Over the past decade, the development of three-dimensional (3D) models has increased exponentially, facilitating the unravelling of fundamental and essential cellular mechanisms by which cells communicate with each other, assemble into tissues and organs and respond to biochemical and biophysical stimuli under both physiological and pathological conditions. This section presents a concise overview of the most recent updates on the significant contribution of different types of 3D cell cultures including spheroids, organoids and organ-on-chip and bio-printed tissues in advancing our understanding of cellular and molecular mechanisms. The case studies presented include the 3D cultures of breast cancer (BC), endometriosis, the liver microenvironment and infections. In BC, the establishment of 3D culture models has permitted the visualization of the role of cancer-associated fibroblasts in the delivery of exosomes, as well as the significance of the physical properties of the extracellular matrix in promoting cell proliferation and invasion. This approach has also become a valuable tool in gaining insight into general and specific mechanisms of drug resistance. Given the considerable heterogeneity of endometriosis, 3D models offer a more accurate representation of the in vivo microenvironment, thereby facilitating the identification and translation of novel targeted therapeutic strategies. The advantages provided by 3D models of the hepatic environment, in conjunction with the high throughput characterizing various platforms, have enabled the elucidation of complex molecular mechanisms underlying various threatening hepatic diseases. A limited number of 3D models for gut and skin infections have been developed. However, a more profound comprehension of the spatial and temporal interactions between microbes, the host and their environment may facilitate the advancement of in vitro, ex vivo and in vivo disease models. Additionally, it may pave the way for the development of novel therapeutic approaches in diverse research fields. The interested reader will also find concluding remarks on the challenges and prospects of using 3D cell cultures for discovering cellular and molecular mechanisms in the research areas covered in this review.

Thermoplastic polyurethane (TPU) is a polymer used in a variety of fields, including medical applications. Here, we aimed to verify if the brush and bar coater deposition techniques did not alter TPU properties. The topography of the TPU-modified surfaces was studied via AFM demonstrating no significant differences between brush and bar coater-modified surfaces, compared to the un-modified TPU (TPU Film). The effect of the surfaces on planktonic bacteria, evaluated by MTT assay, demonstrated their anti-adhesive effect on E. coli, while the bar coater significantly reduced staphylococcal planktonic adhesion and both bacterial biofilms compared to other samples. Interestingly, Pearson’s R coefficient analysis showed that R_a roughness and Haralick’s correlation feature were trend predictors for planktonic bacterial cells adhesion. The surface adhesion property was evaluated against NIH-3T3 murine fibroblasts by MTT and against human fibrinogen and human platelet-rich plasma by ELISA and LDH assay, respectively. An indirect cytotoxicity experiment against NIH-3T3 confirmed the biocompatibility of the TPUs. Overall, the results indicated that the deposition techniques did not alter the antibacterial and anti-adhesive surface properties of modified TPU compared to un-modified TPU, nor its bio- and hemocompatibility, confirming the suitability of TPU brush and bar coater films in the biomedical and pharmaceutical fields.

Calcium phosphate glasses (CPGs) are acquiring great importance in the biomedical field because of their thermomechanical and bioresorbable properties. In this study, optically transparent copper (1 mol %)-doped calcium phosphate glasses (CPGs_Cu) were prepared through the melt-quenching method, and their biocompatibility and antibacterial and antiviral properties were evaluated and compared with undoped CPGs. Biocompatibility was evaluated on murine fibroblast NIH-3T3 cells as a preliminary study of cytocompatibility. The in vitro tests were performed through indirect and direct cytotoxicity analyses by MTT and Alamar Blue assays and supported by electron microscopy observations. Microbiological analyses were performed against the most common Gram-negative and Gram-positive pathogens that cause nosocomial infections: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and the methicillin-resistant Staphylococcus aureus strain. In addition, the bioglass samples were exposed to SARS-CoV-2 to assess their effects on viral survival. The obtained results assessed the biocompatibility of both bioglass types and their ability to reduce the viral load and trap the virus. In addition, Cu²⁺-doped bioglass was found to be antibacterial despite its low content (1 mol %) of copper, making this a promising candidate material for biomedical applications, e.g., surgery probes, drug delivery, and photodynamic therapy.

Human microbiota is increasingly considered to shape health and disease, drawing interest of pharma and biotech industries in advanced models of in vitro human microbiome to streamline drug development. In this context, Universal‐Bac ³ Gel represents a new generation of 3D biomaterials designed to mimic the properties of human mucus and biofilm features, including micro‐gradients that replicate the heterogeneous environments colonized by microorganisms in the human body. To evaluate the suitability of Universal‐Bac ³ Gel for studying clinically relevant species in antimicrobial resistance, the so‐called ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae ) were cultured within this 3D environment. Bacterial growth was monitored at 24‐ and 48‐h post‐inoculation via spot plating, while viability, spatial distribution, and organization were assessed by confocal laser scanning microscopy. All ESKAPE strains successfully grew throughout the structure of Universal‐Bac ³ Gel. Distinct 3D biofilm architectures were observed across species, ranging from diffuse colonization to compact microcolony formation, in agreement with species‐specific biofilm patterns. Ciprofloxacin susceptibility assays revealed reduced susceptibility of bacteria cultured within Universal‐Bac ³ Gel compared with their planktonic counterparts, supporting the development of biofilm‐associated tolerance phenotypes. Consistent with these findings, crystal violet staining confirmed the accumulation of biofilm‐associated biomass within the hydrogel. Notably, the platform's ready‐to‐use 96‐well format allowed direct comparison of these high‐priority pathogens under standardized conditions, highlighting species‐specific biofilm traits that would be difficult to discern in conventional two‐dimensional culture systems. This work highlights the versatility of Universal‐Bac ³ Gel as a biofilm‐relevant in vitro platform for studying pathogen colonization, biofilm development and antimicrobial susceptibility under controlled conditions.

Human microbiota is increasingly considered to shape health and disease, drawing interest of pharma and biotech industries in advanced models of in vitro human microbiome to streamline drug development. In this context, Universal-Bac ³ Gel ^® represents a new generation of 3D biomaterials designed to mimic the properties of human mucus and biofilm features, including micro-gradients that replicate the heterogeneous environments colonized by microorganisms in the human body. To evaluate the suitability of Universal-Bac ³ Gel ^® for studying clinically relevant species in antimicrobial resistance, the so-called ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae ) were cultured within this 3D environment. Bacterial growth was monitored at 24- and 48-hours post-inoculation via spot plating, while viability, distribution and organization were assessed using confocal laser scanning microscopy. All ESKAPE strains successfully grew throughout the structure of Universal-Bac ³ Gel ^® . Distinct 3D biofilm architectures were observed across species, ranging from diffuse colonization to compact microcolony formation, in agreement with species-specific biofilm patterns. Notably, the platform’s ready-to-use 96-well format allowed direct comparison of these high-priority pathogens under identical conditions, highlighting species-specific biofilm traits that would be difficult to discern in traditional 2D or animal models. This work highlights the versatility of Universal-Bac ³ Gel ^® as a platform for studying pathogen colonization under a biofilm-relevant environment and underscores its potential for translational applications, including antimicrobial drug testing, personalized medicine, and reduction of animal model use in preclinical research.