A complex microscopic reconstruction for outgrowth evaluation in a neural spheroid model for screening trophic molecules.

CARRI, NÉSTOR GABRIEL; NOO BERMUDEZ, SEBASTIÁN; SCICOLONE, GABRIEL

La Plata

Workshop; Imaging techniques for biotechnology and biomedical applications ? Whorkshop; 2016

CCT-CONICET

A Complex Microscopic Reconstruction for Outgrowth Evaluation in a Neural Spheroid Model for Screening Trophic Molecules Néstor Gabriel Carri, Sebastián Noo Bermúdez and Gabriel Scicolone Imaging techniques for biotechnology and biomedical applications ? Whorkshop. CCT-CONICET. La Plata, Prov Buenos Aires (30/11-01/12/2016).Our multi-imaging protocol is frequently used with live sample assays under sterile conditions. Disperse cultured cells clump together to form a spheroid. These are organoids of developing neural cells that start differentiation when placed in a 3-D short-term bioassay of type I collagen gel and cultured under basal conditions or with the addition of trophic molecules (like NGF, NT-3, Noggin or FGF9). Spheroids used in these experiments were simple, fast to obtain , with repeatable good results. The distinguishable spheroids placed in the assay under the effect of trophic molecules generated the dense centrifugal outgrowth of neurites from the spheroid. The single neurites never remained alone. It is demonstrated that they always mix with undress of neurites forming a dense core and indistinguishable mesh. Multiview imaging is required for outgrowth measurement. Sequential microscopical sets of z images are obtained in the same frame. With these multiple stacks that could act as set of optical sections, the outgrowth reconstruction will be performed and evaluated. Length could be easily recognized but number would require conventional estimation values from the analysis of the experimental set up- in different windows. NGF: nerve growth factor, NT-3: neurotrophin 3, Noggin and FGF9: fibroblast growth factor 9.