Delahousse, C. | bioRxiv Science

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

Assessing motile cilia coverage and beat frequency in mammalian in-vitro cell culture tissues

Cilia density, distribution and beating frequency are important parameters lung tissues, for example in diagnostics of Primary Ciliary Dyskinesia, and in the study of in vitro models, e.g. derived from induced Pluripotent Stem Cells. Video microscopy can be used to characterise these parameters, but most tools available at the moment are limited in the type of information they can provide, usually only describing the ciliary beat frequency of very small areas, while requiring human intervention and training for their use. We propose a novel and open source method to fully characterise cilia beating frequency and motile cilia coverage in an automated fashion without user intervention. We demonstrate the ability to differentiate between different coverage densities, identifying even small patches of cilia in a larger field of view, and to fully characterise the cilia beating frequency of all moving areas. We also show that the method can be used to combine multiple fields of view to better describe a sample without relying on small pre-selected regions of interest. This is released with a simple graphical user interface for file handling, enabling a full analysis of individual fields of view in a few minutes on a typical personal computer.

biophysics↗