High‐Throughput Measurements of Intra‐Cellular and Secreted Cytokine from Single Spheroids Using Anchored Microfluidic Droplets
Résumé
While many single-cell approaches have been developed to measure secretions from anchorage-independent cells, these protocols cannot be applied to adherent cells, especially when these cells require to be cultured in 3D formats. Here, a platform to measure secretions from individual spheroids of human mesenchymal stem cells, cultured within microfluidic droplets is introduced. The platform allows to quantify the secretions from hundreds of individual spheroids in each device, by using a secondary droplet to bring functionalized micro-beads in proximity to each spheroid. Vascular endothelial growth factor (VEGF-A) is measured on and a broad distribution of secretion levels within the population of spheroids is observed. The intra-cellular level of VEGF-A on each spheroid, measured through immuno-staining, correlates well with the extra-cellular measurement, indicating that the heterogeneities observed at the spheroid level result from variations at the intra-cellular level. Further, the molecular accumulation within the droplets is modeled and it is found that physical confinement is crucial for measurements of protein secretions. The model predicts that the time to achieve a measurement scales with droplet volume. These first measurements of secretions from individual spheroids provide several new biological and technological insights.
Origine : Fichiers produits par l'(les) auteur(s)
