A NEW METHOD FOR IMPROVING STEM CELLS OBTAINMENT FROM THE PORCINE CILIARY BODY

Abstract

Purpose: Ciliary body stem cells (CBSCs) transplantation offers a promising therapy for retinal diseases. However, published isolation techniques are quite rigorous and not very promising to obtain cell spheres. The purpose of this study is to improve and simplify isolation techniques of ciliary body-derived CBSCs from porcine eyes

Methods: Cellular suspensions, obtained from porcine ocular tissue strips containing the ciliary epithelium after tripsin-EDTA incubation, were mechanically triturated and centrifuged. Pallet was suspended, filtered (100μm) and cultured with FBS-DMEM/F12 medium in T25 flasks (2x10⁴ cells/ml). Afer 15 days primary cell spheres were dissociated (0.05% tripsin-EDTA) and cells were suspended and filtered (40μm) (Experimental, n=5). As Control (n=5), CBSCs were isolated as described by Gu et al. (2007) and thus cultured without basement membranes in the same conditions above mentioned. Cells morphology evaluation by phase contrast microscopy and CBSCs immunophenotyping were performed during culture

Results: Spheres with pigmented and non-pigmented cells were observed with both protocols. However, the success rate to obtain cell spheres was higher in Experimental protocol (5/5), where basement membranes were maintained in culture, than in Control (2/5). There were numerous primary cell spheres in clumps in experimental cultures. These clumps were absent in control cultures. CBSCs acquired variable morphology from epithelial- to fibroblast-like and expressed proteins related to different retinal cell types, such as GFAP (astrocytes), recoverin (photoreceptor) and isolectin (microglia)

Conclusions: The modified CBSCs isolation protocol improves cell spheres obtention, due to presence of basement membranes during culture. This simplified and improved protocol will assist to advance in the potentiality of CBSCs therapy for retinal diseases