The design of polymeric systems with intrinsically integrated imaging and responsive functionalities represents a promising strategy to address the complexity of aggressive tumors such as glioblastoma multiforme (GBM). We report a class of star-shaped zinc phthalocyanine-poly(L-glutamic acid) (ZnPc-PGA) nanocarriers in which the photoactive ZnPc core serves as a macroinitiator for the N-carboxyanhydride ring-opening polymerization, enabling control over chain length, architecture, and stoichiometry. ZnPc-PGA nanocarriers exhibited near-infrared absorption, enabling their implementation as imaging agents and photosensitizers for photodynamic therapy. Physicochemical characterization of ZnPc-PGA nanocarriers revealed chain-length- and concentration-dependent supramolecular assembly. ZnPc-PGA nanocarriers demonstrated lysosomal uptake/light-activated cytotoxicity in GBM cells, while evaluation in the GBM-on-a-chip platform confirmed oxygen-dependent therapeutic outcomes. Proteomic profiling revealed a fibrinogen-, cytoskeletal-, and olfactory protein-enriched corona, providing insight into potential biointerface interactions. Post-polymerization conjugation of ZnPc-PGA nanocarriers with paclitaxel facilitated multimodal therapy with enhanced efficacy in GBM cells, while integrating ZnPc-PGA nanocarriers into a hyaluronic acid-based hydrogel enabled intranasal delivery and resulted in brain accumulation. Overall, ZnPc-PGA represents a modular nanoplatform and underscores the role of polymer architecture in dictating behavior in complex biological environments, with potential applicability in the design of multifunctional systems for challenging diseases such as GBM.

The blood-brain barrier remains a critical challenge in CNS drug development. This work presents the preliminary development and characterization of a microfluidic BBB-on-a-chip model, comparing monoculture and triculture configurations against traditional Transwell systems. Additionally, real time monitoring of fundamental metabolites is being implemented.

Se presenta un marco que combina experimentos microfluídicos con modelado basado en agentes (ABM) para caracterizar la dinámica inmune en el microambiente del glioblastoma (GB), estudiando el efecto de los quimioatrayentes tumorales y las propiedades de la matriz extracelular (ECM) en la migración de las células mononucleares de sangre periférica (PBMCs).

Tubuloids offer a powerful tool for advancing renal research and improving patient care. To facilitate this, we have established a robust biobank of 40 adult human kidney organoids derived from fresh or frozen biopsies. These organoids exhibit diverse morphologies, distal and proximal tubules, closely resembling their tissue of origin. We have extensively characterized these organoids using a combination of techniques, including histological staining, immunofluorescence, gene expression analysis (qPCR and RNA-seq), and genetic profiling (STRs). This comprehensive characterization ensures the quality and reliability of the organoids for downstream applications. Molecular analysis revealed the expression of genes characteristic of various kidney cell types. Furthermore, we have successfully cultured these organoids within microfluidic chips, demonstrating their compatibility with advanced culture systems. This platform of well-characterized, living human kidney organoids from fresh and frozen tissues provides a valuable resource for a wide range of biomedical research, including drug screening, disease modeling, and personalized medicine.

Metastasis, the leading cause of cancer-related mortality, is a complex process involving tumor cell detachment from the primary site, survival and dissemination through the circulation, and colonization of distant organs. At each stage, tumor cells face adaptive pressures from successive biological and biomechanical challenges in the local microenvironment, which collectively shape their progression. Traditional in vitro models often fail to replicate these dynamics, while animal models are limited by species differences and restricted real-time monitoring. Microphysiological systems (MPS) have emerged as powerful tools to address these limitations, delivering physiologically relevant cues and precise experimental control to recapitulate step-specific metastatic contexts. This review outlines recent advances in MPS designs for modeling critical hallmarks of metastasis, beginning with matrix interactions, stromal cells, and mechanical forces from the tumor microenvironment that drive epithelial-mesenchymal transition and invasion. The discussion then transitions to MPS that reproduce vascular physiology during intravasation, circulation, and extravasation, and concludes with organ-specific environments for studying colonization and organotropic behavior in the final stages of metastasis. Additionally, common MPS configurations, categorized into horizontal and vertical compartmental arrangements, and strategies for integrating vascularization are explored. Together, these advances highlight the potential of MPS in elucidating metastatic mechanisms and advancing targeted therapies.

