Neuro-Immunomodulatory Potential of Nanoenabled 4D Bioprinted Microtissue for Cartilage Tissue Engineering.

Cartilage defects trigger post-traumatic inflammation, leading to a catabolic metabolism in chondrocytes and exacerbating cartilage degradation. Current treatments aim to relieve pain but fail to target the inflammatory process underlying osteoarthritis progression. Here, we 4D-bioprint a human cartilage microtissue (HCM) nanoenabled with ibuprofen-loaded poly(lactic-co-glycolic acid) nanoparticles (ibu-PLGA NPs) to locally mitigate inflammation and impair nerve sprouting. Under in vitro inflamed environment, the nanoenabled HCM exhibits chondroprotective potential by decreasing the interleukin (IL)1ß and IL6 release, while sustaining extracellular matrix (ECM) production. In vivo assessments utilizing the air pouch mouse model affirmed the nanoenabled HCM non-immunogenicity. Nanoenabled HCM-derived secretome did not elicit a systemic immune response and decreases locally the recruitment of mature dendritic cells and the secretion of multiple inflammatory mediators and matrix metalloproteinases, when compared to inflamed HCM condition. Notably, the nanoenabled HCM secretome had no impact on the innervation profile of the skin above the pouch cavity, suggesting a potential to impede nerve growth. Overall, HCM nanoenabled with ibu-PLGA NPs emerges as a potent strategy to mitigate inflammation and protect ECM without triggering nerve growth, introducing an innovative and promising approach in the cartilage tissue engineering field. This article is protected by copyright. All rights reserved.

Transforming the Niche: The Emerging Role of Extracellular Vesicles in Acute Myeloid Leukaemia Progression.

Acute myeloid leukaemia (AML) management remains a significant challenge in oncology due to its low survival rates and high post-treatment relapse rates, mainly attributed to treatment-resistant leukaemic stem cells (LSCs) residing in bone marrow (BM) niches. This review offers an in-depth analysis of AML progression, highlighting the pivotal role of extracellular vesicles (EVs) in the dynamic remodelling of BM niche intercellular communication. We explore recent advancements elucidating the mechanisms through which EVs facilitate complex crosstalk, effectively promoting AML hallmarks and drug resistance. Adopting a temporal view, we chart the evolving landscape of EV-mediated interactions within the AML niche, underscoring the transformative potential of these insights for therapeutic intervention. Furthermore, the review discusses the emerging understanding of endothelial cell subsets' impact across BM niches in shaping AML disease progression, adding another layer of complexity to the disease progression and treatment resistance. We highlight the potential of cutting-edge methodologies, such as organ-on-chip (OoC) and single-EV analysis technologies, to provide unprecedented insights into AML-niche interactions in a human setting. Leveraging accumulated insights into AML EV signalling to reconfigure BM niches and pioneer novel approaches to decipher the EV signalling networks that fuel AML within the human context could revolutionise the development of niche-targeted therapy for leukaemia eradication.

One-Pot Microfluidics to Engineer Chitosan Nanoparticles Conjugated with Antimicrobial Peptides Using "Photoclick" Chemistry: Validation Using the Gastric Bacterium Helicobacter pylori.

Surface bioconjugation of antimicrobial peptides (AMP) onto nanoparticles (AMP-NP) is a complex, multistep, and time-consuming task. Herein, a microfluidic system for the one-pot production of AMP-NP was developed. Norbornene-modified chitosan was used for NP production (NorChit-NP), and thiolated-AMP was grafted on their surface via thiol-norbornene "photoclick" chemistry over exposure of two parallel UV LEDs. The MSI-78A was the AMP selected due to its high activity against a high priority (level 2) antibiotic-resistant gastric pathogen: Helicobacter pylori (H. pylori). AMP-NP (113 ± 43 nm; zeta potential 14.3 ± 7 mV) were stable in gastric settings without a cross-linker (up to 5 days in pH 1.2) and bactericidal against two highly pathogenic H. pylori strains (1011 NP/mL with 96 µg/mL MSI-78A). Eradication was faster for H. pylori 26695 (30 min) than for H. pylori J99 (24 h), which was explained by the lower minimum bactericidal concentration of soluble MSI-78A for H. pylori 26695 (32 µg/mL) than for H. pylori J99 (128 µg/mL). AMP-NP was bactericidal by inducing H. pylori cell membrane alterations, intracellular reorganization, generation of extracellular vesicles, and leakage of cytoplasmic contents (transmission electron microscopy). Moreover, NP were not cytotoxic against two gastric cell lines (AGS and MKN74, ATCC) at bactericidal concentrations. Overall, the designed microfluidic setup is a greener, simpler, and faster approach than the conventional methods to obtain AMP-NP. This technology can be further explored for the bioconjugation of other thiolated-compounds.

