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Towards Personalized Medicine: Microdevice-Assisted Evaluation of Cancer Stem Cell Dynamics and Treatment Response
(MDPI AG, 2025-06-10)
Eduardo Imanol Agüero
;
Silvia María Gómez López
;
Ana Belén Peñaherrera-Pazmiño
;
Matías Tellado
;
Maximiliano Sebastián Pérez
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.
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Photopolymer Flexographic Printing Plate Mold for PDMS Microfluidic Manufacture
(MDPI AG, 2025-06-20)
Ana Belén Peñaherrera Pazmiño
;
Gustavo Iván Rosero
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Maximiliano Pérez
;
Betiana Lerner
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.
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AQSA—Algorithm for Automatic Quantification of Spheres Derived from Cancer Cells in Microfluidic Devices
(MDPI AG, 2024-11-20)
PEÑAHERRERA PAZMIÑO, ANA BELÉN
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Ramiro Fernando Isa-Jara
;
Elsa Hincapié-Arias
;
Silvia Gómez
;
Denise Belgorosky
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.

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