Long-Term in vitro Expansion of Retinal Progenitor Cells by Culturing Intact Neurospheres in Monolayer

Abstract

This study developed a modified monolayer culture technique for expansion of retinal progenitor cells in vitro. Using this method, spheres collected by low centrifugation from free floating cultures were re-seeded onto poly-D-lysine pre-coated flasks, and cultured in the retinal progenitor cells medium (including fibrous growth factor, epidermal growth factor and leukemia inhibitor factor) plus 5% FBS for the first 24 h, then FBS was removed. These cultured cells were proliferative, and in addition to expressing the neuroectodermal marker nestin they were multipotential. By immunocytochemistry and FACS analysis, we demonstrated that the percentage of nestin-expressing cells in passage 2 and passage 8 was 92.5 and 85.5%, respectively, which was greater than the percentage of their counterparts, 81.9 and 76.2%, in simple adhesive cultures (p < 0.05). Moreover, these cells preferentially differentiated into retinal neurons rather than glial cells.

Characterization of Seeding Conditions for Studies on Differentiation Patterns of Subventricular Zone Derived Neurospheres

“…For the study of differentiation, survival, maturation, and migration of cultivated progenitor cells, attachment to synthetic coating compounds is inevitable for the success of these experiments. PDL and PornT are the two most commonly used coating compounds to ensure better attachment of cells to a preferred underground ( Greilberger et al, 1997 ; Ramsay and Gumbleton, 2002 ; Louis and Reynolds, 2005 ; Lai et al, 2008 ; Ahlenius et al, 2009 ). However, there is no broad consensus as to which of these two synthetic amino acid chains is favorable for the underlying processes of cell actions.…”

“…Poly- D -lysine (PDL) and polyornithine (PornT) are non-specific attachment factors that promote cell adhesion to solid substrates. They are the two most often used coating materials for in vitro or ex vivo experiments and essential for the study of cellular characteristics and processes ( Greilberger et al, 1997 ; Ramsay and Gumbleton, 2002 ; Louis and Reynolds, 2005 ; Lai et al, 2008 ; Ahlenius et al, 2009 ; Yang et al, 2015 ). As in vitro experiments are merely simplified replica of complex cellular in vivo networks, the artificial environment created in an in vitro experiment needs to make use of enrichment factors.…”