Substrate adhesion determines migration during mesenchymal cell condensation in chondrogenesis.

Mesenchymal condensation is a prevalent morphogenetic transition that is essential in chondrogenesis. However, the current understanding of condensation mechanisms is limited. In vivo, progenitor cells directionally migrate from the surrounding loose mesenchyme towards regions of increasing matrix adherence (the condensation centers), which is accompanied by the upregulation of fibronectin. Here, we focused on the mechanisms of cell migration during mesenchymal cell condensation and the effects of matrix adherence. Dendrimer-based nanopatterns of the cell-adhesive peptide arginine-glycine-aspartic acid (RGD), which is present in fibronectin, were used to regulate substrate adhesion. We recorded collective and single-cell migration of mesenchymal stem cells, under chondrogenic induction, using live-cell imaging. Our results show that the cell migration mode of single cells depends on substrate adhesiveness, and that cell directionality controls cell condensation and the fusion of condensates. Inhibition experiments revealed that cell-cell interactions mediated by N-cadherin (also known as CDH2) are also pivotal for directional migration of cell condensates by maintaining cell-cell cohesion, thus suggesting a fine interplay between cell-matrix and cell-cell adhesions. Our results shed light on the role of cell interactions with a fibronectin-depositing matrix during chondrogenesis in vitro, with possible applications in regenerative medicine. This article has an associated First Person interview with the first author of the paper.

BBB-on-a-chip with integrated micro-TEER for permeability evaluation of multi-functionalized gold nanorods against Alzheimer's disease.

BACKGROUND: The lack of predictive models that mimic the blood-brain barrier (BBB) hinders the development of effective drugs for neurodegenerative diseases. Animal models behave differently from humans, are expensive and have ethical constraints. Organ-on-a-chip (OoC) platforms offer several advantages to resembling physiological and pathological conditions in a versatile, reproducible, and animal-free manner. In addition, OoC give us the possibility to incorporate sensors to determine cell culture features such as trans-endothelial electrical resistance (TEER). Here, we developed a BBB-on-a-chip (BBB-oC) platform with a TEER measurement system in close distance to the barrier used for the first time for the evaluation of the permeability performance of targeted gold nanorods for theranostics of Alzheimer's disease. GNR-PEG-Ang2/D1 is a therapeutic nanosystem previously developed by us consisting of gold nanorods (GNR) functionalized with polyethylene glycol (PEG), angiopep-2 peptide (Ang2) to overcome the BBB and the D1 peptide as beta amyloid fibrillation inhibitor, finally obtaining GNR-PEG-Ang2/D1 which showed to be useful for disaggregation of the amyloid in in vitro and in vivo models. In this work, we evaluated its cytotoxicity, permeability, and some indications of its impact on the brain endothelium by employing an animal-free device based on neurovascular human cells. RESULTS: In this work, we fabricated a BBB-oC with human astrocytes, pericytes and endothelial cells and a TEER measuring system (TEER-BBB-oC) integrated at a micrometric distance of the endothelial barrier. The characterization displayed a neurovascular network and the expression of tight junctions in the endothelium. We produced GNR-PEG-Ang2/D1 and determined its non-cytotoxic range (0.05-0.4 nM) for plated cells included in the BBB-oC and confirmed its harmless effect at the highest concentration (0.4 nM) in the microfluidic device. The permeability assays revealed that GNR-PEG-Ang2/D1 cross the BBB and this entry is facilitated by Ang2 peptide. Parallel to the permeability analysis of GNR-PEG-Ang2/D1, an interesting behavior of the TJs expression was observed after its administration probably related to the ligands on the nanoparticle surface. CONCLUSIONS: BBB-oC with a novel TEER integrated setup which allow a correct read-out and cell imaging monitoring was proven as a functional and throughput platform to evaluate the brain permeability performance of nanotherapeutics in a physiological environment with human cells, putting forward a viable alternative to animal experimentation.

The Janus Role of Adhesion in Chondrogenesis.

Tackling the first stages of the chondrogenic commitment is essential to drive chondrogenic differentiation to healthy hyaline cartilage and minimize hypertrophy. During chondrogenesis, the extracellular matrix continuously evolves, adapting to the tissue adhesive requirements at each stage. Here, we take advantage of previously developed nanopatterns, in which local surface adhesiveness can be precisely tuned, to investigate its effects on prechondrogenic condensation. Fluorescence live cell imaging, immunostaining, confocal microscopy and PCR analysis are used to follow the condensation process on the nanopatterns. Cell tracking parameters, condensate morphology, cell-cell interactions, mechanotransduction and chondrogenic commitment are evaluated in response to local surface adhesiveness. Results show that only condensates on the nanopatterns of high local surface adhesiveness are stable in culture and able to enter the chondrogenic pathway, thus highlighting the importance of controlling cell-substrate adhesion in the tissue engineering strategies for cartilage repair.

