Title:Basement Membrane Promotes Ciliary Body Derived Cell Spheres Formation
Volume: 3
Issue: 2
Author(s): G.K. Srivastava, D. Rodriguez-Crespo, A.K. Singh, M.T. Garcia-Gutierrez, I. Fernandez-Bueno and J.C. Pastor
Affiliation:
Keywords:
Cell sphere, ciliary body, epithelial cell, method, retinal disease, stem cell.
Abstract: Controversy still exists whether ciliary body contains stem cells (CBSCs) or epithelial cells (CBECs). CBSCs
were reported for differentiation capacity and CBECs for high reprogramming efficiency to produce induced Pluripotent
Stem (iPS) cells. However, ciliary body derived cells (CBCs) application as a cell therapy needs a guaranteed isolation
and culture protocol from ciliary body tissue strip. Four protocols were compared; one (I) belonged to a published article
(Gu et al., 2007) and the rest three, protocols II, III and IV, were developed. Protocol II, III and IV used trypsin-EDTA
and mechanical scraping to isolate single CBCs, basement membrane (BM) fragments and other debris. Protocols used 40
and/or 100 μm cell strainers at different steps to remove different scraped tissue fragments and isolated single CBCs. Protocol
III and IV contained CBCs attached on BM. Success/failure numbers in out of 5 repeated experiments per protocol
(n=5) were 2/3 (I), 3/2 (II), 4/1 (III) and 5/0 (IV). Cultures were very clean (I), partially clean (II and III) and dirty (IV).
Protocol III and IV based cultures showed clumps of cell spheres. This study results confirmed that BM promotes cell proliferation
and primary cell spheres formation. CBCs are in controversy for recognition as epithelial or stem cells but their
successful isolation and culture, and iPS cells reprogramming protocols can open a new era for reinforcing their applicability.
