Staff member

Tissue engineering holds great promise for biomedical research and healthcare, offering alternatives to animal models and enabling tissue regeneration and organ transplantation. Three-dimensional (3D) bioprinting stands out for its design flexibility and reproducibility. Here, we present an integrated fluorescent light sheet bioprinting and imaging system that combines high printing speed (0.66 mm3 /s) and resolution (9 μm) with light sheet-based imaging. This approach employs direct laser patterning and a static light sheet for confined voxel crosslinking in photocrosslinkable materials. The developed bioprinter enables real-time monitoring of hydrogel crosslinking using fluorescent recovery after photobleaching (FRAP) and brightfield imaging as well as in situ light sheet imaging of cells. Human fibroblasts encapsulated in a thiol-ene click chemistry-based hydrogel exhibited high viability (83% ± 4.34%) and functionality. Furthermore, full-thickness skin constructs displayed characteristics of both epidermal and dermal layers and remained viable for 41 days. The integrated approach demonstrates the capabilities of light sheet bioprinting, offering high speed, resolution, and real-time characterization. Future enhancements involving solid-state laser scanning devices such as acousto-optic deflectors and modulators will further enhance resolution and speed, opening new opportunities in light-based bioprinting and advancing tissue engineering. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

JTD Keywords: Biofabrication, Full-thickness skin model, Light sheet bioprinter, Light sheet fluorescence microscopy, Tissue engineering

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

The small intestine is a complex organ with a characteristic architecture and a major site for drug and nutrient absorption. The three-dimensional (3D) topography organized in finger-like protrusions called villi increases surface area remarkably, granting a more efficient absorption process. The intestinal mucosa, where this process occurs, is a multilayered and multicell-type tissue barrier. In vitro intestinal models are routinely used to study different physiological and pathological processes in the gut, including compound absorption. Still, standard models are typically two-dimensional (2D) and represent only the epithelial barrier, lacking the cues offered by the 3D architecture and the stromal components present in vivo, often leading to inaccurate results. In this work, we studied the impact of the 3D architecture of the gut on drug transport using a bioprinted 3D model of the intestinal mucosa containing both the epithelial and the stromal compartments. Human intestinal fibroblasts were embedded in a previously optimized hydrogel bioink, and enterocytes and goblet cells were seeded on top to mimic the intestinal mucosa. The embedded fibroblasts thrived inside the hydrogel, remodeling the surrounding extracellular matrix. The epithelial cells fully covered the hydrogel scaffolds and formed a uniform cell layer with barrier properties close to in vivo. In particular, the villus-like model revealed overall increased permeability compared to a flat counterpart composed by the same hydrogel and cells. In addition, the efflux activity of the P-glycoprotein (P-gp) transporter was significantly reduced in the villus-like scaffold compared to a flat model, and the genetic expression of other drugs transporters was, in general, more relevant in the villus-like model. Globally, this study corroborates that the presence of the 3D architecture promotes a more physiological differentiation of the epithelial barrier, providing more accurate data on drug absorbance measurements.Copyright © 2023. Published by Elsevier B.V.

JTD Keywords: 3d architecture, alkaline-phosphatase, caco-2 cells, culture, drug development, efflux proteins, gene-expression, human-colon, intestinal absorption, intestinal models, microenvironment, paracellular transport, permeability, photopolymerization, villi, 3d architecture, 3d bioprinting, Drug development, In-vitro, Intestinal absorption, Intestinal models, Photopolymerization, Villi

There is an increasing evidence that the tissue architecture has astrong influence on cell behavior and it is key to obtain functionaltissue replicates (1). Hence, future improvements in the tissue en-gineering field will encompass tailoring three-dimensional (3D) cellmicroenvironments with the precision required to mimic in vivofeatures.3D printing techniques have been positioned as a feasible alter-native to conventional manufacturing techniques to achieve properspatial resolution in a high-throughput manner, mainly due to highadaptability and reduced costs, becoming one of the current hottopics in applied research (2,3). However, requirements in terms ofresolution, mechanical properties, or biocompatibility to obtainbiomimetic 3D tissues demand further innovative approaches.Here we present a fast, low cost and versatile printing methodbased on visible light polymerization for the fabrication of 3Dbioengineered substrates and cell-friendly interfaces using photo-polymerizable hydrogel-based bioinks. These bioinks are highlyS-218ABSTRACTStransparent and have low molecular content, mimicking the softmechanical properties of the tissues.With the appropriate combination of bioink composition, accurateselection of the printing parameters, and optimized designs we suc-ceeded with prints for different applications, including cell culturesubstrates, microfluidic channels and tissue constructs for in vitroassays, compatible with the standard cell culture techniques. As aproof-of-concept, we developed functional scaffolds mimicking the3D microstructure of the small intestine, containing both the epi-thelial and stromal compartments, using cell-laden bioinks.Through this versatile top-down technique we demonstrated thepotential of the light-based 3D bioprinting technology, contributingon providing alternatives beyond tissue engineering state-of-the-art.

