Structural and Functional Mapping of Mesenchymal Bodies.

The formation of spheroids with mesenchymal stem/stromal cells (MSCs), mesenchymal bodies (MBs), is usually performed using bioreactors or conventional well plates. While these methods promote the formation of a large number of spheroids, they provide limited control over their structure or over the regulation of their environment. It has therefore been hard to elucidate the mechanisms orchestrating the structural organization and the induction of the trophic functions of MBs until now. We have recently demonstrated an integrated droplet-based microfluidic platform for the high-density formation and culture of MBs, as well as for the quantitative characterization of the structural and functional organization of cells within them. The protocol starts with a suspension of a few hundred MSCs encapsulated within microfluidic droplets held in capillary traps. After droplet immobilization, MSCs start clustering and form densely packed spherical aggregates that display a tight size distribution. Quantitative imaging is used to provide a robust demonstration that human MSCs self-organize in a hierarchical manner, by taking advantage of the good fit between the microfluidic chip and conventional microscopy techniques. Moreover, the structural organization within the MBs is found to correlate with the induction of osteo-endocrine functions (i.e., COX-2 and VEGF-A expression). Therefore, the present platform provides a unique method to link the structural organization in MBs to their functional properties. Graphic abstract: Droplet microfluidic platform for integrated formation, culture, and characterization of mesenchymal bodies (MBs). The device is equipped with a droplet production area (flow focusing) and a culture chamber that enables the culture of 270 MBs in parallel. A layer-by-layer analysis revealed a hierarchical developmental organization within MBs.

Mapping the structure and biological functions within mesenchymal bodies using microfluidics.

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.

Tracking the Evolution of Transiently Transfected Individual Cells in a Microfluidic Platform.

Transient gene expression (TGE) technology enables the rapid production of large amount of recombinant proteins, without the need of fastidious screening of the producing cells required for stable transfection (ST). However, several barriers must be overcome before reaching the production yields using ST. For optimizing the production yields from suspended cells using TGE, a better understanding of the transfection conditions at the single cell level are required. In this study, a universal droplet microfluidic platform was used to assess the heterogeneities of CHO-S population transiently transfected with cationic liposomes (CL) (lipoplexes) complexed with GFP-coding plasmid DNA (pDNA). A single cell analysis of GFP production kinetics revealed the presence of a subpopulation producing higher levels of GFP compared with the main population. The size of high producing (HP) cells, their relative abundance, and their specific productivity were dependent on the charge and the pDNA content of the different lipoplexes: HPs showed increased cell size in comparison to the average population, lipoplexes with positive charge produced more HPs, and lipoplexes carrying a larger amount of pDNA yielded a higher specific productivity of HPs. This study demonstrates the potential for time-resolved single-cell measurements to explain population dynamics from a microscopic point of view.

Model for the erosion onset of a granular bed sheared by a viscous fluid.

We study theoretically the erosion threshold of a granular bed forced by a viscous fluid. We first introduce a model of interacting particles driven on a rough substrate. It predicts a continuous transition at some threshold forcing θ_{c}, beyond which the particle current grows linearly J∼θ-θ_{c}. The stationary state is reached after a transient time t_{conv} which diverges near the transition as t_{conv}∼|θ-θ_{c}|^{-z} with z≈2.5. Both features are consistent with experiments. The model also makes quantitative testable predictions for the drainage pattern: The distribution P(σ) of local current is found to be extremely broad with P(σ)∼J/σ, and spatial correlations for the current are negligible in the direction transverse to forcing, but long-range parallel to it. We explain some of these features using a scaling argument and a mean-field approximation that builds an analogy with q models. We discuss the relationship between our erosion model and models for the plastic depinning transition of vortex lattices in dirty superconductors, where our results may also apply.