Nanopatterns of Surface-Bound EphrinB1 Produce Multivalent Ligand-Receptor Interactions That Tune EphB2 Receptor Clustering.

Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate-with molecular sensitivity-the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems.

Tuneable hydrogel patterns in pillarless microfluidic devices.

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.

Continuous bone morphogenetic protein-2 gradients for concentration effect studies on C2C12 osteogenic fate.

Cells can respond to small changes in a varying concentration of exogenous signaling molecules. Here we propose the use of continuous surface chemical gradients for the in-depth study of dose-dependent effects on cells. A continuous surface gradient of bone morphogenetic protein-2 (BMP-2) is presented. The gradient covers a narrow range of surface densities (from 1.4 to 2.3 pmol/cm(2)) with a shallow slope (0.9 pmol/cm(3)). These characteristics represent a quasi-homogeneous surface concentration at the cell scale, which is crucial for cell screening studies. Cell fate evaluation at early stages of osteogenesis in C2C12 cells, indicates the potential of continuous gradients for in vitro screening applications. FROM THE CLINICAL EDITOR: The authors propose the use of surface-applied continuous chemical gradients for in-depth study of dose-dependent effects on cells. The method is demonstrated using BMP-2 proteins on C2C12 cells as a model system.

Polyoxometalate-Decorated Gold Nanoparticles Inhibit ß-Amyloid Aggregation and Cross the Blood-Brain Barrier in a µphysiological Model.

Alzheimer's disease is characterized by a combination of several neuropathological hallmarks, such as extracellular aggregates of beta amyloid (Aß). Numerous alternatives have been studied for inhibiting Aß aggregation but, at this time, there are no effective treatments available. Here, we developed the tri-component nanohybrid system AuNPs@POM@PEG based on gold nanoparticles (AuNPs) covered with polyoxometalates (POMs) and polyethylene glycol (PEG). In this work, AuNPs@POM@PEG demonstrated the inhibition of the formation of amyloid fibrils, showing a 75% decrease in Aß aggregation in vitro. As it is a potential candidate for the treatment of Alzheimer's disease, we evaluated the cytotoxicity of AuNPs@POM@PEG and its ability to cross the blood-brain barrier (BBB). We achieved a stable nanosystem that is non-cytotoxic below 2.5 nM to human neurovascular cells. The brain permeability of AuNPs@POM@PEG was analyzed in an in vitro microphysiological model of the BBB (BBB-on-a-chip), containing 3D human neurovascular cell co-cultures and microfluidics. The results show that AuNPs@POM@PEG was able to cross the brain endothelial barrier in the chip and demonstrated that POM does not affect the barrier integrity, giving the green light to further studies into this system as a nanotherapeutic.

Ferulic acid-loaded polymeric nanoparticles prepared from nano-emulsion templates facilitate internalisation across the blood-brain barrier in model membranes.

A hydroxycinnamic acid derivative, namely ferulic acid (FA) has been successfully encapsulated in polymeric nanoparticles (NPs) based on poly(lactic-co-glycolic acid) (PLGA). FA-loaded polymeric NPs were prepared from O/W nano-emulsion templates using the phase inversion composition (PIC) low-energy emulsification method. The obtained PLGA NPs exhibited high colloidal stability, good drug-loading capacity, and particle hydrodynamic diameters in the range of 74 to 117 nm, depending on the FA concentration used. In vitro drug release studies confirmed a diffusion-controlled mechanism through which the amount of released FA reached a plateau at 60% after 6 hours-incubation. Five kinetic models were used to fit the FA release data as a function of time. The Weibull distribution and Korsmeyer-Peppas equation models provided the best fit to our experimental data and suggested quasi-Fickian diffusion behaviour. Moderate dose-response antioxidant and radical scavenging activities of FA-loaded PLGA NPs were demonstrated using the DPPH˙ assay achieving inhibition activities close to 60 and 40%, respectively. Cell culture studies confirmed that FA-loaded NPs were not toxic according to the MTT colorimetric assay, were able to internalise efficiently SH-SY5Y neuronal cells and supressed the intracellular ROS-level induced by H2O2 leading to 52% and 24.7% of cellular viability at 0.082 and 0.041 mg mL-1, respectively. The permeability of the NPs through the blood brain barrier was tested with an in vitro organ-on-a-chip model to evaluate the ability of the FA-loaded PLGA and non-loaded PLGA NPs to penetrate to the brain. NPs were able to penetrate the barrier, but permeability decreased when FA was loaded. These results are promising for the use of loaded PLGA NPs for the management of neurological diseases.