Cell microencapsulation technologies allow non-autologous implantation of therapeutic cells for sustained drug delivery purposes. The perm-selective membrane of these systems provides resistance to rupture, stablishes the upper molecular weight limit in bidirectional diffusion of molecules, and affects biocompatibility. Thus, despite being a decisive factor to succeed in terms of biosafety and therapeutic efficacy, little progress has been made in its optimization so far. Here we show that, compared to other usually used coating designs, genipin-crosslinked double poly-L-lysine (GDP) membranes are able to simultaneously improve mechanical and mass-transport properties of the microcapsules, without causing any significant increase in the foreign body response when implanted in vivo. In particular, we show that GDP membranes confer capsular integrity under high pressures, both internal and external. Furthermore, this membrane design allows for more efficient bidirectional diffusion of molecules in the 20-40 kDa range while preserving the molecular weight cut-off required for exerting an effective immunobarrier. These findings may also be useful for optimizing the membrane characteristics of multiple drug delivery systems.

Glioblastoma (GBM), the most aggressive and lethal primary brain tumor, is characterized by profound intratumoral heterogeneity and a hostile tumor microenvironment (TME) that drives immune evasion, therapeutic resistance, and relentless progression. Among its defining pathological features is the development of a hypoxic-necrotic core, long recognized as a hallmark of poor clinical outcome. This review synthesizes current insights into how hypoxia and necrosis act not merely as pathological markers, but as a spatiotemporal evolution engine of the GBM TME, driving metabolic adaptation, extracellular matrix (ECM) remodeling, and immune evasion. We examine how oxygen and nutrient deprivation activate hypoxia-inducible factors (HIFs), triggering cascades that promote angiogenesis, altered metabolism, and accumulation of immunosuppressive metabolites. These stressors also contribute to the recruitment and polarization of tumor-associated macrophages (TAMs) and neutrophils (TANs), expansion of myeloid-derived suppressor cells (MDSCs), and infiltration of regulatory T cells (Tregs), collectively creating an immune-excluded niche. Furthermore, hypoxia-induced ECM stiffening and degradation enhance tumor invasiveness while limiting immune cell access. By exploring the dynamic interplay between physicochemical stressors and immune modulation within the necrotic core, this review highlights the need for targeting the hypoxia-necrosis axis to overcome current therapeutic limitations. A deeper understanding of these processes will be crucial for the development of precision-targeted therapies in this highly refractory malignancy.

Glioblastoma (GBM) is a highly malignant brain tumor with a poor survival prognosis of 12–15 months despite current therapeutic strategies. Diagnosing GBM is challenging, often requiring invasive techniques such as tissue biopsy and imaging methods that can provide inconclusive results. In this regard, liquid biopsy represents a promising alternative, providing tumor‐derived information from less invasive sources such as blood or cerebrospinal fluid. However, the typically low concentrations of these biomarkers pose challenges for traditional detection techniques, limiting their sensitivity and specificity. Recent advances in microfluidics offer a potential solution by enhancing the isolation and detection of tumor‐derived cells and molecules, thus improving their detectability. This review discusses the latest progress in microfluidic‐based liquid biopsy systems for glioblastoma, laying the basis for future diagnostic practices that are less invasive and more accurate. As these technologies evolve, they hold the potential to transform GBM diagnosis and monitoring, ultimately improving patient outcomes.

