The resazurin assay, also known as the Alamar Blue assay, stands as a cornerstone technique in cell biology, microbiology, and drug development. It assesses the viability of cells through the conversion of resazurin into highly fluorescent resorufin. The resulting fluorescence intensity provides a reliable estimate of viable cell numbers. Cytotoxicity assays, such as the resazurin-based method, play a crucial role in the screening of potential drug candidates and in the assessment of pharmaceutical and chemical toxicity. In recent years, inconsistencies have arisen in pharmacogenomic studies, often due to poorly optimized laboratory protocols. These inconsistencies hinder progress in understanding how substances affect cell health, leading to unreliable findings. Thus, the need for standardized and rigorously optimized protocols is evident to ensure consistent and accurate results in cytotoxicity studies. This manuscript describes a standardized procedure for optimizing resazurin-based viability assays to improve the reliability of cytotoxicity data. This optimization approach focuses on critical experimental parameters and data quality, aiming to achieve a level of measurement imprecision of less than 20%. In conclusion, to address the critical issues of reproducibility and reliability, protocol standardization, such as the one described in this manuscript, can greatly enhance the credibility of cytotoxicity studies, ultimately advancing drug safety assessments.

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.

The irreversible loss of functional cardiomyocytes (CMs) after myocardial infarction (MI) represents one major barrier to heart regeneration and functional recovery. The combination of different cell sources and different biomaterials have been investigated to generate CMs by differentiation or reprogramming approaches although at low efficiency. This critical review article discusses the role of biomaterial platforms integrating biochemical instructive cues as a tool for the effective generation of functional CMs. The report firstly introduces MI and the main cardiac regenerative medicine strategies under investigation. Then, it describes the main stem cell populations and indirect and direct reprogramming approaches for cardiac regenerative medicine. A third section discusses the main techniques for the characterization of stem cell differentiation and fibroblast reprogramming into CMs. Another section describes the main biomaterials investigated for stem cell differentiation and fibroblast reprogramming into CMs. Finally, a critical analysis of the scientific literature is presented for an efficient generation of functional CMs. The authors underline the need for biomimetic, reproducible and scalable biomaterial platforms and their integration with external physical stimuli in controlled culture microenvironments for the generation of functional CMs.

Background: In cell-based therapies, in vitro studies on biomimetic cell–scaffold constructs can facilitate the determination of the cell dose, a key factor in guaranteeing the effectiveness of the treatment. However, highly porous scaffolds do not allow a nondestructive evaluation of the cell number. Our objective was to develop a nondestructive method for human mesenchymal stem cells dose evaluation in a highly porous scaffold for bone regeneration. Materials & measurement method: Proliferation trend of human mesenchymal stem cells on Biocoral® scaffolds was measured by a resazurin-based assay here optimized for 3D cultures. The method allows to noninvasively follow the cell proliferation on biocorals over 3 weeks with very high reproducibility. Conclusion: This reliable method could be a powerful tool in cell-based therapies for cell dose determination. Stem cells regenerate damaged tissues when transplanted into the patient within matrices mimicking the tissues architecture and mechanical properties. Cell number needs to be appropriate to allow the cell survival in the new environment and to stimulate the cell differentiation into the new tissue. In vitro experiments give important hints to determine the appropriate number to transplant in the patient: in this study cells are grown on highly porous matrices for bone regeneration and their number is monitored over time by a method which does not perturb the system and which was here optimized and evaluated as highly reliable.

Cardiac fibrosis is one of the main causes of heart failure, significantly contributing to mortality. The discovery and development of effective therapies able to heal fibrotic pathological symptoms thus remain of paramount importance. Micro‐physiological systems (MPS) are recently introduced as promising platforms able to accelerate this finding. Here a 3D in vitro model of human cardiac fibrosis, named uScar, is developed by imposing a cyclic mechanical stimulation to human atrial cardiac fibroblasts (AHCFs) cultured in a 3D beating heart‐on‐chip and exploited to screen drugs and advanced therapeutics. The sole provision of a cyclic 10% uniaxial strain at 1 Hz to the microtissues is sufficient to trigger fibrotic traits, inducing a consistent fibroblast‐to‐myofibroblast transition and an enhanced expression and production of extracellular matrix (ECM) proteins. Standard of care anti‐fibrotic drugs (i.e., Pirfenidone and Tranilast) are confirmed to be efficient in preventing the onset of fibrotic traits in uScar. Conversely, the mechanical stimulation applied to the microtissues limit the ability of a miRNA therapy to directly reprogram fibroblasts into cardiomyocytes (CMs), despite its proved efficacy in 2D models. Such results demonstrate the importance of incorporating in vivo‐like stimulations to generate more representative 3D in vitro models able to predict the efficacy of therapies in patients.

The restoration of cardiac functionality after myocardial infarction represents a major clinical challenge. Recently, we found that transient transfection with microRNA combination (miRcombo: miR-1, miR-133, miR-208 and 499) is able to trigger direct reprogramming of adult human cardiac fibroblasts (AHCFs) into induced cardiomyocytes (iCMs) in vitro. However, achieving efficient direct reprogramming still remains a challenge. The aim of this study was to investigate the influence of cardiac tissue-like biochemical and biophysical stimuli on direct reprogramming efficiency. Biomatrix (BM), a cardiac-like extracellular matrix (ECM), was produced by in vitro culture of AHCFs for 21 days, followed by decellularization. In a set of experiments, AHCFs were transfected with miRcombo and then cultured for 2 weeks on the surface of uncoated and BM-coated polystyrene (PS) dishes and fibrin hydrogels (2D hydrogel) or embedded into 3D fibrin hydrogels (3D hydrogel). Cell culturing on BM-coated PS dishes and in 3D hydrogels significantly improved direct reprogramming outcomes. Biochemical and biophysical cues were then combined in 3D fibrin hydrogels containing BM (3D BM hydrogel), resulting in a synergistic effect, triggering increased CM gene and cardiac troponin T expression in miRcombo-transfected AHCFs. Hence, biomimetic 3D culture environments may improve direct reprogramming of miRcombo-transfected AHCFs into iCMs, deserving further study.