Mapping the structure and biological functions within mesenchymal bodies using microfluidics

Abstract

Organoids that recapitulate the functional hallmarks of anatomic structures comprise cell populations able to self-organize cohesively in 3D. However, the rules underlying organoid formation in vitro remain poorly understood because a correlative analysis of individual cell fate and spatial organization has been challenging. Here, we use a novel microfluidics platform to investigate the mechanisms determining the formation of organoids by human mesenchymal stromal cells that recapitulate the early steps of condensation initiating bone repair in vivo. We find that heterogeneous mesenchymal stromal cells self-organize in 3D in a developmentally hierarchical manner. We demonstrate a link between structural organization and local regulation of specific molecular signaling pathways such as NF-κB and actin polymerization, which modulate osteo-endocrine functions. This study emphasizes the importance of resolving spatial heterogeneities within cellular aggregates to link organization and functional properties, enabling a better understanding of the mechanisms controlling organoid formation, relevant to organogenesis and tissue repair.

High-throughput measurements of intra-cellular and secreted cytokine from single spheroids using anchored microfluidic droplets

“…Microfabrication. The microfluidic chips were fabricated using the protocols detailed in our previous studies [17,21,25]. Briefly, the chip consists in a top part made with Poly(dimethylsiloxane) (PDMS, SYLGARD 184, Dow Corning, 1:10 (w/w) ratio of curing agent to bulk material) on which is inprinted a flow focusing device connected to an emulsification channel (serpentine), diverging rails, and terminated by a culture chamber;…”

“…The top and bottom parts of the chip were assembled after plasma treatment (Harrick, Ithaca, USA). [17,21]. The intracellular production of VEGF-A was measured by immunocytochemistry, following the protocols detailed in [21].…”

“…[17,21]. The intracellular production of VEGF-A was measured by immunocytochemistry, following the protocols detailed in [21]. Briefly, the cells were fixed in a 4% PFA, then permeabilized with 0.…”

“…After droplet anchoring, the oil flow is stopped and the cells are allowed to settle down at the bottom of the droplets. This initiates cellular clustering, and the cells spontaneously self-arrange to form spheroids (see Figure 1A right) [17,21]. The characteristic time for spheroid formation with hMSCs is about 4 hours (Supp.…”

“…This implies that the behavior in vivo is not a simple superposition of the behavior of individual cells in isolation; instead, the cells can organize into functional units whose secretions are determined by the interactions between them, as it can be exemplified by the maturation of liver functions that is mediated by a complex interplay between hepatic, endothelial and stromal cells [20] The challenge of 3D culture can be addressed by using spheroids, which emerge from the clustering of hundreds of adherent cells into a coherent functional cellular unit. When originating from a heterogeneous population, the cells in the spheroid self-arrange in an organized spatial manner, such that the cellular function is linked with the 3D structure [21].…”