Osteoclast-derived extracellular vesicles are implicated in sensory neurons sprouting through the activation of epidermal growth factor signaling.

BACKGROUND: Different pathologies, affecting the skeletal system, were reported to display altered bone and/or cartilage innervation profiles leading to the deregulation of the tissue homeostasis. The patterning of peripheral innervation is achieved through the tissue-specific expression of attractive or repulsive axonal guidance cues in specific space and time frames. During the last decade, emerging findings attributed to the extracellular vesicles (EV) trading a central role in peripheral tissue innervation. However, to date, the contribution of EV in controlling bone innervation is totally unknown. RESULTS: Here we show that sensory neurons outgrowth induced by the bone resorbing cells-osteoclasts-is promoted by osteoclast-derived EV. The EV induced axonal growth is achieved by targeting epidermal growth factor receptor (EGFR)/ErbB2 signaling/protein kinase C phosphorylation in sensory neurons. In addition, our data also indicate that osteoclasts promote sensory neurons electrophysiological activity reflecting a possible pathway in nerve sensitization in the bone microenvironment, however this effect is EV independent. CONCLUSIONS: Overall, these results identify a new mechanism of sensory bone innervation regulation and shed the light on the role of osteoclast-derived EV in shaping/guiding bone sensory innervation. These findings provide opportunities for exploitation of osteoclast-derived EV based strategies to prevent and/or mitigate pathological uncontrolled bone innervation.

Cutting-Edge Technologies for Inflamed Joints on Chip: How Close Are We?

Osteoarthritis (OA) is a painful and disabling musculoskeletal disorder, with a large impact on the global population, resulting in several limitations on daily activities. In OA, inflammation is frequent and mainly controlled through inflammatory cytokines released by immune cells. These outbalanced inflammatory cytokines cause cartilage extracellular matrix (ECM) degradation and possible growth of neuronal fibers into subchondral bone triggering pain. Even though pain is the major symptom of musculoskeletal diseases, there are still no effective treatments to counteract it and the mechanisms behind these pathologies are not fully understood. Thus, there is an urgent need to establish reliable models for assessing the molecular mechanisms and consequently new therapeutic targets. Models have been established to support this research field by providing reliable tools to replicate the joint tissue in vitro. Studies firstly started with simple 2D culture setups, followed by 3D culture focusing mainly on cell-cell interactions to mimic healthy and inflamed cartilage. Cellular approaches were improved by scaffold-based strategies to enhance cell-matrix interactions as well as contribute to developing mechanically more stable in vitro models. The progression of the cartilage tissue engineering would then profit from the integration of 3D bioprinting technologies as these provide 3D constructs with versatile structural arrangements of the 3D constructs. The upgrade of the available tools with dynamic conditions was then achieved using bioreactors and fluid systems. Finally, the organ-on-a-chip encloses all the state of the art on cartilage tissue engineering by incorporation of different microenvironments, cells and stimuli and pave the way to potentially simulate crucial biological, chemical, and mechanical features of arthritic joint. In this review, we describe the several available tools ranging from simple cartilage pellets to complex organ-on-a-chip platforms, including 3D tissue-engineered constructs and bioprinting tools. Moreover, we provide a fruitful discussion on the possible upgrades to enhance the in vitro systems making them more robust regarding the physiological and pathological modeling of the joint tissue/OA.

A metastasis-on-a-chip approach to explore the sympathetic modulation of breast cancer bone metastasis.

