Biology subjects

Sart, S.

Publications and source records attributed to Sart, S..

6 recordsLinked to original sources

bioRxiv2024

Culture of pluripotent stem cells in microscale droplets modulates differentiation and tissue patterning towards organoids on chip

1The differentiation of pluripotent stem cells (PSCs) and their self-organization into organoids are influenced by cell-cell interactions mediated by contacts and secreted molecules. These interactions are enhanced in microfluidic droplets due to confinement and small culture volumes. However, a comprehensive study on the culture of PSCs within droplets and the impact of this microenvironment has yet to be conducted. In this study, we present a droplet platform for the 3D culture of PSCs at various stages of cellular commitment. We demonstrate PSC differentiation into the three germ layers and the feasibility of organoid formation within droplets. Our findings reveal that culturing PSCs in confined volumes regulates cell fate decisions, promoting tissue patterning in gastruloids through the sequential induction of growth and migration of distinct differentiated cell populations, and facilitating the self-organization of cardiac organoids. This technological approach provides unique insights into the intrinsic factors regulating tissue self-patterning in vitro. 2 Highlights and eTOC blurbO_ST_ABSHighlightsC_ST_ABSO_LIDroplet microfluidics allows expansion and supports the pluripotency of 3D aggregates of PSCs. C_LIO_LIDroplet microfluidics supports and regulates spontaneous differentiation into embryoid bodies. C_LIO_LIDroplet promotes tissue patterning in gastruloids through the sequential induction of growth and migration of mesoderm followed by ectoderm. C_LIO_LIPerfused microfluidic droplets support long term culture and derivation of organoids on chip. C_LI Vertti-Quintero et al. introduces a microfluidic droplet platform for the 3D culture of pluripotent stem cells (PSCs) at various differentiation stages. The format supports the long term 3D culture and the differentiation of PSCs -either spontaneous or directed-. This "microscale culture" can regulate PSCs fate decision, while promoting tissue pattering -as demonstrated in gastruloids polarization- and allowing self-organization towards cardioids formation.

bioengineering↗

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

Griottes: a generalist tool for network generation from segmented tissue images

Microscopy techniques and image segmentation algorithms have improved dramatically this decade, leading to an ever increasing amount of biological images and a greater reliance on imaging to investigate biological questions. This has created a need for methods to extract the relevant information on the behaviors of cells and their interactions, while reducing the amount of computing power required to organize this information. This task can be performed by using a network representation in which the cells and their properties are encoded in the nodes, while the neighborhood interactions are encoded by the links. Here we introduce Griottes, an open-source tool to build the "network twin" of 2D and 3D tissues from segmented microscopy images. We show how the library can provide a wide range of biologically relevant metrics on individual cells and their neighborhoods, with the objective of providing multi-scale biological insights. The librarys capacities are demonstrated on different image and data types. This library is provided as an open-source tool that can be integrated into common image analysis workflows to increase their capacities.

bioinformatics↗

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

Studying 3D cell cultures in a microfluidic droplet array under multiple time-resolved conditions

The relevance of traditional cell cultures to cellular behavior in vivo is limited, since the two-dimensional (2D) format does not appropriately reproduce the microenvironment that regulates cell functions. In this context, spheroids are an appealing 3D cell culture format to complement standard techniques, by combining a high level of biological relevance with simple production protocols. However the methods for spheroid manipulation are still labor intensive, which severely limits the complexity of operations that can be performed on statistically relevant numbers of individual spheroids. Here we show how to apply hundreds of different conditions on spheroids in a single microfluidic chip, where each spheroid is produced and immobilized in an anchored droplet. By using asymmetric anchor shapes, a second drop can be merged with the spheroid-containing drop at a later time. This time-delayed merging uniquely enables two classes of applications that we demonstrate: (1) the initiation of cell-cell interactions on demand, either for building micro-tissues within the device or for observing antagonistic cell-cell interactions with applications in immuno-therapy or host-pathogen interactions, (2) a detailed dose-response curve obtained by exposing an array of hepatocyte-like spheroids to droplets containing a wide range of acetaminophen concentrations. The integrated microfluidic format allows time-resolved measurements of the response of hundreds of spheroids with a single-cell resolution. The data shows an internally regulated evolution of each spheroid, in addition to a heterogeneity of the responses to the drug that the single-cell analysis correlates with the initial presence and location of dead cells within each spheroid.

bioengineering↗