RESUMEN
Nerve growth factor (NGF) plays a crucial role in cellular growth and neurodifferentiation. To achieve significant neuronal regeneration and repair using in vitro NGF delivery, spatiotemporal control that follows the natural neuronal processes must be developed. Notably, a challenge hindering this is the uncontrolled burst release from the growth factor delivery systems. The rapid depletion of NGF reduces treatment efficacy, leading to poor cellular response. To address this, we developed a highly controllable system using graphene oxygen (GO) and GelMA hydrogels modulated by electrical stimulation. Our system showed superior control over the release kinetics, reducing the burst up 30-fold. We demonstrate that the system is also able to sequester and retain NGF up to 10-times more efficiently than GelMA hydrogels alone. Our controlled release system enabled neurodifferentiation, as revealed by gene expression and immunostaining analysis. The increased retention and reduced burst release from our system show a promising pathway for nerve tissue engineering research toward effective regeneration.
Asunto(s)
Materiales Biocompatibles , Estimulación Eléctrica , Grafito , Hidrogeles , Factor de Crecimiento Nervioso , Regeneración Nerviosa , Hidrogeles/química , Hidrogeles/farmacología , Grafito/química , Grafito/farmacología , Regeneración Nerviosa/efectos de los fármacos , Factor de Crecimiento Nervioso/farmacología , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/química , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Animales , Tamaño de la Partícula , Ensayo de Materiales , Ratas , Células PC12 , Ingeniería de TejidosRESUMEN
Analyzing the dynamics of mitochondrial content in developing T cells is crucial for understanding the metabolic state during T cell development. However, monitoring mitochondrial content in real-time needs a balance of cell viability and image resolution. In this chapter, we present experimental protocols for measuring mitochondrial content in developing T cells using three modalities: bulk analysis via flow cytometry, volumetric imaging in laser scanning confocal microscopy, and dynamic live-cell monitoring in spinning disc confocal microscopy. Next, we provide an image segmentation and centroid tracking-based analysis pipeline for automated quantification of a large number of microscopy images. These protocols together offer comprehensive approaches to investigate mitochondrial dynamics in developing T cells, enabling a deeper understanding of their metabolic processes.
