Cell shape and function are intimately linked, in a way that is mediated by the forces exerted between cells and their environment. The relationship between cell shape and forces has been extensively studied for cells seeded on flat 2D substrates, but not for cells in more physiological 3D settings. Here, a technique called 3D micropatterned traction force microscopy (3D-mu TFM) to confine cells in 3D wells of defined shape, while simultaneously measuring the forces transmitted between cells and their microenvironment is demonstrated. This technique is based on the 3D micropatterning of polyacrylamide wells and on the calculation of 3D traction force from their deformation. With 3D-mu TFM, it is shown that MCF10A breast epithelial cells exert defined, reproducible patterns of forces on their microenvironment, which can be both contractile and extensile. Cells switch from a global contractile to extensile behavior as their volume is reduced are further shown. The technique enables the quantitative study of cell mechanobiology with full access to 3D cellular forces while having accurate control over cell morphology and the mechanical conditions of the microenvironment.
Myotonic dystrophy type 1 (DM1) is the most common hereditarymyopathy in adults. The disease is characterized by progressiveskeletal muscle degeneration that produces severe disability. There isstill no effective treatment for DM1 patients, but new therapeuticstrategies are being tested. Animal models and in vitro 2D cell cul-tures have been essential for these advances. However, these modelscannot reproduce the biological complexity of the disease. Biofab-rication tools can be applied to engineer human 3D culture systemsthat complement current preclinical research models.Here, we describe the development of the first in vitro 3D model ofDM1 human skeletal muscle. Patient-derived cells were encapsulatedin micromolded gelatin methacryloyl-carboxymethyl cellulose meth-acrylate (GelMA-CMCMA) hydrogels through photomold patterning.These hydrogels present a microstructured topography that promotesmyoblast alignment and differentiation, resulting in highly alignedmyotubes from healthy and DM1 cells. The DM1 3D microtissuespresent the molecular alterations detected in patient biopsies. Im-portantly, fusion index analyses demonstrate that 3D micropatterningsignificantly improved DM1 cell differentiation into multinucleatedmyotubes compared to standard cell cultures. Moreover, character-ization of the 3D cultures of DM1 myotubes detects a reduced thick-ness of myotubes that can be used for drug screening. Therefore, weevaluated the therapeutic effect of antagomiR-23b administration onbioengineered DM1 skeletal muscle microtissues. AntagomiR-23btreatment rescues both molecular DM1 hallmarks and structural phe-notype, restoring myotube diameter to healthy control sizes. Overall,these new microtissues represent an improvement over conventionalmodels and can be used as biomimetic platforms to establish preclin-ical studies for myotonic dystrophy.
Myotonic dystrophy type 1 (DM1) is the most common hereditary myopathy in the adult population. The disease is characterized by progressive skeletal muscle degeneration that produces severe disability. At present, there is still no effective treatment for DM1 patients, but the breakthroughs in understanding the molecular pathogenic mechanisms in DM1 have allowed the testing of new therapeutic strategies. Animal models and in vitro two-dimensional cell cultures have been essential for these advances. However, serious concerns exist regarding how faithfully these models reproduce the biological complexity of the disease. Biofabrication tools can be applied to engineer human three-dimensional (3D) culture systems that complement current preclinical research models. Here, we describe the development of the first in vitro 3D model of DM1 human skeletal muscle. Transdifferentiated myoblasts from patient-derived fibroblasts were encapsulated in micromolded gelatin methacryloyl-carboxymethyl cellulose methacrylate hydrogels through photomold patterning on functionalized glass coverslips. These hydrogels present a microstructured topography that promotes myoblasts alignment and differentiation resulting in highly aligned myotubes from both healthy and DM1 cells in a long-lasting cell culture. The DM1 3D microtissues recapitulate the molecular alterations detected in patient biopsies. Importantly, fusion index analyses demonstrate that 3D micropatterning significantly improved DM1 cell differentiation into multinucleated myotubes compared to standard cell cultures. Moreover, the characterization of the 3D cultures of DM1 myotubes detects phenotypes as the reduced thickness of myotubes that can be used for drug testing. Finally, we evaluated the therapeutic effect of antagomiR-23b administration on bioengineered DM1 skeletal muscle microtissues. AntagomiR-23b treatment rescues both molecular DM1 hallmarks and structural phenotype, restoring myotube diameter to healthy control sizes. Overall, these new microtissues represent an improvement over conventional cell culture models and can be used as biomimetic platforms to establish preclinical studies for myotonic dystrophy.
