Maximiliano Sebastian Perez

This study addresses some limitations of standard cell encapsulation methods by producing hydrogel beads via UV-induced C,H-insertion crosslinking (CHic) in a microfluidic setup and the simultaneous encapsulation of yeast cells. It describes a simple one-step production of monodisperse beads with consistent dimensions of ~1000 × 500 µm and the encapsulation of Saccharomyces cerevisiae within these beads. Glucose consumption profiles show that the encapsulated yeast consumes glucose at rates comparable to free yeast, indicating that encapsulation does not impair metabolic activity. The encapsulated cells maintain high retained metabolic activity for up to 15 days at 4 °C and even at 30 °C, whereas free yeast stored at this temperature suffers a significant loss of activity. This highlights the potential of the platform for storing cells without refrigeration. Another advantage is that the fermentation solutions remain clear with minimal turbidity, and the beads can be easily separated from the solution by simple sieving, thus avoiding the need to filter the solution after fermentation. The results obtained suggest that the platform is robust and versatile for use in fermentation bioreactors and cell storage.

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.

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.

Flexographic printing, traditionally used in the packaging industry, has emerged as a promising technology for microfluidic device fabrication due to enabling high resolution and being commercially available at a low cost compared to conventional techniques. This review explores the adaptation of a photopolymer flexographic printing plate mold (FMold) for microfluidics, examining its advantages, challenges, and applications. It offers a state-of-the-art view of the application of FMold for microfluidic systems, which offers a unique opportunity in terms of cost-effectiveness, scalability, and rapid prototyping. Applications are diverse: FMold has enabled the fabrication of microfluidic devices used in enhanced oil recovery to prepare rock-on-a-chip models, droplet generation and storage, suspension cell culture, monoclonal antibody production, complex cell differentiation pattern creation, phage screening, drug screening, cell detection, and cancer stem cell culture. Since its first appearance in 2018, FMold has been utilized in 50 publications in different laboratories around the world. Key advancements, current research trends, and future prospects are discussed to provide a comprehensive overview of this evolving tool.

Background/Objectives: Cancer stem cells (CSCs) represent a minor yet critical subpopulation within tumors, endowed with self-renewal and differentiation capacities, and are implicated in tumor initiation, progression, metastasis, therapeutic resistance, and recurrence. Reliable in vitro functional assays to characterize CSCs are pivotal for the development of personalized oncology strategies. This study sought to establish and validate a microfluidic device (MD) platform for the enrichment, functional assessment, and therapeutic evaluation of CSC populations derived from experimental models and primary tumor samples. Methods: Murine (LM38LP) and human (BPR6) breast cancer cell lines were cultured within MDs to promote sphere formation. CSC enrichment was confirmed through the expression analysis of pluripotency-associated genes (Oct4, Sox2, Nanog, and CD44) by quantitative PCR (qPCR) and immunofluorescence. Sphere number, size, and gene expression profiles were quantitatively assessed before (control) and after chemotherapeutic exposure. To validate the MD platform against conventional scale, parallel experiments were performed in 12 well plates. To extend translational relevance, three primary canine tumor samples (solid thyroid carcinoma, simple tubular carcinoma, and reactive lymph node) were mechanically disaggregated and processed within MDs for CSC characterization. Results: The MD platform enabled the consistent enrichment of CSC populations, showing significant modulation of sphere growth parameters and stemness marker expression following chemotherapeutic treatment. Beyond its comparability with conventional culture, the MD also supported immunofluorescence staining and allowed real-time monitoring of individual cell growth. Sphere formation efficiency (SFE) and CSC marker expression were similarly demonstrated in primary veterinary tumor cultures, highlighting the device’s cross-species applicability. Conclusions: Microfluidic-based sphere assays represent a robust, reproducible, and scalable platform for the functional interrogation of CSC dynamics and therapeutic responses. This methodology holds great promise for advancing CSC-targeted therapies and supporting personalized oncology in both human and veterinary settings.

Treatment of cancer patients with autologous T-cells expressing a chimeric antigen receptor (CAR) is one of the most promising therapeutic modalities for hematological malignancy treatment. For this treatment, primary T-cell expansion is needed. Microfluidic technologies can be used to better understand T-cell activation and proliferation. Microfluidics have had a meaningful impact in the way experimental biology and biomedical research are approached in general. Furthermore, microfluidic technology allows the generation of large amounts of data and enables the use of image processing for analysis. However, one of the major technical hurdles involved in growing suspension cells under microfluidic conditions is their immobilization, to avoid washing them out of the microfluidic chip during medium renewal. In this work, we use a multilevel microfluidic chip to successfully capture and immobilize suspension cells. Jurkat cells and T-cells are isolated through traps to microscopically track their development and proliferation after activation over a period of 8 days. The T-cell area of four independent microchannels was compared and there is no statistically significant difference between them (ANOVA p-value = 0.976). These multilevel microfluidic chips provide a new method of studying T-cell activation.

