Two-photon polymerization for production of human iPSC-derived retinal cell grafts

Abstract

Recent advances in induced pluripotent stem cell (iPSC) technology have paved the way for the production of patient-specific neurons that are ideal for autologous cell replacement for treatment of neurodegenerative diseases. In the case of retinal degeneration and associated photoreceptor cell therapy, polymer scaffolds are critical for cellular survival and integration; however, prior attempts to materialize this concept have been unsuccessful in part due to the materials’ inability to guide cell alignment. In this work, we used two-photon polymerization to create 180 μm wide non-degradable prototype photoreceptor scaffolds with varying pore sizes, slicing distances, hatching distances and hatching types. Hatching distance and hatching type were significant factors for the error of vertical pore diameter, while slicing distance and hatching type most affected the integrity and geometry of horizontal pores. We optimized printing parameters in terms of structural integrity and printing time in order to create 1 mm wide scaffolds for cell loading studies. We fabricated these larger structures directly on a porous membrane with 3 μm diameter pores and seeded them with human iPSC-derived retinal progenitor cells. After two days in culture, cells nested in and extended neuronal processes parallel to the vertical pores of the scaffolds, with maximum cell loading occurring in 25 μm diameter pores. These results highlight the feasibility of using this technique as part of an autologous stem cell strategy for restoring vision to patients affected with retinal degenerative diseases.

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

“…For larger structures, these edge effects are not noticeable, and the small distortions are negligible in comparison to the whole. 11 Gelatin stars did not reach the fidelity point at scanning speeds between 20,000 and 24,000 mm/s regardless of laser power, which was an unexpected observation ( Fig. 3).…”

“…Glass coverslips (CS-30R; Warner Instrument, Hamden, CT) that had been prefunctionalized with methacrylate groups, as previously described, 11 were used as substrates for TPP. Briefly, the coverslips were functionalized by exposure to an oxygen plasma (22.5 mL/min at 30 W radio frequency power for 3 min, Plasma Cleaner PCD-011 with PlasmaFlo; Harrick Plasma, Ithaca, NY), submerged in 0.02% 3-(trimethoxysilyl)propyl methacrylate (Sigma-Aldrich, St. Louis, MO) in hexanes (ThermoFisher Scientific) for a minimum of 2 h, rinsed in hexanes, rinsed with acetone, and allowed to dry at room temperature.…”

“…A 3D model for a 50-mm-tall, 180-mm-wide hexagonal scaffold with hexagonally spaced vertical pores (25 mm diameter, spaced 30 mm apart) and elliptical horizontal pores to permit diffusion (25 mm vertical axis and 15 mm horizontal axis for an average diameter of 20 mm) was created in AutoCAD as previously described. 11 This general scaffold was designed to approximate the structure of the retinal ECM and eventually facilitate photoreceptor cell packing while enabling structural integrity of the scaffold during fabrication and processing.…”

“…10 Further, our group has demonstrated that two-photon polymerization (TPP), a form of high-resolution 3D printing, can also be used to create scaffolds (nondegradable) that organize retinal progenitor cells in a pattern that recapitulates the photoreceptor cell layer. 11 Recently, we also applied this fabrication technique to poly(caprolactone), a degradable polyester, and showed that the resulting scaffolds are well tolerated by retinal cells and in vivo retinal tissue. 12 However, high-fidelity disease models should mimic the in vivo environment of the diseased cell type as closely as possible, including both biochemical and biomechanical cues.…”

3D Scaffolds to Study Basic Cell Biology

“…Concerning stem cells, Raimondi and coworkers improved the manufacturing of synthetic 3D niche structures for culturing various types of stem cells, including mesenchymal stem cells . Along this line, Worthington et al demonstrated the potential of DLW to study and control the fate of human induced pluripotent stem cells as a function of substrate interactions …”