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However, <jats:italic>in vitro<\/jats:italic>, CECs display limited proliferation and loss of phenotype via endothelial to mesenchymal transformation (EMT) and cellular senescence. In this study, we demonstrate that continuous supplementary nutrition using a perfusion culture bioreactor and three-dimensional (3D) spheroid culture can be used to improve CEC expansion in culture and to construct a tissue-engineered CEC layer. Compared with static culture, perfusion-derived CECs exhibited an increased proliferative ability as well as formed close cell-cell contact junctions and numerous surface microvilli. We also demonstrated that the CEC spheroid culture significantly down-regulated gene expression of the proliferation marker <jats:italic>Ki67<\/jats:italic> and EMT-related markers <jats:italic>Vimentin<\/jats:italic> and <jats:italic>\u03b1<\/jats:italic>-<jats:italic>SMA<\/jats:italic>, whereas the gene expression level of the CEC marker <jats:italic>ATP1A1<\/jats:italic> was significantly up-regulated. Furthermore, use of the perfusion system in conjunction with a spheroid culture on decellularized corneal scaffolds and collagen sheets promoted the generation of CEC monolayers as well as neo-synthesized ECM formation. This study also confirmed that a CEC spheroid culture on a curved collagen sheet with controlled physiological intraocular pressure could generate a CEC monolayer. Thus, our results show that the use of a perfusion system and 3D spheroid culture can promote CEC expansion and the construction of tissue-engineered corneal endothelial layers <jats:italic>in vitro<\/jats:italic>.<\/jats:p>","DOI":"10.1038\/s41598-017-00914-1","type":"journal-article","created":{"date-parts":[[2017,4,4]],"date-time":"2017-04-04T23:20:17Z","timestamp":1491348017000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["In vitro biomimetic platforms featuring a perfusion system and 3D spheroid culture promote the construction of tissue-engineered corneal endothelial layers"],"prefix":"10.1038","volume":"7","author":[{"given":"Shanyi","family":"Li","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4341-951X","authenticated-orcid":false,"given":"Yuting","family":"Han","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Lei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingxin","family":"Zeng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zekai","family":"Cui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiaolang","family":"Zeng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Deliang","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruiling","family":"Lian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhe","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiansu","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2017,4,10]]},"reference":[{"key":"914_CR1","doi-asserted-by":"publisher","first-page":"359","DOI":"10.1016\/S1350-9462(02)00065-4","volume":"22","author":"NC Joyce","year":"2003","unstructured":"Joyce, N. 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