Facile modification of silica substrates provides a platform for direct-writing surface click chemistry.

Please click here: a facile two-step functionalization strategy for silicon oxide-based substrates generates a stable platform for surface click chemistry via direct writing. The suitability of the obtained substrates is proven by patterning with two different direct-writing techniques and three different molecules.

Cell adhesion and focal contact formation on linear RGD molecular gradients: study of non-linear concentration dependence effects.

Cell adhesion onto bioengineered surfaces is affected by a number of variables, including the former substrate derivatization process. In this investigation, we studied the correlation between cell adhesion and cell-adhesive ligand surface concentration and organization due to substrate modification. For this purpose, Arg-Gly-Asp (RGD) gradient surfaces were created on poly(methyl methacrylate) substrates by continuous hydrolysis and were then grafted with biotin-PEG-RGD molecules. Cell culture showed that adhesion behavior changes in a nonlinear way in the narrow range of RGD surface densities assayed (2.8 to 4.4 pmol/cm(2)), with a threshold value of 4.0 pmol/cm(2) for successful cell attachment and spreading. This nonlinear dependence may be explained by nonhomogeneous RGD surface distribution at the nanometre scale, conditioned by the stochastic nature of the hydrolysis process. Atomic force microscopy analysis of the gradient surface showed an evolution of surface morphology compatible with this hypothesis. FROM THE CLINICAL EDITOR: The authors observed by AFM nonlinear dependence of cell adhesion on RGD gradient surfaces with different surface densities. The nonlinear characteristics may be explained by non-homogeneous RGD surface distribution at the nanometer scale, conditioned by the stochastic nature of the hydrolysis process.

Recent advances in engineering nanotopographic substrates for cell studies.

Cells sense their environment through the cell membrane receptors. Interaction with extracellular ligands induces receptor clustering at the nanoscale, assembly of the signaling complexes in the cytosol and activation of downstream signaling pathways, regulating cell response. Nanoclusters of receptors can be further organized hierarchically in the cell membrane at the meso- and micro-levels to exert different biological functions. To study and guide cell response, cell culture substrates have been engineered with features that can interact with the cells at different scales, eliciting controlled cell responses. In particular, nanoscale features of 1-100 nm in size allow direct interaction between the material and single cell receptors and their nanoclusters. Since the first "contact guidance" experiments on parallel microstructures, many other studies followed with increasing feature resolution and biological complexity. Here we present an overview of the advances in the field summarizing the biological scenario, substrate fabrication techniques and applications, highlighting the most recent developments.

Determination of the nanoscale electrical properties of olfactory receptor hOR1A1 and their dependence on ligand binding: Towards the development of capacitance-operated odorant biosensors.

The transduction of odorant binding into cellular signaling by olfactory receptors (ORs) is not understood and knowing its mechanism would enable developing new pharmacology and biohybrid electronic detectors of volatile organic compounds bearing high sensitivity and selectivity. The electrical characterization of ORs in bulk experiments is subject to microscopic models and assumptions. We have directly determined the nanoscale electrical properties of ORs immobilized in a fixed orientation, and their change upon odorant binding, using electrochemical scanning tunneling microscopy (EC-STM) in near-physiological conditions. Recordings of current versus time, distance, and electrochemical potential allows determining the OR impedance parameters and their dependence with odorant binding. Our results allow validating OR structural-electrostatic models and their functional activation processes, and anticipating a novel macroscopic biosensor based on ORs.

Development of a Custom-Made 3D Printing Protocol with Commercial Resins for Manufacturing Microfluidic Devices.

The combination of microfluidics and photo-polymerization techniques such as stereolithography (SLA) has emerged as a new field which has a lot of potential to influence in such important areas as biological analysis, and chemical detection among others. However, the integration between them is still at an early stage of development. In this article, after analyzing the resolution of a custom SLA 3D printer with commercial resins, microfluidic devices were manufactured using three different approaches. First, printing a mold with the objective of creating a Polydimethylsiloxane (PDMS) replica with the microfluidic channels; secondly, open channels have been printed and then assembled with a flat cover of the same resin material. Finally, a closed microfluidic device has also been produced in a single process of printing. Important results for 3D printing with commercial resins have been achieved by only printing one layer on top of the channel. All microfluidic devices have been tested successfully for pressure-driven fluid flow.

