Jiajun Xie1,2,3, Qi Gao1,2,3, Zelmira Nuñez Del Prado1,2, Nandini Venkateswaran1,4, Hazem M Mousa5, Enrique Salero1,2, Juan Ye3, Elena M De Juan-Pardo6, Alfonso L Sabater1,2, Victor L Perez7,8,9. 1. Department of Ophthalmology, Ocular Surface Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA. 2. Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL, USA. 3. Department of Ophthalmology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. 4. Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA, USA. 5. Department of Ophthalmology, Foster Center for Ocular Immunology, Distinguished Stephen and Frances Foster Chair in Ocular Immunology, Duke Eye Center, 2351 Erwin Road, Durham, NC, 27705, USA. 6. Institute for Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), Brisbane, QLD, Australia. 7. Department of Ophthalmology, Ocular Surface Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA. victor.perez.quinones@duke.edu. 8. Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL, USA. victor.perez.quinones@duke.edu. 9. Department of Ophthalmology, Foster Center for Ocular Immunology, Distinguished Stephen and Frances Foster Chair in Ocular Immunology, Duke Eye Center, 2351 Erwin Road, Durham, NC, 27705, USA. victor.perez.quinones@duke.edu.
Abstract
PURPOSE: To utilize melt electrowriting (MEW) technology using poly-(ε-caprolactone) (PCL) coupled with a 2-step co-culturing strategy for the development of a conjunctival bi-layer synthetic construct. METHODS: Melt electrowritten scaffolds using PCL were fabricated using an in-house-built MEW printer. Human conjunctival stromal cells (CjSCs) and epithelial cells (CjECs) were isolated from donor tissue. A 2-step co-culture method was done by first seeding the CjSCs and culturing for 4 weeks to establish a stromal layer, followed by CjECs and co-culturing for 2 more weeks. Cultured cells were each characterized by morphology and marker expression on immunofluorescence and qPCR. The produced construct was assessed for cellular proliferation using viability assays. The bi-layer morphology was assessed using scanning electron microscopy (SEM), confocal microscopy, and immunofluorescence imaging. The expression of extracellular matrix components and TGF-b was evaluated using qPCR. RESULTS: CjSCs were spindle-shaped and vimentin + while CjECs were polygonal and CK13 + . CjSCs showed consistent proliferation and optimal adherence with the scaffold at the 4-week culture mark. A 2-layered construct consisting of a CjSC-composed stromal layer and a CjEC-composed epithelial layer was appreciated on confocal microscopy, SEM, and immunofluorescence. CjSCs secreted collagens (types I, V, VI) but at differing amounts from natural tissue while TGF-b production was comparable. CONCLUSION: The 3D-printed melt electrowritten PCL scaffold paired with the 2-step co-culturing conditions of the scaffold allowed for the first approximation of a bi-layered stromal and epithelial reconstruction of the conjunctiva that can potentially improve the therapeutic arsenal in ocular surface reconstruction.
PURPOSE: To utilize melt electrowriting (MEW) technology using poly-(ε-caprolactone) (PCL) coupled with a 2-step co-culturing strategy for the development of a conjunctival bi-layer synthetic construct. METHODS: Melt electrowritten scaffolds using PCL were fabricated using an in-house-built MEW printer. Human conjunctival stromal cells (CjSCs) and epithelial cells (CjECs) were isolated from donor tissue. A 2-step co-culture method was done by first seeding the CjSCs and culturing for 4 weeks to establish a stromal layer, followed by CjECs and co-culturing for 2 more weeks. Cultured cells were each characterized by morphology and marker expression on immunofluorescence and qPCR. The produced construct was assessed for cellular proliferation using viability assays. The bi-layer morphology was assessed using scanning electron microscopy (SEM), confocal microscopy, and immunofluorescence imaging. The expression of extracellular matrix components and TGF-b was evaluated using qPCR. RESULTS: CjSCs were spindle-shaped and vimentin + while CjECs were polygonal and CK13 + . CjSCs showed consistent proliferation and optimal adherence with the scaffold at the 4-week culture mark. A 2-layered construct consisting of a CjSC-composed stromal layer and a CjEC-composed epithelial layer was appreciated on confocal microscopy, SEM, and immunofluorescence. CjSCs secreted collagens (types I, V, VI) but at differing amounts from natural tissue while TGF-b production was comparable. CONCLUSION: The 3D-printed melt electrowritten PCL scaffold paired with the 2-step co-culturing conditions of the scaffold allowed for the first approximation of a bi-layered stromal and epithelial reconstruction of the conjunctiva that can potentially improve the therapeutic arsenal in ocular surface reconstruction.