The immune system plays a crucial role in shaping the glioblastoma tumor microenvironment, characterized by its complexity and dynamic interactions. Understanding the tumor-immune crosstalk is essential for advancing cancer research and therapeutic development. Here, a novel multicompartment, barrier-free microfluidic device is presented that overcomes the limitations of existing models by enabling direct tumor-immune interactions without physical barriers, preserving natural immune cell infiltration. This platform supports the independent and simultaneous culture of tumor and immune cells, replicating the healthy-tumoral stroma interface, and allows investigating the effect of matrix stiffness and chemotherapy on both populations. The findings reveal that increased collagen concentration promotes tumor invasiveness while impairing immune cell infiltration. Additionally, temozolomide treatment reduces immune cell motility but enhances anti-tumor immune responses. These insights highlight the critical roles of extracellular matrix mechanics and chemotherapy in tumor progression and immune modulation, establishing this device as a powerful tool for studying glioblastoma-immune dynamics and evaluating therapeutic strategies.

In the pursuit of achieving a more realistic in vitro simulation of human biological tissues, microfluidics has emerged as a promising technology. Organ-on-a-chip (OoC) devices, a product of this technology, contain miniature tissues within microfluidic chips, aiming to closely mimic the in vivo environment. However, a notable drawback is the presence of inert material between compartments, hindering complete contact between biological tissues. Current membranes, often made of PDMS or plastic materials, prevent full interaction between cell types and nutrients. Furthermore, their non-physiological mechanical properties and composition may induce unexpected cell responses. Therefore, it is essential to minimize the contact area between cells and the inert materials while simultaneously maximizing the direct contact between cells and matrices in different compartments. The main objective of this work is to minimize inert materials within the microfluidic chip while preserving proper cellular distribution. Two microfluidic devices were designed, each with a specific focus on maximizing direct cell-matrix or cell-cell interactions. The first chip, designed to increase direct cell-cell interactions, incorporates a nylon mesh with regular pores of 150 microns. The second chip minimizes interference from inert materials, thereby aiming to increase direct cell-matrix contact. It features an inert membrane with optimized macropores of 1 mm of diameter for collagen hydrogel deposition. Biological validation of both devices has been conducted through the implementation of cell migration and cell-to-cell interaction assays, as well as the development of epithelia, from isolated cells or spheroids. This endeavor contributes to the advancement of microfluidic technology, aimed at enhancing the precision and biological relevance of in vitro simulations in pursuit of more biomimetic models.

A kidney-on-a-chip has been developed and optimised to evaluate the effects of exposure to graphene related materials under fluid flow conditions.

Early detection of tumours remains a significant challenge due to their invasive nature and the limitations of current monitoring techniques. Liquid biopsies have emerged as a minimally invasive diagnostic approach, wherein volatile organic compounds (VOCs) show potential as compelling candidates. However, distinguishing tumour-specific VOCs is difficult due to the presence of gases from non-tumour tissues and environmental factors. Therefore, it is essential to develop preclinical models that accurately mimic the intricate tumour microenvironment to induce cellular metabolic changes and secretion of tumour-associated VOCs. In this study, a microfluidic device was used to recreate the ischaemic environment within solid tumours for the detection of tumour-derived VOCs. The system represents a significant advance in understanding the role of VOCs as biomarkers for early tumour detection and holds the potential to improve patient prognosis; particularly for inaccessible and rapidly progressing tumours such as glioblastoma.

Nephrotoxicity stands as one of the most limiting effects in the development and validation of new drugs. The kidney, among the organs evaluated in toxicity assessments, has a higher susceptibility, with nephrotoxic potential frequently evading detection until late in clinical trials. Traditional cell culture, which has been widely used for decades, does not recapitulate the structure and complexity of the native tissue, which can affect cell function, and the response to cytotoxins does not resemble what occurs in the kidney. In the current study, we aimed to address these challenges by creating in vitro kidney models that faithfully biomimic the dynamics of the renal proximal tubule, using the well-established RPTEC/TERT1 cell line. For doing so, two models were developed, one recreating tubule-like structures (2.5D model) and the other using microfluidic technology (kidney-on-a-chip). The 2.5D model allowed tubular structures to be generated in the absence of hydrogels, and the kidney-on-a-chip model allowed shear stress to be applied to the cell culture, which is a physiological stimulus in the renal tissue. After characterization of both models, different nephrotoxic compounds such as cisplatin, tacrolimus, and daunorubicin were used to study cell responses after treatment. The developed models in our study could be a valuable tool for pre-clinical nephrotoxic testing of drugs and new compounds.

