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Kristan S Worthington, Luke A Wiley, Bailey B Banach, Allison E Songstad, Emily E Kaalberg, Robert F Mullins, Edwin M Stone, Budd A Tucker; Human Retinal Engineering using 3D Two-Photon Polymerization and Chitosan Hydrogels. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5321.
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© ARVO (1962-2015); The Authors (2016-present)
Recent advances in induced pluripotent stem cell (iPSC) technology have paved the way for the production of patient-specific photoreceptor precursor cells that are ideal for photoreceptor cell replacement therapy and treatment of retinal degenerative diseases. The use of polymer support scaffolds is critical for cellular survival and integration, but attempts to materialize this concept have ultimately been unsuccessful for two reasons: 1) mechanical mis-matching between material and tissue and 2) improper cell packing. We hypothesize that chitosan hydrogels, which have tunable compressive moduli, can act as suitable substrates for human iPSC-derived photoreceptor cells. We also hypothesize that two-photon polymerization can be used to create precise structures that facilitate dense packing and proper orientation of photoreceptor precursor cells.
Methacrylate-functionalized chitosan (MeCTS) was polymerized at various concentrations and UV light exposure times. The compressive modulus was measured using dynamic mechanical analysis in static mode. Complex prototype structures with varying pore sizes were created using two-photon polymerization of commercially available photoresists. Photoreceptor precursor cells were dissociated from 3D developing eyecups and plated on the hydrogel surface or prototype structure. Cellular survival, identity, morphology and directionality were characterized up to four weeks post-plating using rt-PCR, Western blotting, and immunocytochemistry.
Increasing MeCTS concentration or light exposure time increased compressive modulus of MeCTS hydrogels. These materials supported the attachment and development of photoreceptor precursor cells in vitro. When seeded onto structured materials, photoreceptor precursor cells nested in the vertical pores of the structure, and aligned in a manner similar to naturally occurring photoreceptor cells.
These results lay the foundation for an autologous stem cell-based strategy for restoring vision to patients affected with retinal degenerative diseases. The developed constructs mimic the in vivo retinal environment in terms of stiffness and structure. Importantly, this knowledge will be fundamental to the development of effective cell-based grafts for sub-retinal transplantation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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