Mounting evidence supports the importance of the intestinal epithelial barrier and its permeability both in physiological and pathological conditions. Conventional in vitro models to evaluate intestinal permeability rely on the formation of tightly packed epithelial monolayers grown on hard substrates. These two-dimensional (2D) models lack the cellular and mechanical components of the non-epithelial compartment of the intestinal barrier, the stroma, which are key contributors to the barrier permeability in vivo. Thus, advanced in vitro models approaching the in vivo tissue composition are fundamental to improve precision in drug absorption predictions, to provide a better understanding of the intestinal biology, and to faithfully represent related diseases. Here, we generate photo-crosslinked gelatine methacrylate (GelMA) - poly(ethylene glycol) diacrylate (PEGDA) hydrogel co-networks that provide the required mechanical and biochemical features to mimic both the epithelial and stromal compartments of the intestinal mucosa, i.e., they are soft, cell adhesive and cell-loading friendly, and suitable for long-term culturing. We show that fibroblasts can be embedded in the GelMA-PEGDA hydrogels while epithelial cells can grow on top to form a mature epithelial monolayer that exhibits barrier properties which closely mimic those of the intestinal barrier in vivo, as shown by the physiologically relevant transepithelial electrical resistance (TEER) and permeability values. The presence of fibroblasts in the artificial stroma compartment accelerates the formation of the epithelial monolayer and boosts the recovery of the epithelial integrity upon temporary barrier disruption, demonstrating that our system is capable of successfully reproducing the interaction between different cellular compartments. As such, our hydrogel co-networks offer a technologically simple yet sophisticated approach to produce functional three-dimensional (3D) in vitro models of epithelial barriers with epithelial and stromal cells arranged in a spatially relevant manner and near-physiological functionality.
The stiffness and topography of a cell's extracellular matrix are physical cues that play a key role in regulating processes that determine cellular fate and function. While substrate stiffness can dictate cell differentiation lineage, migration, and self-organization, topographical features can change the cell's differentiation profile or migration ability. Although both physical cues are present and intrinsic to the native tissues in vivo, in vitro studies have been hampered by the lack of technological set-ups that would be compatible with cell culture and characterization. In vitro studies therefore either focused on screening stiffness effects in cells cultured on flat substrates or on determining topography effects in cells cultured onto hard materials. Here, we present a reliable, microfabrication method to obtain well defined topographical structures of micrometer size (5-10 µm) on soft polyacrylamide hydrogels with tunable mechanical stiffness (3-145 kPa) that closely mimic the in vivo situation. Topographically microstructured polyacrylamide hydrogels are polymerized by capillary force lithography using flexible materials as molds. The topographical microstructures are resistant to swelling, can be conformally functionalized by extracellular matrix proteins and sustain the growth of cell lines (fibroblasts and myoblasts) and primary cells (mouse intestinal epithelial cells). Our method can independently control stiffness and topography, which allows to individually assess the contribution of each physical cue to cell response or to explore potential synergistic effects. We anticipate that our fabrication method will be of great utility in tissue engineering and biophysics, especially for applications where the use of complex in vivo-like environments is of paramount importance.