Innovative microfluidic technology aims to create more realistic in vitro simulations of human tissues. Organ-on-a-chip (OoC) devices within microfluidic chips mimic in vivo environments but face limitations due to inert material between compartments, hindering cell interaction. Current membranes made of PDMS or plastic impede proper cell-nutrient exchange and induce unexpected cell responses. This work focuses on minimizing inert materials while maximizing cell-matrix and cell-cell interactions in microfluidic chips.

Organ-on-chip (OOC) technology has recently emerged as a powerful tool to mimic physiological or pathophysiological conditions through cell culture in microfluidic devices. One of its main goals is bypassing animal testing and encouraging more personalized medicine. The recent incorporation of hydrogels as 3D scaffolds into microfluidic devices has changed biomedical research since they provide a biomimetic extracellular matrix to recreate tissue architectures. However, this technology presents some drawbacks such as the necessity for physical structures as pillars to confine these hydrogels, as well as the difficulty in reaching different shapes and patterns to create convoluted gradients or more realistic biological structures. In addition, pillars can also interfere with the fluid flow, altering the local shear forces and, therefore, modifying the mechanical environment in the OOC model. In this work, we present a methodology based on a plasma surface treatment that allows building cell culture chambers with abutment-free patterns capable of producing precise shear stress distributions. Therefore, pillarless devices with arbitrary geometries are needed to obtain more versatile, reliable, and biomimetic experimental models. Through computational simulation studies, these shear stress changes are demonstrated in different designed and fabricated geometries. To prove the versatility of this new technique, a blood-brain barrier model has been recreated, achieving an uninterrupted endothelial barrier that emulates part of the neurovascular network of the brain. Finally, we developed a new technology that could avoid the limitations mentioned above, allowing the development of biomimetic OOC models with complex and adaptable geometries, with cell-to-cell contact if required, and where fluid flow and shear stress conditions could be controlled.

Glioblastoma (GBM) is a highly malignant brain tumour characterised by limited treatment options and poor prognosis. The tumour microenvironment, particularly the central hypoxic region of the tumour, is known to play a pivotal role in GBM progression. Cells within this region adapt to hypoxia by stabilising transcription factor HIF1-α, which promotes cell proliferation, dedifferentiation and chemoresistance. In this study we sought to examine the effects of NNC-55-0396, a tetralol compound which overactivates the unfolded protein response inducing apoptosis, using the organ-on-chip technology. We identified an increased sensitivity of the hypoxic core of the chip to NNC, which correlates with decreasing levels of HIF1-α in vitro. Moreover, NNC blocks the macroautophagic process that is unleashed by hypoxia as revealed by increased levels of autophagosomal constituent LC3-II and autophagy chaperone p62/SQSTM1. The specific effects of NNC in the hypoxic microenvironment unveil additional anti-cancer abilities of this compound and further support investigations on its use in combined therapies against GBM.

A combination of human-induced pluripotent stem cells (hiPSCs) and 3D microtissue culture techniques allows the generation of models that recapitulate the cardiac microenvironment for preclinical research of new treatments. In particular, spheroids represent the simplest approach to culture cells in 3D and generate gradients of cellular access to the media, mimicking the effects of an ischemic event. However, previous models required incubation under low oxygen conditions or deprived nutrient media to recreate ischemia. Here, we describe the generation of large spheroids (i.e., larger than 500 μm diameter) that self-induce an ischemic core. Spheroids were generated by coculture of cardiomyocytes derived from hiPSCs (hiPSC-CMs) and primary human cardiac fibroblast (hCF). In the proper medium, cells formed aggregates that generated an ischemic core 2 days after seeding. Spheroids also showed spontaneous cellular reorganization after 10 days, with hiPSC-CMs located at the center and surrounded by hCFs. This led to an increase in microtissue stiffness, characterized by the implementation of a constriction assay. All in all, these phenomena are hints of the fibrotic tissue remodeling secondary to a cardiac ischemic event, thus demonstrating the suitability of these spheroids for the modeling of human cardiac ischemia and its potential application for new treatments and drug research.

