5 research outputs found
Downstream bioprocessing of human pluripotent stem cellâderived therapeutics
International audienceAbstract With the advancement in lineageâspecific differentiation from human pluripotent stem cells (hPSCs), downstream cell separation has now become a critical step to produce hPSCâderived products. Since differentiation procedures usually result in a heterogeneous cell population, cell separation needs to be performed either to enrich the desired cell population or remove the undesired cell population. This article summarizes recent advances in separation processes for hPSCâderived cells, including the standard separation technologies, such as magneticâactivated cell sorting, as well as the novel separation strategies, such as those based on adhesion strength and metabolic flux. Specifically, the downstream bioprocessing flow and the identification of surface markers for various cell lineages are discussed. While challenges remain for largeâscale downstream bioprocessing of hPSCâderived cells, the rational qualityâbyâdesign approach should be implemented to enhance the understanding of the relationship between process and the product and to ensure the safety of the produced cells
HighâThroughput Measurements of IntraâCellular and Secreted Cytokine from Single Spheroids Using Anchored Microfluidic Droplets
International audienceWhile 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
Structural and Functional Mapping of Mesenchymal Bodies
International audienceThe 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
Griottes: a generalist tool for network generation from segmented tissue images
International audienceBackgroundMicroscopy 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.ResultsThis 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 ânetwork twinâ 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 libraryâs capacities are demonstrated on different image and data types.ConclusionsThis library is provided as an open-source tool that can be integrated into common image analysis workflows to increase their capacities
Griottes: a generalist tool for network generation from segmented tissue images
International audienceBackgroundMicroscopy 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.ResultsThis 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 ânetwork twinâ 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 libraryâs capacities are demonstrated on different image and data types.ConclusionsThis library is provided as an open-source tool that can be integrated into common image analysis workflows to increase their capacities