The effects of mechanical and chemical scaffold surface cues on retinal pigment epithelial cells

摘要

Introduction: Dry Age-related Macular Degeneration (AMD) is one of the leading causes of blindness in developed countries. However, there are few effective treatment options for patients to arrest the degeneration of the retina). The retinal pigment epithelium (RPE), a highly functional cell monolayer, is known to be dysfunctional in this disease causing neural retinal death and eventual blindness. Cell replacement therapy has shown promise in the treatment of this disease, particularly cell transplantation with a support scaffold. However, upon implantation many current scaffolds elicit an inflammatory response, as well as loss of implanted RPE function. We hypothesize that the addition scaffold surface cues, both mechanical and chemical cues, can mitigate this response. We present the development of a mechanically tunable synthetic polymer scaffold, in order to understand how the elasticity of support scaf olds affects cells in culture. We also investigate how the addition of signaling molecules affects these cells and seek to determine which approach is more promising for a translational therapy. Materials and Methods: Mechanical Cues: PEGDA of varying molecular weights from 3.4kDa to 20kDa were used to prepare prepolymer solutions. These solutions were photopolymerized into a hydrogel sheet in a glass mold using UV light. Following swelling for 24 hours, the hydrogels were subjected to tensile testing to determine Young's an immortalized RPE line, and analyzed during culture using microscopy, PrestoBlue metabolic activity assay, and PCR. Chemical Cues: Signaling molecules (epidermal growth factor; Activin A) were conjugated to a hetero-bifunctionalized PEG. This was then incorporated into a scaffold using UV photopolymerization as described above. ARPE-19 cells were seeded on these scaffolds and analyzed throughout culture. Results: By varying polymer concentration and molecular weight, PEGDA hydrogel mechanical properties were tunable and ranged from 60-1200 kPa. Native Bnjch's Membrane has a Young's Modulus of 1000 kPa, which we were able to mimic. Cells cultured on scaffolds of varying moduli demonstrated differences in cell activity and gene expression. To gain insight into how chemical cues affect ARPE-19 cells, the cells were cultured in monolayer with EGF supplemented, PEG conjugated EGF (PEG-EGF) supplemented, or unsupplemented complete medium. The cells were analyzed via microscopy for qualitative morphological differences, PrestoBlue cellular activity assay, and for gene expression using PCR. Results demonstrate that both free EGF and PEG-EGF have significant effects on the activity and gene expression of ARPE cells compared to unsupplemented medium. Results for cells cultured on scaffolds containing immobilized EGF are forthcoming. Conclusions: These studies demonstrated that separately both chemical and mechanical cues affect RPE cells in culture. These two strategies allow for the manipulation of cell expression and function and can potentially be used to overcome the hurdles that current scaffolds face. The presented work contributes to the foundation of knowledge that will aid in the design of a translational scaffold for RPE transplantation.