Organ-on-a-chip models have emerged as a powerful tool to model cancer metastasis and to decipher specific crosstalk between cancer cells and relevant regulators of this particular niche. Recently, the sympathetic nervous system (SNS) was proposed as an important modulator of breast cancer bone metastasis. However, epidemiological studies concerning the benefits of the SNS targeting drugs on breast cancer survival and recurrence remain controversial. Thus, the role of SNS signaling over bone metastatic cancer cellular processes still requires further clarification. Herein, we present a novel humanized organ-on-a-chip model recapitulating neuro-breast cancer crosstalk in a bone metastatic context. We developed and validated an innovative three-dimensional printing based multi-compartment microfluidic platform, allowing both selective and dynamic multicellular paracrine signaling between sympathetic neurons, bone tropic breast cancer cells and osteoclasts. The selective multicellular crosstalk in combination with biochemical, microscopic and proteomic profiling show that synergistic paracrine signaling from sympathetic neurons and osteoclasts increase breast cancer aggressiveness demonstrated by augmented levels of pro-inflammatory cytokines (e.g. interleukin-6 and macrophage inflammatory protein 1α). Overall, this work introduced a novel and versatile platform that could potentially be used to unravel new mechanisms involved in intracellular communication at the bone metastatic niche.

Calcium Signalling in Breast Cancer Associated Bone Pain.

Calcium (Ca2+) is involved as a signalling mediator in a broad variety of physiological processes. Some of the fastest responses in human body like neuronal action potential firing, to the slowest gene transcriptional regulation processes are controlled by pathways involving calcium signalling. Under pathological conditions these mechanisms are also involved in tumoral cells reprogramming, resulting in the altered expression of genes associated with cell proliferation, metastatisation and homing to the secondary metastatic site. On the other hand, calcium exerts a central function in nociception, from cues sensing in distal neurons, to signal modulation and interpretation in the central nervous system leading, in pathological conditions, to hyperalgesia, allodynia and pain chronicization. It is well known the relationship between cancer and pain when tumoral metastatic cells settle in the bones, especially in late breast cancer stage, where they alter the bone micro-environment leading to bone lesions and resulting in pain refractory to the conventional analgesic therapies. The purpose of this review is to address the Ca2+ signalling mechanisms involved in cancer cell metastatisation as well as the function of the same signalling tools in pain regulation and transmission. Finally, the possible interactions between these two cells types cohabiting the same Ca2+ rich environment will be further explored attempting to highlight new possible therapeutical targets.

Micropathological Chip Modeling the Neurovascular Unit Response to Inflammatory Bone Condition.

Organ-on-a-chip in vitro platforms accurately mimic complex microenvironments offering the ability to recapitulate and dissect mechanisms of physiological and pathological settings, revealing their major importance to develop new therapeutic targets. Bone diseases, such as osteoarthritis, are extremely complex, comprising of the action of inflammatory mediators leading to unbalanced bone homeostasis and de-regulation of sensory innervation and angiogenesis. Although there are models to mimic bone vascularization or innervation, in vitro platforms merging the complexity of bone, vasculature, innervation, and inflammation are missing. Therefore, in this study a microfluidic-based neuro-vascularized bone chip (NVB chip) is proposed to 1) model the mechanistic interactions between innervation and angiogenesis in the inflammatory bone niche, and 2) explore, as a screening tool, novel strategies targeting inflammatory diseases, using a nano-based drug delivery system. It is possible to set the design of the platform and achieve the optimized conditions to address the neurovascular network under inflammation. Moreover, this system is validated by delivering anti-inflammatory drug-loaded nanoparticles to counteract the neuronal growth associated with pain perception. This reliable in vitro tool will allow understanding the bone neurovascular system, enlightening novel mechanisms behind the inflammatory bone diseases, bone destruction, and pain opening new avenues for new therapies discovery.

Microfluidic-based models to address the bone marrow metastatic niche complexity.

Bone marrow (BM) is a preferential metastatic site for solid cancers, contributing to higher morbidity and mortality among millions of oncologic patients worldwide. There are no current efficient therapies to minimize this health burden. Microfluidic based in vitro models emerge as powerful alternatives to animal testing, as well as promising tools for the development of personalized medicine solutions. The complexity associated with the BM metastatic niche originated a wide variety of microfluidic platforms designed to mimic this microenvironment. This review gathers the essential parameters to design an accurate in vitro microfluidic device, based on a comparative analysis of existing models created to address the different steps of the metastatic cascade.

Osteoblasts are inherently programmed to repel sensory innervation.

