title: In vitro biomimetic platforms featuring a perfusion system and 3D spheroid culture promote the construction of tissue-engineered corneal endothelial layers.
abstract: Corneal endothelial cells (CECs) are very important for the maintenance of corneal transparency. However, in vitro, 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 Ki67 and EMT-related markers Vimentin and α-SMA, whereas the gene expression level of the CEC marker ATP1A1 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 in vitro.
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, 510632, P.R. China.
authors.forename: Shanyi
authors.initials: S
authors.lastname: Li
authors.affiliation: The Department of Ophthalmology, the First Clinical Medical College, Jinan University, Guangzhou, 510632, P.R. China.
authors.forename: Yuting
authors.identifier: http://orcid.org/0000-0002-4341-951X
authors.initials: Y
authors.lastname: Han
authors.affiliation: Key Laboratory of Optoelectronic Information and Sensing Technologies of Guangdong Higher Educational Institutes, Jinan University, Guangzhou, 510632, P.R. China.
authors.forename: Hao
authors.initials: H
authors.lastname: Lei
authors.affiliation: Key Laboratory of Optoelectronic Information and Sensing Technologies of Guangdong Higher Educational Institutes, Jinan University, Guangzhou, 510632, P.R. China.|Department of Applied Physics, South China Agricultural University, Guangzhou, 510632, P.R. China.
authors.forename: Yingxin
authors.initials: Y
authors.lastname: Zeng
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, 510632, P.R. China.
authors.forename: Zekai
authors.initials: Z
authors.lastname: Cui
authors.affiliation: The Department of Ophthalmology, the First Clinical Medical College, Jinan University, Guangzhou, 510632, P.R. China.
authors.forename: Qiaolang
authors.initials: Q
authors.lastname: Zeng
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, 510632, P.R. China.
authors.forename: Deliang
authors.initials: D
authors.lastname: Zhu
authors.affiliation: The Department of Ophthalmology, the First Clinical Medical College, Jinan University, Guangzhou, 510632, P.R. China.
authors.forename: Ruiling
authors.initials: R
authors.lastname: Lian
authors.affiliation: Key Laboratory of Optoelectronic Information and Sensing Technologies of Guangdong Higher Educational Institutes, Jinan University, Guangzhou, 510632, P.R. China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Optoelectronic Information and Sensing Technologies of Guangdong Higher Educational Institutes, Jinan University, Guangzhou, 510632, P.R. China. thzhechen@jnu.edu.cn.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, 510632, P.R. China. chenjiansu2000@163.com.|Institute of Ophthalmology, Medical College, Jinan University, Jinan University, Guangzhou, 510632, P.R. China. chenjiansu2000@163.com.|The Department of Ophthalmology, the First Clinical Medical College, Jinan University, Guangzhou, 510632, P.R. China. chenjiansu2000@163.com.|Aier Eye Institute, #198 Furong Middle Road, Changsha, 410015, P.R. China. chenjiansu2000@163.com.
authors.forename: Jiansu
authors.initials: J
authors.lastname: Chen
mesh_terms: D032701:Biomimetics / Q000295:instrumentation / Q000379:methods; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology; D058750:Epithelial-Mesenchymal Transition; D010100:Oxygen; D010477:Perfusion; D010641:Phenotype; D011312:Pressure; D018874:Spheroids, Cellular; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods; D054457:Tissue Scaffolds
chemical_list: D003094:Collagen; D010100:Oxygen