Organoids are 3-D cell cultures derived from pluripotent stem cells that mimic the structure, function, and cellular complexity of human organs. These in vitro, miniaturized versions of organs are especially well suited for studying complex multicellular organ structures, such as the brain, retina, kidney, and lungs, and are now widely used to study organ development and disease.

 Researchers often use common 3-D culture techniques to produce spheroids— round cell clusters of primary or immortalized cells that are popularly used in tumour research. Organoids are similar to these structures, except their formation begins with tissue-specific stem cells that self-assemble into microscopic versions of a functioning organ component.

Organoids allow researchers to study matrix-adhered cells and learn about organ development. A process that could take years using live model organisms now takes months in organoid culture. Combined with CRISPR genome editing technology, scientists also use organoid cultures to model genetic diseases in tissues that are otherwise unobtainable, such as the brain. Researchers can produce numerous organoids from tumours or patient-derived induced-pluripotent stem cells (iPSCs), making them ideal models for drug screening and personalized cancer therapies. Organoid culture protocols are continuously improving, giving scientists a chance to focus on novel and targeted treatments while avoiding the risks posed to human subjects. Organoids are derived from either the directed differentiation of human pluripotent stem cells (hPSCs), tissue-specific adult stem cells, or iPSCs. Because organoids come from active stem cell populations, researchers can expand their cultures repeatedly over time. To create an organoid, scientists embed pluripotent cells in an extracellular matrix , which serves to support the cells. Specific growth factors and proteins that mimic the in vivo environment maintain the stem cell phenotype. Based on the initial stem cell population and growth factors chosen for a study, the matrix-embedded cells will self-assemble into 3-D organoid structures that behave similarly to a specific tissue.

 Researchers can adapt most protocols using a typical tissue culture room and standard equipment. When generating organoid cultures with hPSCs, pluripotent cells are initially cultured with a feeder cell population that provides the growth factors needed to maintain stem cell pluripotency. The hPSCs are allowed to form colonies in multi-well plates before they are enzymatically detached from the wells and feeder population. Researchers then dissociate and plate the pluripotent colonies on low-attachment 96 well plates. Over 1-2 weeks, the cells will begin to form embryoid bodies and can be induced towards certain lineages using tissue-specific induction media. Scientists can embed the differentiated embryoid bodies in extracellular matrix droplets and either cryopreserve or continuously culture them for several months using tissue-specific media. There are now also a number of commercial resources that provide pre-generated cryopreserved organoids for purchase.

In vitro systems typically lack key instructions during tissue formation that make in vivo tissue development and maturation extremely reproducible and robust. There are two main control systems in vivo. First, signalling molecule gradients called morphogens present precise instructions for cell type composition in tissues. The cells make different cell fate decisions depending on their distance from the source signal. In vitro organoids lack these messages. The second important control mechanism is physical in nature. The neighbouring tissues that develop together form physical boundaries. Tissues cannot continue to grow unchecked; they must stop because a neighbouring organ must also develop. This environmental influence is absent in vitro, meaning that the cells are growing and building tissues in 3-D space without any constraints. Future research projects are going to be more precision-oriented with these technological enhancements.

Dr Sharique Ahmad

Editor, EJMR