Tissue innervation is a complex process controlled by the expression profile of signaling molecules secreted by tissue-resident cells that dictate the growth and guidance of axons. Sensory innervation is part of the neuronal network of the bone tissue with a defined spatiotemporal occurrence during bone development. Yet, the current understanding of the mechanisms regulating the map of sensory innervation in the bone tissue is still limited. Here, we demonstrated that differentiation of human mesenchymal stem cells to osteoblasts leads to a marked impairment of their ability to promote axonal growth, evidenced under sensory neurons and osteoblastic-lineage cells crosstalk. The mechanisms by which osteoblast lineage cells provide this nonpermissive environment for axons include paracrine-induced repulsion and loss of neurotrophic factors expression. We identified a drastic reduction of NGF and BDNF production and stimulation of Sema3A, Wnt4, and Shh expression culminating at late stage of OB differentiation. We noted a correlation between Shh expression profile, OB differentiation stages, and OB-mediated axonal repulsion. Blockade of Shh activity and signaling reversed the repulsive action of osteoblasts on sensory axons. Finally, to strengthen our model, we localized the expression of Shh by osteoblasts in bone tissue. Overall, our findings provide evidence that the signaling profile associated with osteoblast phenotype differentiating program can regulate the patterning of sensory innervation, and highlight osteoblast-derived Shh as an essential player in this cue-induced regulation.

Human dental pulp stem cells exhibit enhanced properties in comparison to human bone marrow stem cells on neurites outgrowth.

Mesenchymal stem cells (MSCs) have the capacity to self-renew and differentiate into specific cell types and are, therefore, key players during tissue repair and regeneration. The use of MSCs for the regeneration of tissues in vivo is increasingly being explored and already constitutes a promising alternative to existing clinical treatments. MSCs also exert paracrine and trophic functions, including the promotion of innervation that plays fundamental roles in regeneration and in restoration of the function of organs. Human bone marrow stem cells (hBMSCs) and human dental pulp stem cells (hDPSCs) have been used in studies that aimed at the repair and/or regeneration of bone or other tissues of the craniofacial complex. However, the capabilities of hBMSCs and hDPSCs to elicit the growth of specific axons in order to reestablish functional innervation of the healing tissues are not known. Here, we compared the neurotrophic effects of hDPSCs and hBMSCs on trigeminal and dorsal root ganglia neurons using microfluidic organs-on-chips devices. We found that hDPSCs express significantly higher levels of neurotrophins than hBMSCs and consequently neurons cocultured with hDPSCs develop longer axons in the microfluidic co-culture system when compared to neurons cocultured with hBMSCs. Moreover, hDPSCs elicited the formation of extensive axonal networks and established close contacts with neurons, a phenomenon not observed in presence of hBMSCs. Taken together, these findings indicate that hDPSCs constitute a superior option for restoring the functionality of damaged craniofacial tissues, as they are able to support and promote extensive trigeminal innervation.

The lack of neuropeptide Y-Y1 receptor signaling modulates the chemical and mechanical properties of bone matrix.

Genetic and pharmacological functional studies have provided evidence that the lack of Neuropeptide Y-Y1  receptor (Y1 R) signaling pathway induces a high bone mass phenotype in mice. However, clinical observations have shown that drug or genetic mediated improvement of bone mass might be associated to alterations to bone extracellular matrix (ECM) properties, leading to bone fragility. Hence, in this study we propose to characterize the physical, chemical and biomechanical properties of mature bone ECM of germline NPY-Y1 R knockout (Y1 R-/- ) mice, and compare to their wild-type (WT) littermates. Our results demonstrated that the high bone mass phenotype observed in Y1 R-/- mice involves alterations in Y1 R-/-  bone ECM ultrastructure, as a result of accelerated deposition of organic and mineral fractions. In addition, Y1 R-/- bone ECM displays enhanced matrix maturation characterized by greater number of mature/highly packed collagen fibers without pathological accumulation of immature/mature collagen crosslinks nor compromise of mineral crystallinity. These unique features of Y1 R-/-  bone ECM improved the biochemical properties of Y1 R-/-  bones, reflected by mechanically robust bones with diminished propensity to fracture, contributing to greater bone strength. These findings support the future usage of drugs targeting Y1 R signaling as a promising therapeutic strategy to treat bone loss-related pathologies.

µSpikeHunter: An advanced computational tool for the analysis of neuronal communication and action potential propagation in microfluidic platforms.

