Dynamic photopolymerization produces complex microstructures on hydrogels in a moldless approach to generate a 3D intestinal tissue model

Abstract

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.

Imaging the Cell Morphological Response to 3D Topography and Curvature in Engineered Intestinal Tissues

“…The villus-like microstructured scaffolds were fabricated by photolithography, as previously described (Castaño et al, 2019) ( Figure 1A). Briefly, a prepolymer solution containing 6.5% w/v 6 kDa PEGDA, 0.3% w/v acrylic acid (AA), and 1% w/v Irgacure D-2959 photoinitiator in phosphate-buffered saline (PBS) (all from Sigma-Aldrich) was flown into a chip fabricated with a 1 mm thick polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) stencil containing an array of pools of 6.5 mm diameter.…”

“…After UV exposure, unreacted polymer and photoinitiator were washed out with PBS and the hydrogels were kept submerged at 4 • C for at least 3 days to reach equilibrium swelling. Samples fabricated onto PET membranes were assembled on modified Transwell inserts using double-sided pressure-sensitive adhesive rings as detailed in Castaño et al (2019). After swelling, and to provide the scaffolds with cell-adhesion motifs, the PEGDA-AA hydrogels were functionalized with 0.01% w/v collagen type I (Sigma Aldrich) via an N-(3-Dimethylaminopropyl)-Nethylcarbodiimide (EDC)/N-Hydroxysuccinimide (NHS) (Sigma Aldrich) mediated coupling.…”

“…3D villus-like hydrogel scaffolds were fabricated by lithographybased dynamic photopolymerization of PEGDA-AA prepolymer solutions, as described in Castaño et al (2019). The microstructures were fabricated either on glass coverslips or on flexible porous PET membranes ready to be assembled into Transwell insert supports.…”

“…In the case of the small intestine, several strategies have been proposed to fabricate villus-like hydrogels such as replica molding or stereolithography-based 3D printing (Wang et al, 2017;Creff et al, 2019). Using a different approach, we have recently developed a method to fabricate microstructured hydrogel scaffolds mimicking the small intestinal villi by using a photolithography-based microfabrication technique (Castaño et al, 2019). This moldless approach yielded high aspect ratio, finger-like hydrogel microstructures with the physiological shape and dimensions of the native villi.…”

Advanced bioengineering technologies for preclinical research

“…This platform allows for the modeling of kidney diseases such as cystic kidney disease and acute kidney injury as well as drug testing purpose [104]. Finally, the small intestine crypt-villus morphology has also been reproduced by either replica molding procedures [29,105] and, more recently, by reactiondiffusion mediated photolithography ( Figure 6(c)) [106]. In all the cases, a significant improvement of the properties of the epithelial barrier on the 3D structures compared with the 2D monolayers was reported.…”