Publications

The intestine is a complex tissue with a characteristic three-dimensional (3D) crypt-villus architecture, which plays a key role in the intestinal function. This function is also regulated by the intestinal stroma that actively supports the intestinal epithelium, maintaining the homeostasis of the tissue. Efforts to account for the 3D complex structure of the intestinal tissue have been focused mainly in mimicking the epithelial barrier, while solutions to include the stromal compartment are scarce and unpractical to be used in routine experiments. Here we demonstrate that by employing an optimized bioink formulation and the suitable printing parameters it is possible to produce fibroblast-laden crypt-villus structures by means of digital light projection stereolithography (DLP-SLA). This process provides excellent cell viability, accurate spatial resolution, and high printing throughput, resulting in a robust biofabrication approach that yields functional gut mucosa tissues compatible with conventional testing techniques.Copyright © 2023 Elsevier B.V. All rights reserved.

JTD Keywords: 3d microstructure, barrier, cells, epithelial-stromal interactions, gelma-pegda soft hydrogels, growth, hydrogel, intestinal mucosa model, methacrylamide, microfabrication, proliferation, scaffold, stereolithography, 3d bioprinting, 3d microstructure, Epithelial-stromal interactions, Fibroblasts, Gelma-pegda soft hydrogels, Intestinal mucosa model

Organ-on-chip platforms combined with high-throughput sensing technology allow bridging gaps in information presented by 2D cultures modeled on static microphysiological systems. While these platforms do not aim to replicate whole organ systems with all physiological nuances, they try to mimic relevant structural, physiological, and functional features of organoids and tissues to best model disease and/or healthy states. The advent of this platform has not only challenged animal testing but has also presented the opportunity to acquire real-time, high-throughput data about the pathophysiology of disease progression by employing biosensors. Biosensors allow monitoring of the release of relevant biomarkers and metabolites as a result of physicochemical stress. It, therefore, helps conduct quick lead validation to achieve personalized medicine objectives. The organ-on-chip industry is currently embarking on an exponential growth trajectory. Multiple pharmaceutical and biotechnology companies are adopting this technology to enable quick patient-specific data acquisition at substantially low costs.

JTD Keywords: A-chip, Biosensor, Biosensors, Cancer, Cells, Culture, Disease models, Epithelial electrical-resistance, Hydrogel, Microfabrication, Microphysiological systems, Models, Niches, Organ-on-a-chips, Platform

Biomolecular gradients are widely present in multiple biological processes. Historically they were reproduced in vitro by using micropipettes, Boyden and Zigmond chambers, or hydrogels. Despite the great utility of these setups in the study of gradient-related problems such as chemotaxis, they face limitations when trying to translate more complex in vivo-like scenarios to in vitro systems. In the last 20 years, the advances in manufacturing of micromechanical systems (MEMS) had opened the possibility of applying this technology to biology (BioMEMS). In particular, microfluidics has proven extremely efficient in setting-up biomolecular gradients which are stable, controllable, reproducible and at length scales that are relevant to cells. In this chapter, we give an overview of different methods to generate molecular gradients using microfluidics, then we discuss the different steps of the pipeline to fabricate a gradient generator microfluidic device, and at the end, we show an application example of the fabrication of a microfluidic device that can be used to generate a surface-bound biomolecular gradient.© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.

JTD Keywords: biomems, gradient, microfluidics, model, nanotechnology, proteins, Biomems, Gradient, Mechanisms, Microfabrication, Microfluidics, Nanotechnology

Tissue barriers play a crucial role in human physiology by establishing tissue compartmentalization and regulating organ homeostasis. At the interface between the extracellular matrix (ECM) and flowing fluids, epithelial and endothelial barriers are responsible for solute and gas exchange. In the past decade, microfluidic technologies and organ-on-chip devices became popular as in vitro models able to recapitulate these biological barriers. However, in conventional microfluidic devices, cell barriers are primarily grown on hard polymeric membranes within polydimethylsiloxane (PDMS) channels that do not mimic the cell-ECM interactions nor allow the incorporation of other cellular compartments such as stromal tissue or vascular structures. To develop models that accurately account for the different cellular and acellular compartments of tissue barriers, researchers have integrated hydrogels into microfluidic setups for tissue barrier-on-chips, either as cell substrates inside the chip, or as self-contained devices. These biomaterials provide the soft mechanical properties of tissue barriers and allow the embedding of stromal cells. Combining hydrogels with microfluidics technology provides unique opportunities to better recreate in vitro the tissue barrier models including the cellular components and the functionality of the in vivo tissues. Such platforms have the potential of greatly improving the predictive capacities of the in vitro systems in applications such as drug development, or disease modeling. Nevertheless, their development is not without challenges in their microfabrication. In this review, we will discuss the recent advances driving the fabrication of hydrogel microfluidic platforms and their applications in multiple tissue barrier models.