Biosensors Integration in Blood-Brain Barrier-on-a-Chip: Emerging Platform for Monitoring Neurodegenerative Diseases.

Over the most recent decades, the development of new biological platforms to study disease progression and drug efficacy has been of great interest due to the high increase in the rate of neurodegenerative diseases (NDDs). Therefore, blood-brain barrier (BBB) as an organ-on-a-chip (OoC) platform to mimic brain-barrier performance could offer a deeper understanding of NDDs as well as a very valuable tool for drug permeability testing for new treatments. A very attractive improvement of BBB-oC technology is the integration of detection systems to provide continuous monitoring of biomarkers in real time and a fully automated analysis of drug permeably, rendering more efficient platforms for commercialization. In this Perspective, an overview of the main BBB-oC configurations is introduced and a critical vision of the BBB-oC platforms integrating electronic read out systems is detailed, indicating the strengths and weaknesses of current devices, proposing the great potential for biosensors integration in BBB-oC. In this direction, we name potential biomarkers to monitor the evolution of NDDs related to the BBB and/or drug cytotoxicity using biosensor technology in BBB-oC.

Nanoscale ligand density modulates gap junction intercellular communication of cell condensates during chondrogenesis.

Aim: To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis. Materials & methods: Previously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid were used as cell culture substrates to control cell adhesion at the nanoscale. In vitro chondrogenesis of mesenchymal stem cells was conducted on the nanopatterns. Cohesion and GJIC were evaluated in cell condensates. Results: Mechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling. Conclusion: Cell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.

Personalized in vitro Extracellular Matrix Models of Collagen VI-Related Muscular Dystrophies.

Collagen VI-related dystrophies (COL6-RDs) are a group of rare congenital neuromuscular dystrophies that represent a continuum of overlapping clinical phenotypes that go from the milder Bethlem myopathy (BM) to the severe Ullrich congenital muscular dystrophy, for which there is no effective treatment. Mutations in one of the three Collagen VI genes alter the incorporation of this protein into the extracellular matrix (ECM), affecting the assembly and the structural integrity of the whole fibrillar network. Clinical hallmarks of COL6-RDs are secondary to the ECM disruption and include muscle weakness, proximal joint contractures, and distal hyperlaxity. Although some traits have been identified in patients' ECMs, a correlation between the ECM features and the clinical phenotype has not been established, mainly due to the lack of predictive and reliable models of the pathology. Herein, we engineered a new personalized pre-clinical model of COL6-RDs using cell-derived matrices (CDMs) technology to better recapitulate the complexity of the native scenario. We found that CDMs from COL6-RD patients presented alterations in ECM structure and composition, showing a significantly decreased Collagen VI secretion, especially in the more severe phenotypes, and a decrease in Fibrillin-1 inclusion. Next, we examined the Collagen VI-mediated deposition of Fibronectin in the ECM, finding a higher alignment, length, width, and straightness than in patients with COL6-RDs. Overall, these results indicate that CDMs models are promising tools to explore the alterations that arise in the composition and fibrillar architecture due to mutations in Collagen VI genes, especially in early stages of matrix organization. Ultimately, CDMs derived from COL6-RD patients may become relevant pre-clinical models, which may help identifying novel biomarkers to be employed in the clinics and to investigate novel therapeutic targets and treatments.

Phosphorylation disrupts long-distance electron transport in cytochrome c.

It has been recently shown that electron transfer between mitochondrial cytochrome c and the cytochrome c1 subunit of the cytochrome bc1 can proceed at long-distance through the aqueous solution. Cytochrome c is thought to adjust its activity by changing the affinity for its partners via Tyr48 phosphorylation, but it is unknown how it impacts the nanoscopic environment, interaction forces, and long-range electron transfer. Here, we constrain the orientation and separation between cytochrome c1 and cytochrome c or the phosphomimetic Y48pCMF cytochrome c, and deploy an array of single-molecule, bulk, and computational methods to investigate the molecular mechanism of electron transfer regulation by cytochrome c phosphorylation. We demonstrate that phosphorylation impairs long-range electron transfer, shortens the long-distance charge conduit between the partners, strengthens their interaction, and departs it from equilibrium. These results unveil a nanoscopic view of the interaction between redox protein partners in electron transport chains and its mechanisms of regulation.

