Engineering poly (ethylene glycol) diacrylate-based microstructures to develop an in vitro model of small intestinal epithelium

Abstract

Most of the current in vitro cell culture models do not reproduce the anatomy of tissues and physiological behavior of cells in vivo and provide misleading results when compared to the real tissue. Microfabrication technologies can be used to go one step beyond the conventional 2D in vitro tissue culture models and reliably reproduce in vivo tissue microenvironments.1 For the small intestinal epithelium, this approach has been explored through the fabrication of collagen and poly (lactic-co-glycolic acid) villi-like scaffolds, using sequential micromolding.2–4 Although that has been an innovative and interesting approach, the nature of the employed materials does not allow fine-tuning of the system properties and the molding method is laborious, requiring several intermediate molding steps. Here we describe a simple and cost-effective method to fabricate soft 3D villi-like microstructures with the anatomical architecture. We use poly (ethylene glycol) diacrylated (PEGDA) and acrylic acid that copolymerize and form soft hydrogels upon crosslinking through UV photolithography. And we demonstrate that our technology allows producing functional monolayers of polarized Caco-2 epithelial cells. Hydrogels are networked materials with high water content, which allow easy diffusion of soluble factors and oxygen.5,6 PEG-based hydrogels possess highly tunable chemical and mechanical properties and have become trendy materials to mimic and are the extracellular matrix and tissue basement membranes.7,8 To get 3D villi-like microstructures, 2D photomasks were used in a UV light-dependent polymerization process. During this process, several variable factors, such as UV exposure time, photoinitiator concentration, and PEGDA molecular weight and concentration, were studied to understand the mechanisms that allow the formation of high aspect ratio soft microstructures. Then, these variables were adjusted to control the microstructure dimensions and to obtain villi-like microstructures resembling the in vivo villi dimensions, physical and mechanical properties. To allow cell adhesion and growth, we copolymerize PEGDA (non-bioactive) with acrylic acid and we took advantage of the exposed carboxylic groups of the acrylic acid to covalently incorporate laminin through the EDC/NHS coupling reaction. We have stablished the ratio between PEGDA and acrylic acid to obtain on the one hand, a successful copolymerization and villi-like structures formation and, on the other a proper protein functionalization of the hydrogel. Our results show that the amount of covalent bound protein to the hydrogel depends on the amount of acrylic acid employed which offers a way to control ligand density on the hydrogel, independently on the mechanical properties Functionalized villi-like microstructured hydrogels have proven suitable for cell growth. First, cell adhesion on the hydrogels was tested by culturing fibroblasts. Then, MDCK cells were used to determine the suitability of the microstructured hydrogels of growing epithelial monolayers. Finally, we used Caco-2 cells (widely used in intestinal barrier models)9 to validate the functionality of our construct. Caco-2 cells were seeded on

the villi-like hydrogel and maintained for 21 days. Our results show that Caco-2 cells were able to adhere and divide, lining the villi-like microstructures and forming a monolayer of polarized epithelial cells, as shown by the expression of actin and villin in the apical part of the monolayer and the shape, position and orientation of cell nuclei. We have also been able to successfully transfer the hydrogel platform to polycarbonate membranes and Transwell® inserts. With this technology, we are currently working on performing paracellular and transcellular permeability tests to evaluate the performance of our system in comparison with standard Caco-2 monolayers. Furthermore, we have evaluated the transepithelial electrical resistance (TEER) of the resulting monolayers, and our TEER measurements results confirmed the formation of functional epithelial cell barriers.

La major part dels actuals models in vitro de cultiu cel·lular no reprodueixen l'anatomia dels teixits i el comportament fisiològic de les cèl·lules in vivo. Les tècniques de microfabricació es poden utilitzar per anar un pas més enllà dels models in vitro convencionals, basats en cultius 2D, i reproduir de forma fiable microambients del teixit viu. Per l'epiteli de l'intestí prim, aquest enfocament s'ha explorat a través de la fabricació de scaffolds de col·lagen i poly (làctic-co àcid glicòlic) que imiten les vellositats, utilitzant micromolding seqüencial. Encara que ha estat un enfocament innovador i interessant, la naturalesa dels materials emprats no permet l'ajust de les propietats del sistema i el mètode d'emmotllament és laboriós, requerint diverses etapes de intermedies. Aquí es descriu un mètode senzill i ràpid per fabricar microestructures 3D similars a les vellositats. Utilitzem poli (etilenglicol) diacrylat (PEGDA) i àcid acrílic per formar hidrogels tous mitjançant fotolitografia. Hem demostrat que el nostre mètode permet produir monocapes funcionals de celules epitelials polaritzades(Caco-2). Els hidrogels són materials polimèrics que formen xarxes 3D amb un alt contingut d'aigua, que permeten una fàcil difusió de factors solubles i oxygen. Els hidrogels fets de PEG posseeixen propietats químiques i mecàniques que es poden ajustar i s'han convertit en materials molt itilitzats com a substrat per cultivar cèl·lules. Per obtenir microestructures 3D similars a les vellositats, s’han utilitzat fotomáscares en un procés de polimerització depenent de llum UV. Per permetre l'adhesió cel·lular i el seu creixement, el PEGDA es va copolimeritzar amb l’àcid acrílic per treure profit dels grups carboxílics de l'àcid acrílic i incorporar-hi covalentment proteines de la matrix extracel·lular. Després de la fabricació i caracterització dels hidrogels microestructurats, es va optimitzar la fabricació per obtenir microestructures similars a les vellositats. Per validar la funcionalitat del nostre sistema, es van utilitzar cèl·lules epitelials (Caco-2). Els nostres resultats mostren que les Caco-2 van ser capaces d'adherir-se i dividir-se, i formar monocapes de cèl·lules epitelials polaritzades. També hem estat capaços de transferir amb èxit la plataforma dels hidrogels microestructurats als sistemes convencionals (Transwell®) per fer mesures quantitatives de la integritat de la barrera epitelial.