Connexin Communication Compartments and Wound Repair in Epithelial Tissue.

Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.

High Temporal Resolution Detection of Patient-Specific Glucose Uptake from Human ex Vivo Adipose Tissue On-Chip.

Human tissue in vitro models on-chip are highly desirable to dissect the complexity of a physio-pathological in vivo response because of their advantages compared to traditional static culture systems in terms of high control of microenvironmental conditions, including accurate perturbations and high temporal resolution analyses of medium outflow. Human adipose tissue (hAT) is a key player in metabolic disorders, such as Type 2 Diabetes Mellitus (T2DM). It is involved in the overall energy homeostasis not only as passive energy storage but also as an important metabolic regulator. Here, we aim at developing a large scale microfluidic platform for generating high temporal resolution of glucose uptake profiles, and consequently insulin sensitivity, under physio-pathological stimulations in ex vivo adipose tissues from nondiabetic and T2DM individuals. A multiscale mathematical model that integrates fluid dynamics and an intracellular insulin signaling pathway description was used for assisting microfluidic design in order to maximize measurement accuracy of tissue metabolic activity in response to perturbations. An automated microfluidic injection system was included on-chip for performing precise dynamic biochemical stimulations. The temporal evolution of culture conditions could be monitored for days, before and after perturbation, measuring glucose concentration in the outflow with high temporal resolution. As a proof of concept for detection of insulin resistance, we measured insulin-dependent glucose uptake by hAT from nondiabetic and T2DM subjects, mimicking the postprandial response. The system presented thus represents an important tool in dissecting the role of single tissues, such as hAT, in the complex interwoven picture of metabolic diseases.

Three-Dimensional Microfibrous Scaffold with Aligned Topography Produced via a Combination of Melt-Extrusion Additive Manufacturing and Porogen Leaching for In Vitro Skeletal Muscle Modeling.

Skeletal muscle tissue (SMT) has a highly hierarchical and anisotropic morphology, featuring aligned and parallel structures at multiple levels. Various factors, including trauma and disease conditions, can compromise the functionality of skeletal muscle. The in vitro modeling of SMT represents a useful tool for testing novel drugs and therapies. The successful replication of SMT native morphology demands scaffolds with an aligned anisotropic 3D architecture. In this work, a 3D PCL fibrous scaffold with aligned morphology was developed through the synergistic combination of Melt-Extrusion Additive Manufacturing (MEAM) and porogen leaching, utilizing PCL as the bulk material and PEG as the porogen. PCL/PEG blends with different polymer ratios (60/40, 50/50, 40/60) were produced and characterized through a DSC analysis. The MEAM process parameters and porogen leaching in bi-distilled water allowed for the development of a micrometric anisotropic fibrous structure with fiber diameters ranging from 10 to 100 µm, depending on PCL/PEG blend ratios. The fibrous scaffolds were coated with Gelatin type A to achieve a biomimetic coating for an in vitro cell culture and mechanically characterized via AFM. The 40/60 PCL/PEG scaffolds yielded the most homogeneous and smallest fibers and the greatest physiological stiffness. In vitro cell culture studies were performed by seeding C2C12 cells onto a selected scaffold, enabling their attachment, alignment, and myotube formation along the PCL fibers during a 14-day culture period. The resultant anisotropic scaffold morphology promoted SMT-like cell conformation, establishing a versatile platform for developing in vitro models of tissues with anisotropic morphology.

Biomimetic Electrospun Scaffold-Based In Vitro Model Resembling the Hallmarks of Human Myocardial Fibrotic Tissue.

