotes. Comparison with previously reported intracellular amastigote data revealed marked stage-dependent differences in compound activity, while pre-treatment with both libraries reduced parasite infectivity by approximately 50%. Overall, the integrated LOC–TR_KF platform provides pharmacological responses comparable to conventional assays while reducing assay-solution requirements, manual handling and enabling automated, label-free phenotypic screening of T. cruzi trypomastigotes.

Reliable world-to-chip connections remain a critical challenge in PDMS-based microfluidic devices, particularly under high-pressure flow conditions. In this work, we present a simple, low-cost, and reproducible method to directly mold male Luer cone fittings into PDMS devices during fabrication, without compromising the geometry of microchannels. To evaluate the effectiveness of this approach, we compared the performance of three connection types—PTFE tubing, metal pipes, and molded Luer adaptors—by measuring the infusion pressure at which fluid leakage occurred. Devices with integrated Luer adaptors consistently withstood higher pressures (up to 1555 mbar) with reduced variability across repeated connection cycles, while PTFE and metal connections failed at lower pressures and showed inconsistent leakage behavior. As a biological proof of concept, Staphylococcus aureus biofilms were grown in the microfluidic channels under continuous perfusion and subsequently exposed to flow rates of 50, 83.3, and 116.7 µL/min. Image analysis showed an initial biofilm-covered area of 14.22% of the analyzed channel area, while the biofilm areas remaining after exposure to 50, 83.3, and 116.7 µL/min were 1.39, 0.85, and 0.32%, respectively. The final measurement represented a reduction of approximately 97.7% relative to the initial biofilm coverage. The molded Luer connections maintained stable fluid delivery during biofilm cultivation and flow-induced detachment, supporting their use in biological microfluidic applications. Overall, this molding strategy provides a robust and reusable Luer-compatible interface for integrating standardized connections into soft-lithography-based microfluidic platforms.

Microfluidics enables precise control of fluid movement within microchannels, facilitating the generation of microdroplets at high frequencies. This technology provides a unique platform for conducting biological and chemical experiments, enhancing throughput and sensitivity, particularly in single-cell analysis. The microdroplet environment enhances interactions between cells and gene delivery materials, resulting in greater contact area, higher reagent concentration, and improved diffusion for both eukaryotic and prokaryotic cells. This review discusses the advantages and limitations of transfection and transformation within microdroplet technologies, highlighting their potential to improve gene editing efficiency while addressing challenges related to delivery mechanisms and cellular uptake rates. The integration of microdroplet technology with advanced gene editing tools, such as CRISPR/Cas9, promises to streamline processes and improve outcomes in various applications, including therapeutic interventions, vaccine development, regenerative medicine, and personalized medicine. These advancements could lead to more precise targeting of genetic modifications, resulting in tailored therapies that better meet individual patient needs. Overall, the integration of gene delivery in microdroplets represents a significant leap in biotechnology, enhancing the efficacy of gene delivery systems and opening new avenues for research and development in precision medicine.

T cells play a pivotal role in cancer research, particularly in immunotherapy, which harnesses the immune system to target malignancies. However, conventional expansion methods face limitations such as high reagent consumption, contamination risks, and difficulties in maintaining suspension cells in dynamic culture environments. This study presents a microfluidic system for long-term culture of non-adherent cells, featuring automated perfusion and image acquisition. The system integrates deep learning-based image analysis, which quantifies cell coverage and estimates cell numbers, and efficiently processes large volumes of data. The performance of this deep learning approach was benchmarked against the widely used Trainable Weka Segmentation (TWS) plugin for Fiji. Additionally, two distinct lab-on-a-chip (LOC) devices were evaluated independently: the commercial ibidi^® LOC and a custom-made PDMS LOC. The setup supported the proliferation of Jurkat cells and primary human T cells without significant loss during medium exchange. Each platform proved suitable for long-term expansion while offering distinct advantages in terms of design, cell seeding and recovery, and reusability. This integrated approach enables extended experiments with minimal manual intervention, stable perfusion, and supports multi-reagent administration, offering a powerful platform for advancing suspension cell research in immunotherapy.