Development of High-Resolution Three-Dimensional-Printed Extracellular Matrix Scaffolds and Their Compatibility with Pluripotent Stem Cells and Early Retinal Cells

Abstract

Purpose: Widely used approaches for retinal disease modeling and in vitro therapeutic testing can be augmented by using tissue-engineered scaffolds with a precise 3-dimensional structure. However, the materials currently used for these scaffolds are poorly matched to the biochemical and mechanical properties of the in vivo retina. Here, we create biopolymer-based scaffolds with a structure that is amenable to retinal tissue engineering and modeling. Methods: Optimal two-photon polymerization (TPP) settings, including laser power and scanning speed, are identified for 4 methacrylated biopolymer formulations: collagen, gelatin, hyaluronic acid (HA), and a 50/50 mixture of gelatin/HA, each with methylene blue as a photoinitiator. For select formulations, fabrication accuracy and swelling are determined and biocompatibility is evaluated by using human induced pluripotent stem cells and rat postnatal retinal cells. Results: TPP is feasible for each biopolymer formulation, but it is the most reliable for mixtures containing gelatin and the least reliable for HA alone. The mean size of microscaffold pores is within several microns of the intended value but the overall structure size is several times greater than the modeled volume. The addition of HA to gelatin scaffolds increases cell viability and promotes neuronal phenotype, including Tuj-1 expression and characteristic morphology. Conclusion: We successfully determined a useful range of TPP settings for 4 methacrylated biopolymer formulations. When crosslinked, these extracellular matrix-derived molecules support the growth and attachment of retinal cells. We anticipate that when combined with existing patient-specific approaches, this technique will enable more efficient and accurate retinal disease modeling and therapeutic testing in vitro than current techniques allow.

Ultrathin micromolded 3D scaffolds for high-density photoreceptor layer reconstruction

“…Solid and porous polymeric thin-film scaffolds supporting hPSCderived neural retina cell growth have been developed in recent years to overcome problems inherent to bolus cell suspension injections (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Despite these advances, two of the core engineering challenges these scaffolds are intended to address-cell disorganization and low or unpredictable cell payloads-have remained largely unsolved.…”

Three-dimensional printing in ophthalmology and eye care: current applications and future developments

“…This could facilitate in vitro studies of photoreceptor cell behaviour, disease pathogenesis and novel treatments for retinal degeneration. In another recent article, 61 a high-resolution 3D-printed extracellular matrix was developed that is compatible with pluripotent stem cells and early retinal cells. This biopolymer-based scaffold supported the growth and attachment of retinal cells.…”

Aplicações da manufatura aditiva em oftalmologia

“…Biopolímeros de colágeno, ácido hialurônico e gelatina se mostraram biocompatíveis e promissores para o desenvolvimento de células pluripotentes de humanos e células retinianas de ratos. (36) Kim et al (37) desenvolveram substratos que mimetizam uma membrana de Bruch utilizando uma biotinta derivada de membrana de Bruch suína, com o objetivo de cultivar células do epitélio pigmentado da retina (EPR) com funções que podem ser úteis para um modelo in vitro e possível transplante de EPR. A escassez de córneas de doadores é um grave problema global, e, portanto, o desenvolvimento de substitutos viáveis para córneas doadoras é alvo de equipes de pesquisa em todo o mundo.…”