Adverse remodeling post-myocardial infarction is hallmarked by the phenotypic change of cardiac fibroblasts (CFs) into myofibroblasts (MyoFs) and over-deposition of the fibrotic extracellular matrix (ECM) mainly composed by fibronectin and collagens, with the loss of tissue anisotropy and tissue stiffening. Reversing cardiac fibrosis represents a key challenge in cardiac regenerative medicine. Reliable in vitro models of human cardiac fibrotic tissue could be useful for preclinical testing of new advanced therapies, addressing the limited predictivity of traditional 2D cell cultures and animal in vivo models. In this work, we engineered a biomimetic in vitro model, reproducing the morphological, mechanical, and chemical cues of native cardiac fibrotic tissue. Polycaprolactone (PCL)-based scaffolds with randomly oriented fibers were fabricated by solution electrospinning technique, showing homogeneous nanofibers with an average size of 131 ± 39 nm. PCL scaffolds were then surface-functionalized with human type I collagen (C1) and fibronectin (F) by dihydroxyphenylalanine (DOPA)-mediated mussel-inspired approach (PCL/polyDOPA/C1F), in order to mimic fibrotic cardiac tissue-like ECM composition and support human CF culture. BCA assay confirmed the successful deposition of the biomimetic coating and its stability during 5 days of incubation in phosphate-buffered saline. Immunostaining for C1 and F demonstrated their homogeneous distribution in the coating. AFM mechanical characterization showed that PCL/polyDOPA/C1F scaffolds, in wet conditions, resembled fibrotic tissue stiffness with an average Young's modulus of about 50 kPa. PCL/polyDOPA/C1F membranes supported human CF (HCF) adhesion and proliferation. Immunostaining for α-SMA and quantification of α-SMA-positive cells showed HCF activation into MyoFs in the absence of a transforming growth factor ß (TGF-ß) profibrotic stimulus, suggesting the intrinsic ability of biomimetic PCL/polyDOPA/C1F scaffolds to sustain the development of cardiac fibrotic tissue. A proof-of-concept study making use of a commercially available antifibrotic drug confirmed the potentialities of the developed in vitro model for drug efficacy testing. In conclusion, the proposed model was able to replicate the main hallmarks of early-stage cardiac fibrosis, appearing as a promising tool for future preclinical testing of advanced regenerative therapies.

Micropatterning topology on soft substrates affects myoblast proliferation and differentiation.

Micropatterning techniques and substrate engineering are becoming useful tools to investigate several aspects of cell-cell interaction biology. In this work, we rationally study how different micropatterning geometries can affect myoblast behavior in the early stage of in vitro myogenesis. Soft hydrogels with physiological elastic modulus (E = 15 kPa) were micropatterned in parallel lanes (100, 300, and 500 µm width) resulting in different local and global myoblast densities. Proliferation and differentiation into multinucleated myotubes were evaluated for murine and human myoblasts. Wider lanes showed a decrease in murine myoblast proliferation: (69 ± 8)% in 100 µm wide lanes compared to (39 ± 7)% in 500 µm lanes. Conversely, fusion index increased in wider lanes: from (46 ± 7)% to (66 ± 7)% for murine myoblasts, and from (15 ± 3)% to (36 ± 2)% for human primary myoblasts, using a patterning width of 100 and 500 µm, respectively. These results are consistent with both computational modeling data and conditioned medium experiments, which demonstrated that wider lanes favor the accumulation of endogenous secreted factors. Interestingly, human primary myoblast proliferation is not affected by patterning width, which may be because the high serum content of their culture medium overrides the effect of secreted factors. These data highlight the role of micropatterning in shaping the cellular niche through secreted factor accumulation, and are of paramount importance in rationally understanding myogenesis in vitro for the correct design of in vitro skeletal muscle models.

Electroconductive Photo-Curable PEGDA-Gelatin/PEDOT:PSS Hydrogels for Prospective Cardiac Tissue Engineering Application.

Electroconductive hydrogels (ECHs) have attracted interest for tissue engineering applications due to their ability to promote the regeneration of electroactive tissues. Hence, ECHs with tunable electrical and mechanical properties, bioactivity, biocompatibility and biodegradability are demanded. In this work, ECHs based on photo-crosslinked blends of polyethylene glycol diacrylate (PEGDA) and gelatin with different PEGDA:gelatin ratios (1:1, 1.5:1 and 2:1 wt./wt.), and containing poly (3,4-ethylenedioxythiophene):poly (styrene sulfonate) (PEDOT:PSS) (0.0, 0.1, 0,3 and 0.5% w/v%) were prepared. Main novelty was the use of gelatin as bioactive component and co-initiator in the photo-crosslinking process, leading to its successful incorporation in the hydrogel network. Physical properties could be modulated by the initial PEGDA:gelatin weight ratio. Pristine hydrogels with increasing PEGDA:gelatin ratio showed: (i) an increasing compressive elastic modulus from 5 to 28 kPa; (ii) a decreasing weight loss from 62% to 43% after 2 weeks incubation in phosphate buffered saline at 37°C; (iii) reduced crosslinking time; (iv) higher crosslinking density and (v) lower water absorption. The addition of PEDOT:PSS in the hydrogels reduced photo-crosslinking time (from 60 to 10 s) increasing their surface and bulk electrical properties. Finally, in vitro tests with human cardiac fibroblasts showed that hydrogels were cytocompatible and samples with 1.5:1 initial PEGDA:gelatin ratio promoted the highest cell adhesion at 24 h. Results from this work suggested the potential of electroconductive photo-curable PEGDA-gelatin/PEDOT:PSS hydrogels for prospective cardiac tissue engineering applications.