Universal chemical gradient platforms using poly(methyl methacrylate) based on the biotin-streptavidin interaction for biological applications.

This article describes a simple method for the construction of a universal surface chemical gradient platform based on the biotin-streptavidin model. In this approach, surface chemical gradients were prepared in poly(methyl methacrylate) (PMMA), a biocompatible polymer, by a controlled hydrolysis procedure. The physicochemical properties of the resulting modified surfaces were extensively characterized. Chemical analysis carried out via time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) showed the formation of a smooth, highly controllable carboxylic acid gradient of increasing concentration along the sample surface. Atomic force microscopy (AFM) and contact angle (CA) results indicate that, in contrast with most of the chemical gradient methods published in the literature, the chemical modification of the polymer surface barely affects its physical properties. The introduction of carboxylic acid functionality along the surface was then used for biomolecule anchoring. For this purpose, the surface was activated and derivatized first with biotin and finally with streptavidin (SAV) in a directed orientation fashion. The SAV gradient was qualitatively assessed by fluorescence microscopy analysis and quantified by surface plasmon resonance (SPR) in order to establish a quantitative relationship between SAV surface densities and the surface location. The usefulness of the fabrication method described for biological applications was tested by immobilizing biotinylated bradykinin onto the SAV gradient. This proof-of-concept application shows the effectiveness of the concentration range of the gradient because the effects of bradykinin on cell morphology were observed to increase gradually with increasing drug concentrations. The intrinsic characteristics of the fabricated gradient platform (absence of physicochemical modifications other than those due to the biomolecules included) allow us to attribute cell behavior unequivocally to the biomolecule surface density changes.

RGD-Dendrimer-Poly(L-lactic) Acid Nanopatterned Substrates for the Early Chondrogenesis of Human Mesenchymal Stromal Cells Derived from Osteoarthritic and Healthy Donors.

Aiming to address a stable chondrogenesis derived from mesenchymal stromal cells (MSCs) to be applied in cartilage repair strategies at the onset of osteoarthritis (OA), we analyzed the effect of arginine-glycine-aspartate (RGD) density on cell condensation that occurs during the initial phase of chondrogenesis. For this, we seeded MSC-derived from OA and healthy (H) donors in RGD-dendrimer-poly(L-lactic) acid (PLLA) nanopatterned substrates (RGD concentrations of 4 × 10-9, 10-8, 2.5 × 10-8, and 10-2 w/w), during three days and compared to a cell pellet conventional three-dimensional culture system. Molecular gene expression (collagens type-I and II-COL1A1 and COL2A1, tenascin-TNC, sex determining region Y-box9-SOX9, and gap junction protein alpha 1-GJA1) was determined as well as the cell aggregates and pellet size, collagen type-II and connexin 43 proteins synthesis. This study showed that RGD-tailored first generation dendrimer (RGD-Cys-D1) PLLA nanopatterned substrates supported the formation of pre-chondrogenic condensates from OA- and H-derived human bone marrow-MSCs with enhanced chondrogenesis regarding the cell pellet conventional system (presence of collagen type-II and connexin 43, both at the gene and protein level). A RGD-density dependent trend was observed for aggregates size, in concordance with previous studies. Moreover, the nanopatterns' had a higher effect on OA-derived MSC morphology, leading to the formation of bigger and more compact aggregates with improved expression of early chondrogenic markers.

Large-Area Biomolecule Nanopatterns on Diblock Copolymer Surfaces for Cell Adhesion Studies.

Cell membrane receptors bind to extracellular ligands, triggering intracellular signal transduction pathways that result in specific cell function. Some receptors require to be associated forming clusters for effective signaling. Increasing evidences suggest that receptor clustering is subjected to spatially controlled ligand distribution at the nanoscale. Herein we present a method to produce in an easy, straightforward process, nanopatterns of biomolecular ligands to study ligand⁻receptor processes involving multivalent interactions. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that form PMMA nanodomains in a closed-packed hexagonal arrangement. Upon PMMA selective functionalization, biomolecular nanopatterns over large areas are produced. Nanopattern size and spacing can be controlled by the composition of the block-copolymer selected. Nanopatterns of cell adhesive peptides of different size and spacing were produced, and their impact in integrin receptor clustering and the formation of cell focal adhesions was studied. Cells on ligand nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that our methodology is a suitable, versatile tool to study and control receptor clustering signaling and downstream cell behavior through a surface-based ligand patterning technique.