Fig. 5.

Sphericity, volume, viability and histological evaluation of organotypic multicellular spheroid (OMS). (A) Sphericity Index, non significant differences were observed between the spheres cultivated without and with Flt3L, (B) Volumen, similarly there are no differences between the experimental conditions (p>0.05). 3D structures were treated with exogenous Flt3L (100 ng/ml) (C, D) or without cytokine (E–F). Viability was evaluated with LIVE/DEAD^TM Cell Imaging Kit (Invitrogen) and confocal microscope FV1000 (Olympus) using an UPLSAPO 20× (1.35 NA oil immersion objective), 488 nm argon laser line for green fluorescence (living cells) and HeNe 543 nm laser for red fluorescence (dead cells). Data analysis was performed with the FlowView and Fiji software (green cells: alive, red cells: dead) (Scale bar: 100 um). For histological evaluation, after 15 days of culture, cells without pyknotic nuclei are observed in the OMS (G) without Flt3L (×40, hematoxylin & eosin. Scale bar: 5 mm) and (H) with Flt3L (×40, hematoxylin & eosin. Scale bar: 5 mm) (I) Negative control of vimentin (brain tissue) (×40. Scale bar: 5 mm), (J) Positive control of vimentin (lymph node) (×40. Scale bar: 5 mm), (K) Vimentina in OMS without Flt3L (×40. Scale bar: 5 mm) and (L) OMS with Flt3L (×40. Scale bar: 5 mm).