JTD

The intestinal epithelium is formed by villi and crypts. Intestinalstem cells (ISCs) located at the crypt base divide giving rise toproliferative cells that migrate up along the villi while differentiating,ultimately dying at the tips of the villi. This homeostasis is tightlycontrolled by biomolecular gradients of EGF, Wnt and BMP sig-naling pathways along the crypt-villus axis1. Intestinal organoids,despite including many physiologically relevant features, are notvalid cultures when access to the lumen is required. Here we present aculture platform that overcomes this limitation while comprising allkey features of the intestinal epithelium: 3D architecture, prolifera-tive and differentiated cell domains, and gradients of ISCs nichebiomolecules.Employing a simple photolithographic technique2, we fabricatedpoly(ethylene) glycol diacrylate (PEGDA) 3D villus-like scaffolds.We developed in silico models to simulate gradients of ISCs nichebiomolecules, we created them through the hydrogels by free diffusionand we characterized them by Light-sheet fluorescence microscopy.Organoid-derived intestinal epithelial cells covered the wholescaffold surface. The gradients profile and composition, constantover time, impacted on cell behavior by modifying the proportionand positioning of the different intestinal epithelial cell types alongthe vertical axis of our scaffold, faithfully recreating in vivo cellcompartmentalization.We have developed an apically accessible and 3D in vitro intes-tinal epithelial model, which bears biomolecular ISC niche gradientsand all relevant epithelial cell types. Therefore, we believe our modelcan be employed in many applications, particularly in the study ofintestinal epithelium biology in physiological and pathologicalconditions.

JTD

Three-dimensional bioprinting (3D bioprinting) has been at theforefront of tissue engineering research in the past years, with evermore efficient systems reaching the market(1). While existing 3Dbioprinting techniques are numerous and varied, they are limited bylong printing times when used at high resolution(2). The techniquedescribed in this work aims at enabling fast and accurate productionof monolayered skin constructs.To achieve shorter production times, a digital scanned light sheetis used to produce patterns of polymerized hydrogel, which enablesthe printing of a full three-dimensional plane in a matter of a fewhundred milliseconds. The high resolution resides in the properties ofthe light sheet itself – the width of the light sheet represents the z-axial resolution of the system (as low as 10mm) and the x-axialresolution is determined by the intensity profile of the gaussian beam(around 50mm). In order to fully exploit this system, the hydrogelused to encapsulate the cells must therefore be tailor-made for pho-toactivated cross-linking.As a proof of concept, a light sheet microscope is used as a po-lymerization source for novel photosensitive hydrogels. The up-coming hardware, software, chemical and biological improvementsneeded to reach the full potential of this system are expected toeventually be sufficient to print a complete skin construct, whichcould be used in the drug development industry, or as a graft forregenerative medicine therapy. Additionally, the constructs can beused to reduce and even replace animal testing for drug or cosmetictesting.

JTD Keywords: 3d bioprinting, Light sheet microscopy, Stereolithography

Skin is the largest tissue of the human body and represents ourdaily barrier against the external environment. Nowadays, mimick-ing the complex skin structure with high spatial resolution is one ofthe most important challenges of tissue engineering, for skin re-placement in transplantations, drug testing and disease modelling(1,2). Here we present a novel approach based on visible-light 3Dbio-printing to produce skin tissue models as faithful as possible tothe in-vivo structure. Hydrogels scaffolds are the best choice to echothe perfect cell microenvironment due to their biocompatibility andtheir tunable properties. A Direct Laser Writing system (DLW),equipped with a 405 nm wavelength laser diode and automatedtranslational stages, is used to print photo-polymerizable hydrogels,based on Norbornene thiol-based reactions (3), preserving bothmechanical and biochemical properties of cell microenvironmentand reproducing skin heterogenous architecture. Different polymercompositions were evaluated in terms of printing morphology andstability. Cell-laden scaffolds with embedded human skin fibroblastswere fabricated and kept in standard culture conditions for severaldays, showing cell spreading and migration, resulting in high cellviability values. Co-culture systems studies, including both fibro-blasts and keratinocytes, containing RGD cell-friendly motifs topromote cell attachment on the scaffolds’ surface, have been per-formed showing cellular compatibility. Furthermore, immunostain-ing results revealed an enhanced production of extracellular matrix(ECM) proteins compared to single fibroblasts cultures, proving thatthe combination of photo-crosslinkable hydrogels based on thiol-Norbornene chemistry and co-culture systems provides promisingskin-like models.