JTD Keywords: hydrogel, microfabrication, microfluidics, organ-on-chip, tissue barrier, Hydrogel, Microfabrication, Microfluidics, Organ-on-chip, Tissue barrier

The fabrication of hierarchical and bioactive self-supporting membranes, which integrate physical and biomolecular elements, using a single-step process that combines molecular self-assembly with soft lithography is reported. A positively charged multidomain peptide (with or without the cell-adhesive sequence arginine-glycine-aspartic acid-serine (RGDS)) self-assembles with hyaluronic acid (HA), an anionic biopolymer. Optimization of the assembling conditions enables the realization of membranes with well-controlled and easily tunable features at multiple size scales including peptide sequence, building-block co-assembly, membrane thickness, bioactive epitope availability, and topographical pattern morphology. Membrane structure, morphology, and bioactivity are investigated according to temperature, assembly time, and variations in the experimental setup. Furthermore, to evaluate the physical and biomolecular signaling of the self-assembled microfabricated membranes, rat mesenchymal stem cells are cultured on membranes exhibiting various densities of RGDS and different topographical patterns. Cell adhesion, spreading, and morphology are significantly affected by the surface topographical patterns and the different concentrations of RGDS. The versatility of the combined bottom-up and top-down fabrication processes described may permit the development of hierarchical macrostructures with precise biomolecular and physical properties and the opportunity to fine tune them with spatiotemporal control.

JTD Keywords: Membrane scaffolds, Mesenchymal stem cells, Microfabrication, Self-assembly, Topography

A method for the simultaneous (bio)chemical and topographical patterning of enclosed structures in poly(dimethyl siloxane) (PDMS) is presented. The simultaneous chemical and topography transference uses a water-soluble chitosan sacrificial mold to impart a predefined pattern with micrometric accuracy to a PDMS replica. The method is compared to conventional soft-lithography techniques on planar surfaces. Its functionality is demonstrated by the transference of streptavidin directly to the surface of the three-dimensional PDMS structures as well as indirectly using streptavidin-loaded latex nanoparticles. The streptavidin immobilized on the PDMS is tested for bioactivity by coupling with fluorescently labeled biotin. This proves that the streptavidin is immobilized on the PDMS surface, not in the bulk of the polymer, and is therefore accessible for use as signaling/binding element in micro and bioengineering. The use of a biocompatible polymer and processes enables the technique to be used for the chemical patterning of tissue constructions.

JTD Keywords: Biotechnology, Chitosan, Microfabrication, MEMs, Soft lithography

The shape and dimensions of an atomic force microscope tip are crucial factors to obtain high resolution images at the nanoscale. When measuring samples with narrow trenches, inclined sidewalls near 90 or nanoscaled structures, standard silicon atomic force microscopy (AFM) tips do not provide satisfactory results. We have combined deep reactive ion etching (DRIE) and focused ion beam (FIB) lithography techniques in order to produce probes with sharp rocket-shaped silicon AFM tips for high resolution imaging. The cantilevers were shaped and the bulk micromachining was performed using the same DRIE equipment. To improve the tip aspect ratio we used FIB nanolithography technique. The tips were tested on narrow silicon trenches and over biological samples showing a better resolution when compared with standard AFM tips, which enables nanocharacterization and nanometrology of high-aspect-ratio structures and nanoscaled biological elements to be completed, and provides an alternative to commercial high aspect ratio AFM tips.

JTD Keywords: Atomic-Force Microscope, Carbon nanotube tips, Probes, Roughness, Cells, Microfabrication, Calibration, Surfaces