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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\u00a0weeks 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.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>CjSCs were spindle-shaped and vimentin\u2009+\u2009while CjECs were polygonal and CK13\u2009+\u2009. 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.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusion<\/jats:title>\n                <jats:p>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.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1007\/s10792-022-02418-y","type":"journal-article","created":{"date-parts":[[2022,8,6]],"date-time":"2022-08-06T10:02:32Z","timestamp":1659780152000},"page":"215-232","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Establishment of a bi-layered tissue engineered conjunctiva using a 3D-printed melt electrowritten poly-(\u03b5-caprolactone) scaffold"],"prefix":"10.1007","volume":"43","author":[{"given":"Jiajun","family":"Xie","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qi","family":"Gao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zelmira Nu\u00f1ez","family":"del Prado","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nandini","family":"Venkateswaran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hazem M.","family":"Mousa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Enrique","family":"Salero","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Ye","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena M.","family":"De Juan-Pardo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alfonso L.","family":"Sabater","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Victor L.","family":"Perez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,8,6]]},"reference":[{"key":"2418_CR1","first-page":"40","volume-title":"Ophthalmic Pathology","year":"1996","unstructured":"Spencer W (ed) (1996) Ophthalmic Pathology. 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The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be seen as a potential conflict of interest","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}},{"value":"The study was in strict accordance with the tenets of the Declaration of Helsinki. The research protocol was evaluated and approved by the Institutional Review Boards of the University of Miami, Miami, Florida, USA.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethical approval"}}]}}