Organoids are miniaturized and simplified models of tissues, typically generated from stem cells, or primary tissue samples. Stem cells are fed specific factors to induce differentiation into the cell type(s) of interest. Organoids are used to more accurately model tissue microenvironments that cannot be recapitulated in 2D cell culture alone.
Are there differences between organoids and spheroids?
Organoids typically consist of multiple cell types to replicate the variety of cell types within tissues and the microenvironment between those cells. Spheroids are typically a cluster of a single cell type that is generated by aggregating the cells on special culture plates/conditions. Spheroids are simpler in nature and do not mimic the extracellular matrix in tissues.
How are organoids established?
Using stem cells, the cells are suspended in a basement membrane extract (BME) and exposed to differentiation factors to replicate the various cell types within the tissue of interest. When starting from primary tissue, the tissue is both mechanically and enzymatically digested to produce small cell clusters. The clusters are suspended in BME and cultured to allow the propagation of the organoids.
How are organoids cultured?
Typically, organoids are cultured within BME domes or layers in multi-well plates surrounded or layered with culture media. The three-dimensional culture in BME more closely mimics the three-dimensional microenvironment found within tissues. BME is porous enough that culture media, growth factors, and signaling factors can diffuse to the cell clusters, and waste can diffuse away from the cell clusters. Tissue specific medias are used so that factors normally found in the tissue microenvironment are present in the culture media to promote growth.
What are BME domes?
BME refers to basement membrane extract, which is a gelatinous mixture of compounds and factors typically found within tissue extracellular matrices and are important signaling factors in cell proliferation and maintaining health. The domes allow for three-dimensional growth of organoids.
What media or supplements are required for organoid culture?
There are some components of media that are universally used; however, the supplements usually vary depending on the tissue type and model organism. Optimization over the course of years contributes to updated composition of media and represents an ongoing effort to continually make the products better.
How long can organoids be expanded in culture?
This is dependent on many factors, including tissue type, culture conditions, initial tissue source health, etc. Organoids can generally be cultured for weeks to months.
Can organoids be frozen for long term storage?
Yes, similar to cell lines, organoids can be frozen and later thawed for later usage. Like cell lines, a cryoprotectant must be used to ensure that the organoids are protected from the formation of ice crystals. Cryopreservation allows researchers to store organoids for an extended period and revive them when needed, reducing the need for continuous culture and passage.