Development of an Innovative Soft Piezoresistive Biomaterial Based on the Interconnection of Elastomeric PDMS Networks and Electrically-Conductive PEDOT:PSS Sponges.

A deeply interconnected flexible transducer of polydimethylsiloxane (PDMS) and poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) was obtained as a material for the application of soft robotics. Firstly, transducers were developed by crosslinking PEDOT:PSS with 3-glycidyloxypropryl-trimethoxysilane (GPTMS) (1, 2 and 3% v/v) and using freeze-drying to obtain porous sponges. The PEDOT:PSS sponges were morphologically characterized, showing porosities mainly between 200 and 600 µm2; such surface area dimensions tend to decrease with increasing degrees of crosslinking. A stability test confirmed a good endurance for up to 28 days for the higher concentrations of the crosslinker tested. Consecutively, the sponges were electromechanically characterized, showing a repeatable and linear resistance variation by the pressure triggers within the limits of their working range (∆RR0 max = 80% for 1-2% v/v of GPTMS). The sponges containing 1% v/v of GPTMS were intertwined with a silicon elastomer to increase their elasticity and water stability. The flexible transducer obtained with this method exhibited moderately lower sensibility and repeatability than the PEDOT:PSS sponges, but the piezoresistive response remained stable under mechanical compression. Furthermore, the transducer displayed a linear behavior when stressed within the limits of its working range. Therefore, it is still valid for pressure sensing and contact detection applications. Lastly, the flexible transducer was submitted to preliminary biological tests that indicate a potential for safe, in vivo sensing applications.

Biomimetic design of bioartificial scaffolds for the in vitro modelling of human cardiac fibrosis.

In vitro models of pathological cardiac tissue have attracted interest as predictive platforms for preclinical validation of therapies. However, models reproducing specific pathological features, such as cardiac fibrosis size (i.e., thickness and width) and stage of development are missing. This research was aimed at engineering 2D and 3D models of early-stage post-infarct fibrotic tissue (i.e., characterized by non-aligned tissue organization) on bioartificial scaffolds with biomimetic composition, design, and surface stiffness. 2D scaffolds with random nanofibrous structure and 3D scaffolds with 150 µm square-meshed architecture were fabricated from polycaprolactone, surface-grafted with gelatin by mussel-inspired approach and coated with cardiac extracellular matrix (ECM) by 3 weeks culture of human cardiac fibroblasts. Scaffold physicochemical properties were thoroughly investigated. AFM analysis of scaffolds in wet state, before cell culture, confirmed their close surface stiffness to human cardiac fibrotic tissue. Following 3 weeks culture, biomimetic biophysical and biochemical scaffold properties triggered the activation of myofibroblast phenotype. Upon decellularization, immunostaining, SEM and two-photon excitation fluorescence microscopy showed homogeneous decoration of both 2D and 3D scaffolds with cardiac ECM. The versatility of the approach was demonstrated by culturing ventricular or atrial cardiac fibroblasts on scaffolds, thus suggesting the possibility to use the same scaffold platforms to model both ventricular and atrial cardiac fibrosis. In the future, herein developed in vitro models of cardiac fibrotic tissue, reproducing specific pathological features, will be exploited for a fine preclinical tuning of therapies.

Immune response of polarized cystic fibrosis airway epithelial cells infected with Influenza A virus.