Drug resistance is one of the biggest challenges in the fight against cancer. In particular, in the case of glioblastoma, the most lethal brain tumour, resistance to temozolomide (the standard of care drug for chemotherapy in this tumour) is one of the main reasons behind treatment failure and hence responsible for the poor prognosis of patients diagnosed with this disease.In this work, we combine the power of three-dimensional in vitro experiments of treated glioblastoma spheroids with mathematical models of tumour evolution and adaptation. We use a novel approach based on internal variables for modelling the acquisition of resistance to temozolomide that was observed in experiments for a group of treated spheroids. These internal variables describe the cell’s phenotypic state, which depends on the history of drug exposure and affects cell behaviour. We use model selection to determine the most parsimonious model and calibrate it to reproduce the experimental data, obtaining a high level of agreement between the in vitro and in silico outcomes. A sensitivity analysis is carried out to investigate the impact of each model parameter in the predictions. More importantly, we show how the model is useful for answering biological questions, such as what is the intrinsic adaptation mechanism, or for separating the sensitive and resistant populations. We conclude that the proposed in silico framework, in combination with experiments, can be useful to improve our understanding of the mechanisms behind drug resistance in glioblastoma and to eventually set some guidelines for the design of new treatment schemes.

Primary cilia act as cellular sensors for multiple extracellular stimuli and regulate many intracellular signaling pathways in response. Here we investigate whether the cold-shock proteins (CSPs), CIRP and RBM3, are present in the primary cilia and the physiological consequences of such a relationship. R28, an immortalized retinal precursor cell line, was stained with antibodies against CIRP, RBM3, and ciliary markers. Both CSPs were found in intimate contact with the basal body of the cilium during all stages of the cell cycle, including migrating with the centrosome during mitosis. In addition, the morphological and physiological manifestations of exposing the cells to hypothermia and shear stress were investigated. Exposure to moderately cold (32°C) temperatures, the hypothermia mimetic small molecule zr17-2, or to shear stress resulted in a significant reduction in the number and length of primary cilia. In addition, shear stress induced expression of CIRP and RBM3 in a complex pattern depending on the specific protein, flow intensity, and type of flow (laminar versus oscillatory). Flow-mediated CSP overexpression was detected by qRT-PCR and confirmed by Western blot, at least for CIRP. Furthermore, analysis of public RNA Seq databases on flow experiments confirmed an increase of CIRP and RBM3 expression following exposure to shear stress in renal cell lines. In conclusion, we found that CSPs are integral components of the centrosome and that they participate in cold and shear stress sensing.

Biomimetic 3D models emerged some decades ago to address 2D cell culture limitations in the field of replicating biological phenomena, structures or functions found in nature. The fabrication of hydrogels for cancer disease research enables the study of cell processes including growth, proliferation and migration and their 3D design is based on the encapsulation of tumoral cells within a tunable matrix. In this work, a platform of gelatin methacrylamide (GelMA)-based photocrosslinked scaffolds with embedded colorectal (HCT-116) or pancreatic (MIA PaCa-2) cancer cells is presented. Prior to cell culture, the mechanical characterization of hydrogels was assessed in terms of stiffness and swelling behavior. Modifications of the UV curing time enabled a fine tuning of the mechanical properties, which at the same time, showed susceptibility to the chemical composition and crosslinking mechanism. All scaffolds displayed excellent cytocompatibility with both tumoral cells while eliciting various cell responses depending on the microenvironment features. Individual and collective cell migration were observed for HCT-116 and MIA PaCa-2 cell lines, highlighting the ability of the colorectal cancer cells to cluster into aggregates of different sizes governed by the surrounding matrix. Additionally, metabolic activity results pointed out to the development of a more proliferative phenotype within stiffer networks. These findings confirm the suitability of the presented platform of GelMA-based hydrogels to conduct 3D cell culture experiments and explore biological processes associated with colorectal and pancreatic cancer.