Epithelial tissues contain three-dimensional (3D) complex microtopographies that are essential for proper performance. These microstructures provide cells with the physicochemical cues needed to guide their self-organization into functional tissue structures. However, most in vitro models do not implement these 3D architectural features. The main problem is the availability of simple fabrication techniques that can reproduce the complex geometries found in native tissues on the soft polymeric materials required as cell culture substrates. In this study reaction-diffusion mediated photolithography is used to fabricate 3D microstructures with complex geometries on poly(ethylene glycol)-based hydrogels in a single step and moldless approach. By controlling fabrication parameters such as the oxygen diffusion/depletion timescales, the distance to the light source and the exposure dose, the dimensions and geometry of the microstructures can be well-defined. In addition, copolymerization of poly(ethylene glycol) with acrylic acid improves control of the dynamic reaction-diffusion processes that govern the free-radical polymerization of highly-diluted polymeric solutions. Moreover, acrylic acid allows adjusting the density of cell adhesive ligands while preserving the mechanical properties of the hydrogels. The method proposed is a simple, single-step, and cost-effective strategy for producing models of intestinal epithelium that can be easily integrated into standard cell culture platforms.
Valls-Margarit, M., Iglesias-García, O., Di Guglielmo, C., Sarlabous, L., Tadevosyan, K., Paoli, R., Comelles, J., Blanco-Almazán, D., Jiménez-Delgado, S., Castillo-Fernández, O., Samitier, J., Jané, R., Martínez, Elena, Raya, Á., (2019). Engineered macroscale cardiac constructs elicit human myocardial tissue-like functionalityStem Cell Reports 13, (1), 207-220
In vitro surrogate models of human cardiac tissue hold great promise in disease modeling, cardiotoxicity testing, and future applications in regenerative medicine. However, the generation of engineered human cardiac constructs with tissue-like functionality is currently thwarted by difficulties in achieving efficient maturation at the cellular and/or tissular level. Here, we report on the design and implementation of a platform for the production of engineered cardiac macrotissues from human pluripotent stem cells (PSCs), which we term “CardioSlice.” PSC-derived cardiomyocytes, together with human fibroblasts, are seeded into large 3D porous scaffolds and cultured using a parallelized perfusion bioreactor with custom-made culture chambers. Continuous electrical stimulation for 2 weeks promotes cardiomyocyte alignment and synchronization, and the emergence of cardiac tissue-like properties. These include electrocardiogram-like signals that can be readily measured on the surface of CardioSlice constructs, and a response to proarrhythmic drugs that is predictive of their effect in human patients.
Cell motility is an important phenomenon in cell biology, developmental biology, and cancer. Here we report methods that we designed to identify and characterize external factors which direct cell motions by breaking locally the symmetry. We used microfabrication and microfluidics techniques to impose and combine mechanical and chemical cues to moving fibroblasts. Gradients can thereby be engineered at the cellular scale and this approach has allowed to disentangle roles of the nucleus and protrusion activity in setting cell directions.
Cell migration is a crucial event during development and in disease. Mechanical constraints and chemical gradients can contribute to the establishment of cell direction, but their respective roles remain poorly understood. Using a microfabricated topographical ratchet, we show that the nucleus dictates the direction of cell movement through mechanical guidance by its environment. We demonstrate that this direction can be tuned by combining the topographical ratchet with a biochemical gradient of fibronectin adhesion. We report competition and cooperation between the two external cues. We also quantitatively compare the measurements associated with the trajectory of a model that treats cells as fluctuating particles trapped in a periodic asymmetric potential. We show that the cell nucleus contributes to the strength of the trap, whereas cell protrusions guided by the adhesive gradients add a constant tunable bias to the direction of cell motion.
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/cm2) with a shallow slope (0.9 pmol/cm3). 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.
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/cm2), with a threshold value of 4.0 pmol/cm2 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.