BACKGROUND: Cystic fibrosis (CF), a genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is characterized by dysfunction of the immune response in the airway epithelium that leads to prolonged infection, colonization and exacerbated inflammation. In this study, we determined the gene expression profile of airway epithelial cells knockdown for CFTR (CFTR KD) in response to bacterial and viral challenges. METHODS: In a first approach, polarized CFTR KD and their control counterpart (CFTR CTL) cells were stimulated with P. aeruginosa-derived virulence factor flagellin. Next, we developed a model of Influenza A virus (IAV) infection in CTL and CFTR KD polarized cells. mRNA was collected for transcriptome analysis. RESULTS: Beside the expected pro-inflammatory response, Gene Set Enrichment Analysis highlighted key molecular pathways and players involved in IAV and anti-viral interferon signaling. Although IAV replication was similar in both cell types, multiplex gene expression analysis revealed changes of key immune genes dependent on time of infection that were found to be CFTR-dependent and/or IAV-dependent. Interferons are key signaling proteins/cytokines in the antibacterial and antiviral response. To evaluate their impact on the altered gene expression profile in CFTR responses to pathogens, we measured transcriptome changes after exposure to Type I-, Type II- and Type III-interferons. CONCLUSIONS: Our findings reveal target genes in understanding the defective immune response in the CF airway epithelium in the context of viral infection. Information provided in this study would be useful to understand the dysfunctional immune response of the CF airway epithelium during infection.

Dual stimuli-responsive polyurethane-based hydrogels as smart drug delivery carriers for the advanced treatment of chronic skin wounds.

The design of multi-stimuli-responsive vehicles for the controlled and localized release of drugs is a challenging issue increasingly catching the attention of many research groups working on the advanced treatment of hard-to-close wounds. In this work, a thermo- and pH-responsive hydrogel (P-CHP407) was prepared from an ad hoc synthesized amphiphilic poly(ether urethane) (CHP407) exposing a significant amount of -COOH groups (8.8 ± 0.9 nmol/gpolymer). The exposure of acid moieties in P-CHP407 hydrogel led to slightly lower initial gelation temperature (12.1 °C vs. 14.6 °C, respectively) and gelation rate than CHP407 hydrogel, as rheologically assessed. Nanoscale hydrogel characterization by Low Field NMR (LF-NMR) spectroscopy suggested that the presence of carboxylic groups in P-CHP407 caused the formation of bigger micelles with a thicker hydrated shell than CHP407 hydrogels, as further proved by Dynamic Light Scattering analyses. In addition, P-CHP407 hydrogel showed improved capability to change its internal pH compared to CHP407 one when incubated with an alkaline buffer (pH 8) (e.g., pHchange_5min = 3.76 and 1.32, respectively). Moreover, LF-NMR characterization suggested a stronger alkaline-pH-induced interaction of water molecules with micelles exposing -COOH groups. Lastly, the hydrogels were found biocompatible according to ISO 10993 and able to load and release Ibuprofen: delivery kinetics of Ibuprofen was enhanced by P-CHP407 hydrogels at alkaline pH, suggesting their potential use as smart delivery systems in the treatment of chronic infected wounds.

Vav3 Mediates Pseudomonas aeruginosa Adhesion to the Cystic Fibrosis Airway Epithelium.

Pseudomonas aeruginosa (Pa) represents the leading cause of airway infection in cystic fibrosis (CF). Early airways colonization can be explained by enhanced adhesion of Pa to the respiratory epithelium. RNA sequencing (RNA-seq) on fully differentiated primary cultures of airway epithelial cells from CF and non-CF donors predict that VAV3, ß1 INTEGRIN, and FIBRONECTIN genes are significantly enriched in CF. Indeed, Vav3 is apically overexpressed in CF, associates with active ß1 integrin luminally exposed, and increases fibronectin deposition. These luminal microdomains, rich in fibronectin and ß1 integrin and regulated by Vav3, mediate the increased Pa adhesion to the CF epithelium. Interestingly, Vav3 inhibition normalizes the CF-dependent fibronectin and ß1-integrin ectopic expression, improves the CF epithelial integrity, and prevents the enhanced Pa trapping to the CF epithelium. Through its capacity to promote a luminal complex with active ß1 integrin and fibronectin that favors bacteria trapping, Vav3 may represent a new target in CF.