Understanding neuronal communication is fundamental in neuroscience, but there are few methodologies offering detailed analysis for well-controlled conditions. By interfacing microElectrode arrays with microFluidics (µEF devices), it is possible to compartmentalize neuronal cultures with a specified alignment of axons and microelectrodes. This setup allows the extracellular recording of spike propagation with a high signal-to-noise ratio over the course of several weeks. Addressing these µEF devices, we developed an advanced yet easy-to-use publically available computational tool, µSpikeHunter, which provides a detailed quantification of several communication-related properties such as propagation velocity, conduction failure, spike timings, and coding mechanisms. The combination of µEF devices and µSpikeHunter can be used in the context of standard neuronal cultures or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. The ability to analyze axonal signals (in a user-friendly, time-efficient, high-throughput manner) opens the door to new approaches in studies of peripheral innervation, neural coding, and neuroregeneration, among many others. We demonstrate the use of µSpikeHunter in dorsal root ganglion neurons where we analyze the presence of both anterograde and retrograde signals in µEF devices. A fully functional version of µSpikeHunter is publically available for download from https://github.com/uSpikeHunter .

The alliance between nerve fibers and stem cell populations in bone marrow: life partners in sickness and health.

The bone marrow (BM) is the central hematopoietic organ in adult mammals, with great potential to be used as a tool to improve the efficacy of the body's response to a number of malignancies and stressful conditions. The nervous system emerges as a critical regulatory player of the BM both under homeostatic and pathologic settings, with essential roles in cellular anchorage and egress, stem cell differentiation, and endothelial cell permeability. This review collects the current knowledge on the interplay between the nervous system and the BM cell populations, with a focus on how the nervous system modulates hematopoietic stem and progenitor cell, mesenchymal stromal cell, and endothelial progenitor cell activity in BM. We have also highlighted the pathologies that have been associated with disturbances in the neuronal signaling in BM and discussed if targeting the nervous system, either by modulating the activity of specific neuronal circuits or by pharmacologically leveling the activity of sympathetic and sensorial signaling-responsive cells in BM, is a promising therapeutic approach to tackling pathologies from BM origin.-Leitão, L., Alves, C. J., Sousa, D. M., Neto, E., Conceição, F., Lamghari, M. The alliance between nerve fibers and stem cell populations in bone marrow: life partners in sickness and health.

Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Selectively recruiting bone marrow (BM)-derived stem and progenitor cells to injury sites is a promising therapeutic approach. The coordinated action of soluble factors is thought to trigger the mobilization of stem cells from the BM and recruit them to lesions to contribute to tissue regeneration. Nevertheless, the temporal response profile of the major cellular players and soluble factors involved in priming the BM and recruiting BM-derived cells to promote regeneration is unknown. We show that injury alters the BM cellular composition, introducing population-specific fluctuations during tissue regeneration. We demonstrate that injury causes an immediate, transient response of mesenchymal stromal cells and endothelial cells followed by a nonoverlapping increase in hematopoietic stem and progenitor cells. Moreover, BM reaction is identical whether the injury is inflicted on skin and muscle or also involves a bone defect, but these 2 injury paradigms trigger distinct systemic cytokine responses. Together, our results indicate that the BM response to injury in the early stages of regeneration is independent of the tissue-of-injury based on the 2 models used, but the injured tissue dictates the systemic cytokine response.-Leitão, L., Alves, C. J., Alencastre, I. S., Sousa, D. M., Neto, E., Conceição, F., Leitão, C., Aguiar, P., Almeida-Porada, G., Lamghari, M. Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Axonal outgrowth, neuropeptides expression and receptors tyrosine kinase phosphorylation in 3D organotypic cultures of adult dorsal root ganglia.

Limited knowledge from mechanistic studies on adult sensory neuronal activity was generated, to some extent, in recapitulated adult in vivo 3D microenvironment. To fill this gap there is a real need to better characterize the adult dorsal root ganglia (aDRG) organotypic cultures to make these in vitro systems exploitable for different approaches, ranging from basic neurobiology to regenerative therapies, to address the sensory nervous system in adult stage. We conducted a direct head-to-head comparison of aDRG and embryonic DRG (eDRG) organotypic culture focusing on axonal growth, neuropeptides expression and receptors tyrosine kinase (RTK) activation associated with neuronal survival, proliferation and differentiation. To identify alterations related to culture conditions, these parameters were also addressed in retrieved aDRG and eDRG and compared with organotypic cultures. Under similar neurotrophic stimulation, aDRG organotypic cultures displayed lower axonal outgrowth rate supported by reduced expression of growth associated protein-43 and high levels of RhoA and glycogen synthase kinase 3 beta mRNA transcripts. In addition, differential alteration in sensory neuropeptides expression, namely calcitonin gene-related peptide and substance P, was detected and was mainly pronounced at gene expression levels. Among 39 different RTK, five receptors from three RTK families were emphasized: tropomyosin receptor kinase A (TrkA), epidermal growth factor receptors (EGFR, ErbB2 and ErbB3) and platelet-derived growth factor receptor (PDGFR). Of note, except for EGFR, the phosphorylation of these receptors was dependent on DRG developmental stage and/or culture condition. In addition, EGFR and PDGFR displayed alterations in their cellular expression pattern in cultured DRG. Overall we provided valuable information particularly important when addressing in vitro the molecular mechanisms associated with development, maturation and regeneration of the sensory nervous system.

