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

GABRIEL SCICOLONE; CARRI NÉSTOR GABRIEL

La Plata

Workshop; Imaging Techniques for Biotechnology and Biomedical Applications; 2016

CINDEFI-INIFTA-CCT La Plata Argentina

Primary neural stem cells obtainedfrom embryonic brain and retinal tissues were dissected and cultured in stemcell media. Explanted tissue contains viable cells that form spheroids inculture (Rojas Mayorquin et al., 2010). Mitosis of the peripheral cells intospheroids has been attributed to stimulation of bFGF within the stem cellmedium (Reynolds et al., 1992). Disperse cultured cells clump together to form aspheroid. These are organoids ofdeveloping neural cells that start differentiation when placed in a 3-Dshort-term bioassay of type I collagen gel and cultured under basal conditionsor with the addition of trophic molecules (like NGF, NT-3, Noggin or FGF9).Spheroids used in these experiments were simple, fast to obtain , withrepeatable good results. Developing tissues are easy to dissociate by simplemechanical procedures without any enzyme treatment. This is advantageous forneural stem cells and results show a good number of proliferating stem cellsfew hours after culture. These proliferative cells seemed to be the same asthose reported by Dayer et al. (2005). The distinguishable spheroids placed inthe assay under the effect of trophic molecules generated the dense centrifugaloutgrowth of neurites from the spheroid. The outgrowth obtained was measured(length and number) at 24 and 48 h of culture for subsequent analysis. Thesingle neurites never remained alone. It is demonstrated that they always mixwith undress of neurites forming a dense core and indistinguishable mesh. Someclusters of proliferative cells remained of very small size with quite reasonableoutgrowth and were used to set up our protocol. Though this assay showed highreproducibility and good resolution, further difficult microscopicaladjustments will be necessary to estimate outgrowth after few hours of culture.Multiview imaging is required for outgrowth measurement. Sequentialmicroscopical sets of z images are obtained in the same frame. With these multiple stacks that could act as set of optical sections, theoutgrowth reconstruction will be performed and evaluated. Length could be easilyrecognized but number would require conventional estimation values from theanalysis of the experimental set up- in different windows. Our multi-imagingprotocol is frequently used with live sample assays under sterile conditionsand can be registered several times, during hours of incubation, to plot dose-respondecurves that allow a complete set of data.Further information could be obtained with this procedure in fix tissues with immunohistochemistry. NGF: nerve growth factor, NT-3: neurotrophin 3, Noggin and FGF9:fibroblast growth factor 9.Dayer, A., Cleaver, K., Abouantoun, T., Cameron, H.,2005. New GABAergic interneu-rons in the adult neocortex and striatum aregenerated from different precursors.J. Cell Biol. 168, 415?427.  Reynolds, B., Tetzlaff, W., Weiss, S., 1992.AmultipotentEGF-responsive striatalembryonic progenitor cell produces neuronsand astrocytes. J. Neurosci. 12,4565?4574. Rojas Mayorquin, A.E., Torres Ruiz, N.M., GudiñoCabrera, G., Ortuño-Sahagún,D., 2010. Substrative hybridization identified gendifferentially expressed byolfactory ensheating cells and neural stem cells.Int. J. Dev. Neurosci. 28,75?82.  Images of spheroid culture in bioassay of collagen gelsand stimulated by Noggin at 20 ng/ml.