Transcriptomic profile of cystic fibrosis airway epithelial cells undergoing repair.

Pathological remodeling of the airway epithelium is commonly observed in Cystic Fibrosis (CF). The different cell types that constitute the airway epithelium are regenerated upon injury to restore integrity and maintenance of the epithelium barrier function. The molecular signature of tissue repair in CF airway epithelial cells has, however, not well been investigated in primary cultures. We therefore collected RNA-seq data from well-differentiated primary cultures of bronchial human airway epithelial cells (HAECs) of CF (F508del/F508del) and non-CF (NCF) origins before and after mechanical wounding, exposed or not to flagellin. We identified the expression changes with time of repair of genes, the products of which are markers of the different cell types that constitute the airway epithelium (basal, suprabasal, intermediate, secretory, goblet and ciliated cells as well as ionocytes). Researchers in the CF field may benefit from this transcriptomic profile, which covers the initial steps of wound repair and revealed differences in this process between CF and NCF cultures.

Cross-talk of healthy and impaired human tissues for dissection of disease pathogenesis.

Systemic diseases affect multiple tissues that interact with each other within a network difficult to explore at the body level. However, understanding the interdependences between tissues could be of high relevance for drug target identification, especially at the first stages of disease development. In vitro systems have the advantages of accessibility to measurements and precise controllability of culture conditions, but currently have limitations in mimicking human in vivo systemic tissue response. In this work, we present an in vitro model of cross-talk between an ex vivo culture of adipose tissue from an obese donor and a skeletal muscle in vitro model from a healthy donor. This is relevant to understand type 2 diabetes mellitus pathogenesis, as obesity is one of its main risk factors. The human adipose tissue biopsy was maintained as a three-dimensional culture for 48 h. Its conditioned culture medium was used to stimulate a human skeletal muscle-on-chip, developed by differentiating primary cells of a patient's biopsy under topological cues and molecular self-regulation. This system has been characterized to demonstrate its ability to mimic important features of the normal skeletal muscle response in vivo. We then found that the conditioned medium from a diseased adipose tissue is able to perturb the normal insulin sensitivity of a healthy skeletal muscle, as reported in the early stages of diabetes onset. In perspective, this work represents an important step toward the development of technological platforms that allow to study and dissect the systemic interaction between unhealthy and healthy tissues in vitro. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2766, 2019.

Animal and model systems for studying cystic fibrosis.

The cystic fibrosis (CF) field is the beneficiary of five species of animal models that lack functional cystic fibrosis transmembrane conductance regulator (CFTR) channel. These models are rapidly informing mechanisms of disease pathogenesis and CFTR function regardless of how faithfully a given organ reproduces the human CF phenotype. New approaches of genetic engineering with RNA-guided nucleases are rapidly expanding both the potential types of models available and the approaches to correct the CFTR defect. The application of new CRISPR/Cas9 genome editing techniques are similarly increasing capabilities for in vitro modeling of CFTR functions in cell lines and primary cells using air-liquid interface cultures and organoids. Gene editing of CFTR mutations in somatic stem cells and induced pluripotent stem cells is also transforming gene therapy approaches for CF. This short review evaluates several areas that are key to building animal and cell systems capable of modeling CF disease and testing potential treatments.

Dendritic Cell Migration Toward CCL21 Gradient Requires Functional Cx43.

Dendritic cells (DCs) travel through lymphatic vessels to transport antigens and present them to T cells in lymph nodes. DCs move directionally toward lymphatics by virtue of their CCR7 and a CCL21 chemotactic gradient. We evaluated in vivo and in bone marrow-derived dendritic cells (BMDCs) whether the gap junction protein Cx43 contributes to CCL21/CCR7-dependent DC migration in wild-type (WT) mice, heterozygous (Cx43+/-) mice and mice expressing a truncated form of Cx43 lacking its regulatory C-terminus (Cx43K258/-). In a model of flank skin inflammation, we found that the recruitment of myeloid DCs (mDCs) to skin draining lymph nodes was reduced in Cx43K258/- mice as compared to WT and Cx43+/- mice. In addition, the migration of Cx43K258/- BMDCs toward CCL21 was abolished in an in vitro chemotactic assay while it was only reduced in Cx43+/- cells. Both mutant genotypes showed defects in the directionality of BMDC migration as compared to WT BMDCs. No difference was found between the three populations of BMDCs in terms of expression of surface markers (CD11c, CD86, CD80, CD40, MHC-II, and CCR7) after differentiation and TLR activation. Finally, examination of the CCR7-induced signaling pathways in BMDCs revealed normal receptor-induced mobilization of intracellular Ca2+. These results demonstrate that full expression of an intact Cx43 is critical to the directionality and rate of DC migration, which may be amenable to regulation of the immune response.

