Hydrogel co-networks of gelatine methacrylate and poly(ethylene glycol) diacrylate sustain 3D functional in vitro models of intestinal mucosa

Abstract

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 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.

A simple DLP-bioprinting strategy produces cell-laden crypt-villous structures for an advanced 3D gut model

“…GelMA-PEGDA hydrogels bioprinted with the low cost DLP-SLP instrument sustain cell culture effectively NIH-3T3 mouse embryonic fibroblasts were chosen to validate the suitability of our setup for bioprinting, since they are commonly used for cell encapsulation experiments (13,56). In addition, these cells represent the fibroblast cell population within the stromal compartment of our intestinal model.…”

“…While in recent years there has been a lot of efforts to produce engineered tissues that recapitulate the architecture of the intestinal tissue, in most of the cases those have been limited to represent only the epithelium. Mostly, this is due to the use of complex fabrication methods based on replica moulding that are not friendly with embedding cells within the matrix (11, 12) or the use of relatively high dense matrices that do not allow cell survival (13, 14). Recently, some attempts have been made to include fibroblasts to mimic the stromal tissue in flat constructs (15), demonstrating the relevance of testing drug absorption in the presence of a full mucosa tissue.…”

“…Despite its good properties, when used at low concentrations, GelMA hydrogels are not mechanically stable, compromising the geometrical shape of the designs and lifespan of the printed constructs (36). Previous works have highlighted the benefits of combining PEG and GelMA polymers for the fabrication of cell-laden scaffolds for in vitro studies (13). Such combinations have also been successfully used as bioinks in 3D printing (37, 38).…”

Advanced Hydrogels as Exosome Delivery Systems for Osteogenic Differentiation of MSCs: Application in Bone Regeneration

“…Nevertheless, the incorporation and amplification of various cells in GelMA hydrogels have been used extensively due to their combined properties of mechanical characteristics, the presence of bioactive peptide sequences, and biocompatibility [31]. It has been shown that a combination of poly ethylene glycol diacrylate and GelMA (PEGDA/GelMA) creates a hydrogel with stronger mechanical properties and appropriate degradation rates compared with pure GelMA hydrogel [32]. Moreover, the incorporation and proliferation of various cell types in GelMA hydrogels have been extensively used.…”

Nanoconfined Crosslinked Poly(ionic liquid)s with Unprecedented Selective Swelling Properties Obtained by Alkylation in Nanophase-Separated Poly(1-vinylimidazole)-l-poly(tetrahydrofuran) Conetworks

“…Nanoconfined material structures, including polymers as well, due to the wide range of unique structural features, properties, and effects arising under nanocompartmentalized conditions, have created significant worldwide interest over the last couple of years (see e.g., references [ 1 , 2 , 3 , 4 , 5 , 6 ] and references therein). The recently emerged polymer conetworks, especially amphiphilic conetworks (APCNs), composed of covalently or supramolecularly bonded, otherwise immiscible, hydrophilic and hydrophobic polymer chains, belong to such nanophase-separated materials with mutually nanoconfined macromolecular components [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ]. Considering that polymers containing imidazole rings, which also play a pivotal role in the major biopolymers and compounds of living organisms, such as DNA, RNA, proteins, enzymes, hormones, vitamins, etc., provide various advantageous properties, like water solubility, strong metal ion coordinating ability, protonation, and alkylation possibilities, we have recently explored the synthesis, nanophasic structure, and properties of poly(1-vinylimidazole)- l -poly(tetrahydrofuran) (PVIm- l -PTHF) and poly(1-vinylimidazole)- l -poly(propylene...…”