Biology subjects

Baroud, C. N.

Publications and source records attributed to Baroud, C. N..

5 recordsLinked to original sources

bioRxiv2023

Using a micro-device with a deformable ceiling to probe stiffness heterogeneities within 3D cell aggregates

Recent advances in the field of mechanobiology have led to the development of methods to characterize single-cell or monolayer mechanical properties and link them to their functional behaviour. However, there remains a strong need to establish this link for three-dimensional multicellular aggregates, which better mimic tissue function. Here we present a platform to actuate and observe many such aggregates within one deformable micro-device. The platform consists of a single PDMS piece cast on a 3D-printed mold and bonded to a glass slide or coverslip. It consists of a chamber containing cell spheroids, which is adjacent to air cavities that are fluidically independent. Controlling the air pressure in these air cavities leads to a vertical displacement of the chambers ceiling. The device can be used in static or dynamic modes over time-scales of seconds to hours, with displacement amplitudes from a few m to several tens of microns. Further, we show how the compression protocols can be used to obtain measurements of stiffness heterogeneities within individual co-culture spheroids, by comparing image correlations of spheroids at different levels of compression with finite element simulations. The labeling of the cells and their cytoskeleton is combined with image correlation methods to relate the structure of the co-culture spheroid with its mechanical properties at different locations. The device is compatible with various microscopy techniques, including confocal microscopy, which can be used to observe the displacements and rearrangements of single cells and neighborhoods within the aggregate. The complete experimental and imaging platform can now be used to provide multi-scale measurements that link single-cell behavior with the global mechanical response of the aggregates.

bioengineering↗

bioRxiv2022

Single-cell microfluidic analysis unravels individual cellular fates during Double-Strand Break Repair

Trinucleotide repeat expansions are responsible for two dozen human disorders. Contracting expanded repeats by Double-Strand Break Repair (DSBR) might be a therapeutic approach. Given the complexity of manipulating human cells, recent assays were made to quantify DSBR efficacy in yeast, using a fluorescent reporter. In this study DSBR is characterized with an interdisciplinary approach, linking large population dynamics and individual cells. Time-resolved molecular measurements of changes in the population are first confronted to a coupled differential equation model to obtain repair processes rates. Comparisons with measurements in microfluidic devices, where the progeny of 80-150 individual cells are followed, show good agreement between individual trajectories and mathematical and molecular results. Further analysis of individual progenies shows the heterogeneity of individual cell contributions to global repair efficacy. Three different categories of repair are identified: high-efficacy error-free, low-efficacy error-free and low-efficacy error-prone. These categories depend on the type of endonuclease used and on the target sequence.

bioengineering↗

bioRxiv2020

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

While many single-cell approaches have been developed to measure secretion from anchorage-independent cells, these protocols cannot be applied to adherent cells, especially when these cells requires to be cultured in 3D formats. Here we demonstrate a platform to measure the secretions from individual spheroids of human mesenchymal stem cells, cultured within microfluidic droplets. The platform allows us to quantify the secretion from hundreds of individual spheroids in each device, by using a secondary droplet to bring functionalized micro-beads into proximity with each spheroid. We focus on vascular endothelial growth factor (VEGF) and measure a distribution of secretion levels that presents broad heterogeneity within the population of spheroids. Moreover, 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 spheroids level result from variations at the scale of individual cells. Finally, we model the molecular accumulation within the droplets and find that physical confinement is crucial for measurements of protein secretions. The model predicts the time to achieve a measurement, which scales with droplet volume. Therefore these first measurements of secretions from individual spheroids provide several new biological insights.

bioengineering↗

bioRxiv2019

Mapping 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-{kappa}B 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.

cell biology↗

bioRxiv2018

Tetrahymena RIB72A and RIB72B are Microtubule Inner Proteins in the ciliary doublet microtubules

Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia and flagella. In contrast to dynamic cytoplasmic microtubules, their luminal surface is coated with regularly arranged Microtubule Inner Proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical and imaging techniques we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins co-localize to Tetrahymena cilia and basal bodies, but assemble independently. Cryo-electron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4 and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.\n\nSUMMARYMicrotubule Inner Proteins (MIPs) bind to the luminal surface of highly stable microtubules. Combining cell biology and cryo-electron tomography, Stoddard et al. show that RIB72A and RIB72B are conserved MIPs in ciliary doublet microtubules and that they are important for proper ciliary motility.

cell biology↗