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Lilangi S. Ediriwickrema, Shaomin Peng, Nina Kristofik, Tavé A. van Zyl, Geliang Gan, Lina Li, Caihong Qiu, Mark Saltzman, Ron A. Adelman, Lawrence J. Rizzolo; Reconstructing an Outer Retina using Electrospun Polycaprolactone (PCL) and human embryonic stem cell-derived Retinal Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):337.
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As a first step towards engineering an outer retina suitable for transplantation, we designed a biocompatible, biodegradable scaffold that will allow retinal progenitor cells (RPCs) to form flat, laminar structures. The scaffolds minimize the exposure of retinal progenitors to extracellular matrix components that are not found in the retina, and will allow for co-culture with the RPE.
Scaffold sheets were formed from electrospun fibers of polycaprolactone (PCL). Duration of collection determined polymer thickness and solvent concentration dictated porosity and pore distribution of the scaffold. The thickness and porosity of the sheets were varied to optimize cell culture and rate of degradation of the PCL. Eight mm circular sheets were cut with a biopsy punch and coated with laminin. Retinal progenitors, derived from human embryonic stem cells (hESC-derived RPCs), were cultured to form neurospheres. The neurospheres were dissociated into single cell suspensions, seeded onto PCL sheets, and maintained in serum free medium. Fluorescence labeling and confocal microscopy were used to assess the morphology of the cultures.
Individual PCL fibers measured ~3 µm in thickness. The thickness of the sheet could be varied from 20-200 µm. The porosity could be adjusted to form 5 to 100 µm diameter pores. The scaffolds degraded via surface erosion, within a slightly basic pH range at 7.4 to 7.9, over four to seven weeks, depending upon porosity. Scaffolds with pores 25 to 50 µm in diameter were subsequently chosen for culture experiments. Confocal imaging confirmed that the RPCs penetrated the thickness of the scaffold and continued to express retinal markers such as Pax6, recoverin, and N-cadherin. There was evidence of polarity, as the N-cadherin was expressed only along one surface of the polymer.
Electrospun PCL polymers are biodegradable, biocompatible, and sustain the differentiated properties acquired by the neurospheres. It appears to be a suitable scaffold for reconstructing an outer retinal layer from hESC-derived RPCs that can enhance cell survival and delivery to a diseased sub-retinal space via transplantation.
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