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MINERVA BIOTECNOLOGICA

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Minerva Biotecnologica 2006 September;18(3):129-35

language: English

A fibrin-based substrate for in vitro reconstruction of cultured corneal endothelial cells

Pagani P. 1, Campanile G. 2, Bricola G. 1,3, Csutak A. 1,4, Traverso C. E. 1,3

1 Fondazione Banca degli Occhi Melvin Jones, Genoa, Italy
2 Laboratory of Regenerative Medicine Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy
3 Clinica Oculistica, Centro di Ricerca Clinica e Laboratorio per il Glaucoma e la Cornea, Di.N.O.G., University of Genoa, Az. Ospedaliera Universitaria “San Martino”, Genoa, Italy
4 Department of Ophthalmology Medical and Health Science Center Faculty of Medicine University of Debrecen, Debrecen, Hungary


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Aim. Purpose of our work is to define an appropriate environment and a biological substrate for cultured corneal endothelial cells (CECs) proliferation in vitro.
Methods. Primary cultures of human CECs were established from donor corneas (40 corneas, mean age: 60 y; range 24-75); primary cultures of six bovine, ten porcine and eight rabbit corneas were established from whole corneas. Explants of the endothelial - Descemet’s membrane, removed mechanically, were seeded in wells coated with a substrate of extracellular matrix secreted by bovine CECs. Endothelial cells were separated from keratocytes and epithelial cells by the polygonal cell morphology as well as by immunohistochemistry. After confluence on extracellular matrix, cells were transferred on the fibrin film; the resulting composite was cut, lifted and transferred with a sterile spoon from the Petri dish onto the recipient cornea, with the endothelial cells facing the Descemet membrane.
Results. Vimentin and pan-cytokeratin were expressed by the endothelial cells on all fresh corneal section, while no expression was detected with anti-collagen type I. Cells from the study animals reached confluence within 15 days. Human CECs were slower and reached confluence within 45 days. Histological analyses confirmed that the endothelial cells adhered and grew on the fibrin film, maintaining proper morphology and monolayer disposition.
Conclusions. Our fibrin-based substrate demonstrated to be viable for growth and transportation of CECs. Each type of CECs studied required an appropriate environment to start the activation of proliferation.

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