Organoids

Step 1) 2D preculture

Organoids are derived from either primary cells (i.e., intestine, lung, or kidney) or induced pluripotent stem cells. Stem cells are able to differentiate and self-assemble into a variety of tissue-specific organoids.

Step 2) Developing 3D organoids

Typically cells are premixed with Matrigel and droplets are placed into a 24-well plate at room temperature. The plates are then placed into an incubator to form a solid droplet dome. Media is then added for seven or more days to promote cell growth and differentiation into specific tissue (brain, gut, lungs, etc.). Media includes extracellular matrix (ECM), proteins, and different growth factors, which will vary depending on the type of tissue that is being developed.

Step 3) Organoid culture

Organoid culture is a long process and may include several steps with different media. During this process, cell phenotype needs to be monitored (PCR, imaging), typically used for understanding developmental biology and tissues.

Step 4) Monitoring and readouts

Before experiments are conducted, organoids need to be monitored and characterized to ensure that they have the appropriate tissue structure and differentiation. High-content imaging allows for monitoring and visualizing the growth and differentiation of organoids, 3D reconstitution of the structures, complex analysis of organoids structure, cell morphology and viability, as well as the expression of different cell markers.

Step 5) Confocal imaging and 3D analysis

Confocal imaging and 3D analysis of organoids allow visualization and quantitation of the number, size distribution, cell number, cell content, cell viability, volume, as well as quantitation of cell proliferation and expression of specific markers. Characterization of multiple quantitative descriptors of organoids could be used for studying disease phenotypes and compound effects.