Therapeutic Drugs in Bone Loss-Associated Disorders: Clinical Outcomes and Challenges.

Current therapeutic drugs for the treatment of bone loss-associated disorders such as osteoporosis and metastatic bone disease have limited clinical outcomes, namely in terms of efficiency and sustainability. Given the ageing of population in developed countries and the cumulative costs with treatment, bone loss-associated disorders represent a major socioeconomic burden worldwide. In this review, the therapeutic agents targeting bone loss tested in clinical and pre-clinical trials are summarized, as well as the challenges encountered by clinicians and patients. In an effort to attain costeffective clinical outcomes, potential cellular and signalling targets are disclosed.

Microfluidic-based platform to mimic the in vivo peripheral administration of neurotropic nanoparticles.

AIM: Propose a nanoparticle for neuron-targeted retrograde gene delivery and describe a microfluidic-based culture system to provide insight into vector performance and safety. METHODS: Using compartmentalized neuron cultures we dissected nanoparticle bioactivity upon delivery taking advantage of (quantitative) bioimaging tools. RESULTS: Targeted and nontargeted nanoparticles were internalized at axon terminals and retrogradely transported to cell bodies at similar average velocities but the former have shown an axonal flux 2.7-times superior to nontargeted nanoparticles, suggesting an improved cargo-transportation efficiency. The peripheral administration of nanoparticles to axon terminals is nontoxic as compared with their direct administration to the cell body or whole neuron. CONCLUSION: A neuron-targeted nanoparticle system was put forward. Microfluidic-based neuron cultures are proposed as a powerful tool to investigate nanoparticle bio-performance.

Bone Injury and Repair Trigger Central and Peripheral NPY Neuronal Pathways.

Bone repair is a specialized type of wound repair controlled by complex multi-factorial events. The nervous system is recognized as one of the key regulators of bone mass, thereby suggesting a role for neuronal pathways in bone homeostasis. However, in the context of bone injury and repair, little is known on the interplay between the nervous system and bone. Here, we addressed the neuropeptide Y (NPY) neuronal arm during the initial stages of bone repair encompassing the inflammatory response and ossification phases in femoral-defect mouse model. Spatial and temporal analysis of transcriptional and protein levels of NPY and its receptors, Y1R and Y2R, reported to be involved in bone homeostasis, was performed in bone, dorsal root ganglia (DRG) and hypothalamus after femoral injury. The results showed that NPY system activity is increased in a time- and space-dependent manner during bone repair. Y1R expression was trigged in both bone and DRG throughout the inflammatory phase, while a Y2R response was restricted to the hypothalamus and at a later stage, during the ossification step. Our results provide new insights into the involvement of NPY neuronal pathways in bone repair.

Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In Vitro Tools for Neurobiological Research.

Microfluidic technology has become a valuable tool to the scientific community, allowing researchers to study fine cellular mechanisms with higher variable control compared with conventional systems. It has evolved tremendously, and its applicability and flexibility made its usage grow exponentially and transversely to several research fields. This has been particularly noticeable in neuroscience research, where microfluidic platforms made it possible to address specific questions extending from axonal guidance, synapse formation, or axonal transport to the development of 3D models of the CNS to allow pharmacological testing and drug screening. Furthermore, the continuous upgrade of microfluidic platforms has allowed a deeper study of the communication occurring between different neuronal and glial cells or between neurons and other peripheral tissues, both in physiological and pathological conditions. Importantly, the evolution of microfluidic technology has always been accompanied by the development of new computational tools addressing data acquisition, analysis, and modeling.