Abstract
Purpose :
Delivering cells to replace degenerate cells in degenerative disease of the outer retina in the effort to restore vision carries great hope. Many approaches for optimizing the 3D scaffold for improving the spatial orientation, function, viability and potentially the synaptic formation of transplanted cells have been pursued, and are the focus of many studies. Here we investigated, in vitro, the morphological characterization and viability of single human photoreceptors precursors cells (hPRP) cells in microwells array.
Methods :
– hPRP were differentiated form human embryonic stem cells following a protocol optimized by our group. GFP labeled PRP cells were seeded on an SU8 scaffold array fabricated by lithography containing 17,424 wells 20µm in diameter and 17 µm in height suitable for a single cell dimension. SU8 surfaces were treated with serum, plasma, matrigel, collagen and PEI to enhance cell attachment. Cells morphology and creation of focal adhesions were monitored for several days after seeding by confocal microscopy and staining for actin and vinculin. Viability of the cells was further evaluated by PI staining. High resolution imaging of the membrane scaffold interface was studied by focused ion beam and scanning electron microscopy (FIB/SEM) technique.
Results :
Surface functionalization of SU8 with serum and plasma increased the wettability of SU8 surface and improved the cell attachment. PI staining revealed that SU8 polymer is biocompatible for PRP cells. Centrifugation of the microwell (1400 rpm, 4min) yielded high percentage (> 70%) of cells within the wells. Both confocal and FIB/SEM microscopy showed that cells seeded in microwells filled the entire volume of the well with the gap detected between the cell membrane and the well wall being at the nanometric scale.
Conclusions :
SU8 polymer is biocompatible for scaffold fabrication for PRP cells. Surface functionalization is needed to improve cell attachment to SU8 surfaces. Single PRP cells seeded in the wells survived in-vitro and occupied the entire well space.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.