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Steffi Matthyssen, Kurt Coppens, Eleonora Ferraris, Jennifer Patterson, Marie-José Tassignon, Nadia Zakaria; Cellularization of 3D printed Recombinant Human Collagen type III scaffolds using corneal mesenchymal stem cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2361. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate cellularization of 3D printed human recombinant collagen type III (RHC III) scaffolds in vitro, using corneal mesenchymal stem cells (MSCs).
Corneal MSC cultures were established using either a collagenase digestion (1.5hrs vs 4hrs) of the stroma or by plating stromal explants. Cells were seeded in either DMEM + 10% FBS or Epilife +5% FBS. At passage three, cells were characterized using Flow Cytometry and trilineage differentiation was performed using specific differentiation media. MSCs were seeded onto 3D printed (n=6) and non-printed (n=6) samples at a density of 20,000 cells/sample. For controls, cells were seeded on glass (n=6). At day 10, scaffolds were processed for immunocytochemistry (n=15) and Scanning Electron Microscopy (SEM) (n=3).
Flow cytometry showed that corneal MSCs were positive for CD73, CD90, CD105, CD13, CD29, CD44, CD166 and negative for CD11b, CD14, CD19, CD34, CD45, CD79a and HLA-DR. Corneal MSCs were able to differentiate into osteocytes, chondrocytes and adipocytes. There was no significant difference between the MSC phenotypes using the different isolation or culture protocols. Light microscopy showed that corneal MSCs proliferated on both printed and non-printed RHC III scaffolds and SEM showed the circular print pattern of the RHC III. On immunocytochemistry we observed collagen type III fibril alignment in the printed samples but not in the non-printed samples. The MSCs actin filaments of the cytoskeleton stained positive for phalloidin and we observed alignment orthogonally to the direction of the collagen.
Our results demonstrate that 3D printed RHC III is a suitable substrate for cultivating corneal MSCs.
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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