The A549 cell line has become a cornerstone in biomedical research, particularly in cancer studies and serves as a critical tool in cytotoxicity studies and drug screening where it is used to evaluate the impact of pharmaceutical compounds on cellular viability. One of the most widely adopted methods for viability assessment, which is also used in evaluating drug cytotoxicity, is the resazurin-based assay. This assay exploits the ability of living cells to convert resazurin into fluorescent resorufin, providing a reliable indicator of metabolic activity. By measuring this conversion, cell viability can be estimated. Resazurin assay is extensively used for evaluating cytotoxic effects on various cell lines, including A549 cells, thereby bridging the gap between in vitro experimentation and drug development. However, frequent data inconsistencies in pre-clinical drug screening highlight the critical need for standardization to ensure reliability and reproducibility. This manuscript addresses these challenges by describing the optimization of resazurin-based viability assays for A549 cells in both 2D cultures and 3D fibrin gel models. By optimizing this test, the study aims to enhance the reliability of cytotoxicity results and introduces a new standard operating procedure, thus providing consistent results with minimal measurement uncertainty. This standardization is crucial for advancing drug screening and ensuring robust research findings.

Fibrin gel, a protein-based polymer naturally generated during coagulation, has garnered attention in the biomedical field for applications such as fibrin glue, due to its specific physical and biological properties. Despite it, low mechanical strength and rapid degradation limited its utilization for biomedical applications. This study presents a reproducible protocol for the synthesis of pure fibrin hydrogels, aimed at achieving predictable structural properties through the precise calibration of fibrinogen and thrombin concentrations. By examining the mechanical and morphological characteristics, as well as the relationship between reagent concentrations and structural integrity, this research assesses impacts on swelling behavior, water absorption, and overall stability. Through a comprehensive analytical approach, we identified an optimal formulation, specifically 2.25 mg/mL fibrinogen and 1.375 U/mL thrombin, that effectively balances structural integrity with high cytocompatibility. The results demonstrate that this calibrated approach ensures high procedural reproducibility and a well-defined hydrogel architecture without the need for exogenous chemical cross-linkers. This work provides a robust methodological framework to overcome the common lack of reproducibility in fibrin-based hydrogel studies, positioning these materials as highly reliable candidates for advanced 3D in vitro models and biomedical applications.

Accurate and comparable quantification of somatic mutations is essential for precision oncology, as clinical decision-making increasingly relies on the quantification of molecular biomarkers. Despite major technological advances, inter-laboratory variability and the lack of metrological traceability remain significant barriers to harmonization and confidence in mutation testing results. Reference Measurement Procedures (RMPs) represent a critical framework to address these challenges by anchoring molecular measurements to common quantitative standards. Here, we describe the development and validation of a candidate RMP for the detection and quantification of the clinically relevant NRAS p.Q61R mutation using digital PCR (dPCR). The assay was systematically optimized to maximize specificity and minimize cross-reactivity between wild-type and mutant alleles. Analytical characterization demonstrated excellent linearity across a broad range of variant allele frequencies (vAF), with a limit of detection of 0.1 %. Precision studies performed on commercially available circulating tumour DNA reference materials (RM) showed good repeatability and intermediate precision, while a full measurement uncertainty budget confirmed the robustness of the approach. Comparison with a commercial dPCR assay provided independent support for assay comparability and consistent vAF estimates across the investigated range. Preliminary inter-laboratory assessment supported transferability of the candidate RMP and comparability of the resulting measurements. Overall, this work establishes a metrologically characterized and transferable dPCR-based RMP for NRAS p.Q61R quantification. Its implementation can support the harmonization of molecular measurements, the value assignment of RM, and the alignment of routine and secondary methods, thereby strengthening the reliability of quantitative biomarker assessment in precision oncology.

Hydrogels represent a unique class of materials characterized by their three-dimensional, hydrophilic polymer networks, which enable them to absorb and retain large quantities of aqueous solutions. While the literature is abundant with excellent works on the classification, preparation, and applications of hydrogels, a notable lack remains in comprehensive studies focused on their characterization. This gap contributes to heterogeneous results and conflicting fields. In this context, this review aims to provide a detailed and systematic overview of hydrogel characterization techniques. It begins with an exploration of physicochemical, morphological, and mechanical properties, followed by an examination of distinctive hydrogel features, biocompatibility, and application-based properties, particularly for testing applications within the biomedical field. By highlighting the significance of various characterization methods, this review seeks to enhance the understanding of hydrogels and guide future research efforts, ultimately facilitating the development of more effective hydrogel-based materials for several applications.