JTD

The intestinal epithelium is characterized by a complex micro-scale topography formed by finger-like protrusions called villi andinvaginations called crypts. This organized three-dimensional (3D)architecture together with the soft mechanical properties of the in-testinal tissue are essential for cell behavior and tissue function.However, the experimental in vitro modeling of the delicate intes-tinal architecture is limited due to the difficulty in microfabricatingsoft materials with complex 3D geometries, high aspect ratio andcurvature using efficient and simple methods. In this work, we fab-ricated soft hydrogel scaffolds that accurately mimic the villus andcrypt morphologies of the intestinal epithelium using a simple andmoldless approach (1).We used mixtures of polyethylene glycol diacrylate (PEGDA)and acrylic acid (AA), or PEGDA and gelatin methacryloyl (Gel-MA) to fabricate 3D hydrogels by a single-step photolithographyprocess. Different photomasks were used for the fabrication of themicropillars mimicking the intestinal villi and the crypt-like in-vaginations. The dimensions of the villi and crypts were easilyfine-tuned just by changing the energy dose. The scaffolds weredirectly microfabricated onto permeable membranes, enablingtheir assembly into cell culture inserts. Intestinal epithelial cellslines or organoid-derived primary cells covered the biomimeticscaffolds and form polarized monolayers with proper barrierproperties. The use of PEGDA-GelMA blends also allowed thefabrication of cell-laden scaffolds that mimic the intestinal stromalcompartment.Through these results, we demonstrated that this microfabricationtechnology is a promising tool to faithfully replicate the intestinalmucosa topography with improved performance, while keepingcompatibility with standard cell culture techniques.

JTD

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

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.

JTD

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.

JTD

Epithelial tissues are composed of layers of tightly connected cells shaped into complex three-dimensional (3D) structures such as cysts, tubules, or invaginations. These complex 3D structures are important for organ-specific functions and often create biochemical gradients that guide cell positioning and compartmentalization within the organ. One of the main functions of epithelia is to act as physical barriers that protect the underlying tissues from external insults. In vitro, epithelial barriers are usually mimicked by oversimplified models based on cell lines grown as monolayers on flat surfaces. While useful to answer certain questions, these models cannot fully capture the in vivo organ physiology and often yield poor predictions. In order to progress further in basic and translational research, disease modeling, drug discovery, and regenerative medicine, it is essential to advance the development of new in vitro predictive models of epithelial tissues that are capable of representing the in vivo-like structures and organ functionality more accurately. Here, we review current strategies for obtaining biomimetic systems in the form of advanced in vitro models that allow for more reliable and safer preclinical tests. The current state of the art and potential applications of self-organized cell-based systems, organ-on-a-chip devices that incorporate sensors and monitoring capabilities, as well as microfabrication techniques including bioprinting and photolithography, are discussed. These techniques could be combined to help provide highly predictive drug tests for patient-specific conditions in the near future.

JTD Keywords: 3D cell culture models, Biofabrication, Disease modeling, Drug screening, Epithelial barriers, Microengineered tissues, Organ-on-a-chip, Organoids

Suspended planar-array (SPA) chips embody millions of individual miniaturized arrays to work in extremely small volumes. Here, the basis of a robust methodology for the fabrication of SPA silicon chips with on-demand physical and chemical anisotropies is demonstrated. Specifically, physical traits are defined during the fabrication process with special focus on the aspect ratio, branching, faceting, and size gradient of the final chips. Additionally, the chemical attributes augment the functionality of the chips with the inclusion of complete coverage or patterns of selected biomolecules on the surface of the chips with contact printing techniques, offering an extremely high versatility, not only with the choice of the pattern shape and distribution but also in the choice of biomolecular inks to pattern. This approach increases the miniaturization of printed arrays in 3D structures by two orders of magnitude compared to those previously demonstrated. Finally, functional micrometric and sub-micrometric patterned features are demonstrated with an antibody binding assay with the recognition of the printed spots with labeled antibodies from solution. The selective addition of physical and chemical attributes on the suspended chips represents the basis for future biomedical assays performed within extremely small volumes.

JTD Keywords: Microcontact printing, Microparticles, Molecular multiplexing, Polymer pen lithography, Silicon chip technology

A novel suspended planar-array chips technology is described, which effectively allows molecular multiplexing using a single suspended chip to analyze extraordinarily small volumes. The suspended chips are fabricated by combining silicon-based technology and polymer-pen lithography, obtaining increased molecular pattern flexibility, and improving miniaturization and parallel production. The chip miniaturization is so dramatic that it permits the intracellular analysis of living cells.

JTD Keywords: Chip-in-a-cell, Suspended-arrays, Planar-arrays, Silicon chips, Single-cell analysis