The excellent self-assembling properties of DNA and the excellent specificity of the antibodies to detect analytes of small molecular weight under competitive conditions have been combined in this study. Three oligonucleotide sequences (N(1)up, N(2)up, and N(3)up) have been covalently attached to three steroidal haptens (8, hG, and 13) of three anabolic-androgenic steroids (AAS), stanozolol (ST), tetrahydrogestrinone (THG), and boldenone (B), respectively. The synthesis of steroid oligonucleotide conjugates has been performed by the reaction of oligonucleotides carrying amino groups with carboxyl acid derivatives of steroidal haptens. Due to the chemical nature of the steroid derivatives, two methods for coupling the haptens and the ssDNA have been studied: a solid-phase coupling strategy and a solution-phase coupling strategy. Specific antibodies against ST, THG, and B have been used in this study to asses the possibility of using the self-assembling properties of the DNA to prepare biofunctional SPR gold chips based on the immobilization of haptens, by hybridization with the complementary oligonucleotide strands possessing SH groups previously immobilized. The capture of the steroid oligonucleotide conjugates and subsequent binding of the specific antibodies can be monitored on the sensogram due to variations produced on the refractive index on top of the gold chip. The resulting steroid oligonucleotide conjugates retain the hybridization and specific binding properties of oligonucleotides and haptens as demonstrated by thermal denaturation experiments and surface plasmon resonance (SPR).
In this work, we propose an easy method to produce highly tunable gradients of covalently bound proteins on topographically modified poly(methyl methacrylate). We used a rnicrofluidic approach to obtain linear gradients with high slope (0.5 pmol.cm(-2).mm(-1)), relevant at the single-cell level. These protein gradients were characterized using fluorescence microscopy and surface plasmon resonance. Both experimental results and theoretical modeling on the protein gradients generated have proved them to be highly reproducible, stable up to 7 days, and easily tunable. This method enables formation of versatile cell culture platforms combining both complex biochemical and physical cues in an attempt to approach in vitro cell culture methods to in vivo cellular microenvironments.
Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. However, novel applications in the biosensor field require materials to be compatible with cell growth and at the same time be suitable for technological processing. Technological polymers are key materials in the fabrication of disposable parts and other sensing elements. As such, it is essential to characterize the surface properties of technological polymers, especially after processing and sterilization. It is also important to understand how technological polymers affect cell behavior when in contact with polymer materials. Therefore, the aim of this research was to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly (methyl methacrylate), polystyrene, and poly(dimethylsiloxane). Glass was used as the control material. From the Clinical Editor: Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. The aim of this research is to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly(methylmethacrylate) (PMMA), polystyrene (PS), and poly(dimethylsiloxane) (PDMS).
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) (PM MA), 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 (ToRSIMS) 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 (SA V) 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.
New fabrication technologies and, in particular, new nanotechnologies have provided biomaterial and biomedical scientists with enormous possibilities when designing customized supports and scaffolds with controlled nanoscale topography and chemistry. The main issue now is how to effectively design these components and choose the appropriate combination of structure and chemistry to tailor towards applications as challenging and complex as stem cell differentiation. Occasionally, an incomplete knowledge of the fundamentals of biological differentiation process has hampered this issue. However, the recent technological advances in creating controlled cellular microenvironments can be seen as a powerful tool for furthering fundamental biology studies. This article reviews the main strategies followed to achieve solutions to this challenge, particularly emphasizing the working hypothesis followed by the authors to elucidate the mechanisms behind the observed effects of structured surfaces on cell behavior.
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Faure, LM, Gómez-González,, M, Baguer, O, Comelles, J, Martínez, E, Arroyo, M, Trepat, X, Roca-Cusachs, P, (2024). 3D Micropatterned Traction Force Microscopy: A Technique to Control 3D Cell Shape While Measuring Cell-Substrate Force Transmission Advanced Science 11, 2406932 
Methods in Cell Biology - Biophysical Methods in Cell Biology (ed. Wilson, L., Tran, P.), Academic Press (Santa Barbara, USA) 125, 437-452