Sphere formation assay is an accepted cancer stem cell (CSC) enrichment method. CSCs play a crucial role in chemoresistance and cancer recurrence. Therefore, CSC growth is studied in plates and microdevices to develop prediction chemotherapy assays in cancer. As counting spheres cultured in devices is laborious, time-consuming, and operator-dependent, a computational program called the Automatic Quantification of Spheres Algorithm (ASQA) that detects, identifies, counts, and measures spheres automatically was developed. The algorithm and manual counts were compared, and there was no statistically significant difference (p = 0.167). The performance of the AQSA is better when the input image has a uniform background, whereas, with a nonuniform background, artifacts can be interpreted as spheres according to image characteristics. The areas of spheres derived from LN229 cells and CSCs from primary cultures were measured. For images with one sphere, area measurements obtained with the AQSA and SpheroidJ were compared, and there was no statistically significant difference between them (p = 0.173). Notably, the AQSA detects more than one sphere, compared to other approaches available in the literature, and computes the sphere area automatically, which enables the observation of treatment response in the sphere derived from the human glioblastoma LN229 cell line. In addition, the algorithm identifies spheres with numbers to identify each one over time. The AQSA analyzes many images in 0.3 s per image with a low computational cost, enabling laboratories from developing countries to perform sphere counts and area measurements without needing a powerful computer. Consequently, it can be a useful tool for automated CSC quantification from cancer cell lines, and it can be adjusted to quantify CSCs from primary culture cells. CSC-derived sphere detection is highly relevant as it avoids expensive treatments and unnecessary toxicity.

Microfluidic models have become essential instruments for studying enhanced oil recovery techniques through fluid and chemical injection into micromodels to observe interactions with pore structures and resident fluids. The widespread use of cost-effective lab-on-a-chip devices, known for efficient data extraction and minimal reagent usage, has driven demand for efficient data management methods crucial for high-performance data and image analyses. This article introduces a semiautomatic method for calculating oil recovery in polymeric nanofluid flooding experiments based on the background subtraction (BSEO). It employs the background subtraction technique, generating a foreground binary mask to detect injected fluids represented as pixel areas. The pixel difference is then compared to a threshold value to determine whether the given pixel is foreground or background. Moreover, the proposed method compares its performance with two other representative methods: the ground truth (manual segmentation) and Fiji-ImageJ software. The experiments yielded promising results. Low values of mean-squared error (MSE), mean absolute error (MAE), and root-mean-squared error (RMSE) indicate minimal prediction errors, while a substantial coefficient of determination ( R 2 ) of 98% highlights the strong correlation between the method’s predictions and the observed outcomes. In conclusion, the presented method emphasizes the viability of BSEO as a robust alternative, offering the advantages of reduced computational resource usage and faster processing times.

Gene editing tools have triggered a revolutionary transformation in the realms of cellular and molecular physiology, serving as a fundamental cornerstone for the evolution of disease models and assays in cell culture reactions, marked by various enhancements. Concurrently, microfluidics has emerged over recent decades as a versatile technology capable of elevating performance and reducing costs in daily experiments across diverse scientific disciplines, with a pronounced impact on cell biology. The amalgamation of these groundbreaking techniques holds the potential to amplify the generation of stable cell lines and the production of extracellular matrix hydrogels. These hydrogels, assuming a pivotal role in isolating cells at the single-cell level, facilitate a myriad of analyses. This study presents a novel method that seamlessly integrates CRISPR-Cas9 gene editing techniques with single-cell isolation methods in induced pluripotent stem cell (hiPSC) lines, utilizing the combined power of droplets and hydrogels. This innovative approach is designed to optimize clonal selection, thereby concurrently reducing costs and the time required for generating a stable genetically modified cell line. By bridging the advancements in gene editing and microfluidic technologies, our approach not only holds significant promise for the development of disease models and assays but also addresses the crucial need for efficient single-cell isolation. This integration contributes to streamlining processes, making it a transformative method with implications for enhancing the efficiency and cost-effectiveness of stable cell line generation. As we navigate the intersection of gene editing and microfluidics, our study marks a significant stride toward innovative methodologies in the dynamic landscape of cellular and molecular physiology research.