Skeletal Muscle Differentiation on a Chip Shows Human Donor Mesoangioblasts' Efficiency in Restoring Dystrophin in a Duchenne Muscular Dystrophy Model.

: Restoration of the protein dystrophin on muscle membrane is the goal of many research lines aimed at curing Duchenne muscular dystrophy (DMD). Results of ongoing preclinical and clinical trials suggest that partial restoration of dystrophin might be sufficient to significantly reduce muscle damage. Different myogenic progenitors are candidates for cell therapy of muscular dystrophies, but only satellite cells and pericytes have already entered clinical experimentation. This study aimed to provide in vitro quantitative evidence of the ability of mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from DMD patients, using a microengineered model. We designed an ad hoc experimental strategy to miniaturize on a chip the standard process of muscle regeneration independent of variables such as inflammation and fibrosis. It is based on the coculture, at different ratios, of human dystrophin-positive myogenic progenitors and dystrophin-negative myoblasts in a substrate with muscle-like physiological stiffness and cell micropatterns. Results showed that both healthy myoblasts and mesoangioblasts restored dystrophin expression in DMD myotubes. However, mesoangioblasts showed unexpected efficiency with respect to myoblasts in dystrophin production in terms of the amount of protein produced (40% vs. 15%) and length of the dystrophin membrane domain (210-240 µm vs. 40-70 µm). These results show that our microscaled in vitro model of human DMD skeletal muscle validated previous in vivo preclinical work and may be used to predict efficacy of new methods aimed at enhancing dystrophin accumulation and distribution before they are tested in vivo, reducing time, costs, and variability of clinical experimentation. SIGNIFICANCE: This study aimed to provide in vitro quantitative evidence of the ability of human mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from patients with Duchenne muscular dystrophy (DMD), using a microengineered model. An ad hoc experimental strategy was designed to miniaturize on a chip the standard process of muscle regeneration independent of variables such as inflammation and fibrosis. This microscaled in vitro model, which validated previous in vivo preclinical work, revealed that mesoangioblasts showed unexpected efficiency as compared with myoblasts in dystrophin production. Consequently, this model may be used to predict efficacy of new drugs or therapies aimed at enhancing dystrophin accumulation and distribution before they are tested in vivo.

Determination of glucose flux in live myoblasts by microfluidic nanosensing and mathematical modeling.

Glucose is the main energy source for cells in an organism and its blood concentration is tightly regulated in healthy individuals. However, impaired blood glucose control has been found in diseases such as metabolic syndrome and diabetes, and anomalous glucose utilization in cancer tissues. Dissecting the dynamics of the different phenomena involved in glucose handling (extracellular mass transport, membrane diffusion, and intracellular phosphorylation) is very relevant to identify which mechanisms are disrupted under disease conditions. In this work, we developed an effective methodology for quantitatively analyzing these phenomena in living cells. A measurement of steady-state glucose uptake is, by itself, insufficient to determine the dynamics of intracellular glucose. For this purpose, we integrated two types of measurements: cytosolic glucose concentration at the single-cell level, obtained using a cytosolic FRET nanosensor, and cell population glucose uptake, obtained without perturbing culture conditions using a microfluidic perfusion system. Microfluidics enabled accurate temporal stimulation of cells through cyclic pulses of glucose concentration at defined flow rates. We found that both, glucose uptake and phosphorylation, are linearly dependent on glucose concentration in the physiological range. Mathematical modeling enabled precise determination of the kinetic constants of membrane transport (0.27 s(-1)) and intracellular phosphorylation (2.01 s(-1)).