Organoid and three-dimensional (3-D) cell culture are emerging as pivotal systems for understanding human organ development, modeling disease, screening for drug efficacy or toxicity, and investigating personalized medicine. The reagents and protocols needed to culture these advanced multi-cellular in vitro tissues vary by organ, species, and whether they are being generated from tissue or pluripotent stem cells. This page serves as a reagent and technical resource to help researchers build robust and consistent organoid cultures designed to provide you with a central location to access protocols, view webinars, stay up to date on organoid recipes and blogs, and discover new products relevant to your work in organoid research. Navigate below to find information for culturing organoids from all tissue types.
Stomach epithelial organoids
Small intestine epithelial organoids
|Chromogranin A||Enteroendocrine cells|
|Periodic acid-Schiff staining||Goblet cells|
|LGR5||Intestinal stem cell marker|
|ASCL2/Mash||Crypt cell marker|
Colon epithelial organoids
|Cytokeratin 7||Centrilobular hepatocytes|
|Glutamine Synthetase||Hepatocyte progenitors|
|SP5||Hepatocyte transcription factor|
|Integrin beta 1/CD29||Hepatocytes|
|Lgr5/GPR49||Liver stem cells|
|PROM1||Liver stem cells|
|Cytokeratin 5||Basal prostate cell|
|p63||Basal prostate cell|
|Integrin alpha 6/CD49f||Epithelial stem cells|
|TROP-2||Epithelial stem cells|
|Cytokeratin 8||Luminal prostate cell|
|Androgen R/NR3C4||Prostate cells|
|Cytokeratin 18||Prostate cells|
|NKX3.1||Prostate-specific transcription factor|
|Laminin gamma 1||Stromal cells|
|EMX1||Dorsal cortex marker|
|EOMES||Intermediate progenitor marker|
|Pax6||Neural induction marker/ventral cortex marker|
|beta-II Tubulin (clone TuJ-1)||Neuronal marker|
|p63||Basal airway cells|
|NKX2.1||Lung lineage marker|
|ID2||Lung progenitor marker|
|Lgr5/GPR49||Lung stem cells|
|alpha-Smooth Muscle Actin||Smooth muscle cells|
|HOPX||Type 1 airway epithelial cells|
|SFTPC||Type 2 airway epithelial cells|
Inner Ear Organoids
|Cancer organoids should be cultured in similar conditions as to those for which the parent organoid can be grown. In some instances mutations occur in cancer stem cells that allow them to grow in the absence of normal growth factors, such as EGF or FGF. Depending on the cancer organoid it may be possible to grow them in a medium where one or more factors are removed from the normal organoids growth medium.|
Additional Background Information on Organoids
An organoid is a miniaturized version of an organ produced in vitro that shows realistic micro-anatomy, is capable of self-renewal and self-organization, and exhibits similar functionality as the tissue of origin. While their size is small (typically < 3 mm in diameter), organoids are stable model systems of organs and tissues that are amenable to long-term cultivation and manipulation. In conjunction with advances in cell reprogramming technology and gene editing methods, organoids allow unprecedented insight into human development, act as disease models, and can be utilized for drug screening platforms or even cell transplantation.
Organoids can be classified into those that are tissue-derived and those that are stem cell-derived. Tissue-derived organoids typically originate from adult tissues while stem cell-derived organoids are established from pluripotent stem cells. Researchers have devised methods to generate physiologically relevant organoid models for organs such as the brain, liver, thymus, thyroid, lung, pancreas, and heart that can be used for drug discovery and toxicology. However, the utility of organoids may be impacted by their degree of in vitro maturation. Stem cell-derived organoids may only recapitulate the first few months of development, but not the stages beyond, therefore they potentially lack some cell types of interest for researchers. In this case, tissue organoids generated from isolated adult stem/progenitor cells or resected fragments of organ tissues (i.e. intestinal crypts, liver, or pancreatic ducts) may be more suitable.
The formation of organoids is facilitated by the presence of biological or synthetic scaffolds. The most common are biological scaffolds derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (i.e. Cultrex® Basement Membrane Extracts). These protein-rich extracellular matrices not only provide a scaffold for the cells to attach and organize into 3-D structures, but they can provide additional microenvironmental cues, such as growth factors and hormones, that help define the growth and organization of the 3-D tissue. More defined scaffolds of polymeric or synthetic origin have also been used, but they lack endogenous factors that help promote cell development. The scaffold of choice is ultimately driven by the research application. Currently, EHS-derived matrices are the most robust method for generating viable and sustainable organoids used in drug screening and toxicological studies.
The promise of organoid technology is widely recognized in the biomedical field, but it is still in its infancy. There are practical challenges to overcome before it can be widely implemented in disease modeling, drug discovery, and toxicological applications. Of course, data generated using these methods needs to be validated against established assays to confirm the accuracy of in vivo responses. At present, protocols for organoid generation result in heterogeneous cultures (size and shape), which can produce unwanted experimental variability. Tissue access within an organoid is another hurdle to be addressed. For intestinal organoids, the apical (luminal) surface of the epithelium, which faces the interior of the structure, is important for testing compounds for drug toxicity, permeability, and absorption. However, the spheroidal architecture of the organoid restricts access to the lumen, presenting challenges that could require time-consuming and labor-intensive procedures, such as microinjection of compounds. Additionally, there is a push in the field to generate organoids of increased size and complexity because larger organoids are thought to be more biologically relevant and predictive of native tissues, but the current maximum size is constrained by the gas and nutrient diffusion rates.
Larger organoids may be possible in the future as the methods for nutrient supply improve (i.e. spinning bioreactors to facilitate higher extents of diffusion or coculture with endothelial cells). Another consideration is that current 3-D culture methods limit the use of organoids in established drug discovery pipelines that have been designed and optimized for cells grown as 2-D monolayers. Organoid cultures are not yet easily scalable for high-throughput screening protocols or amenable to automation in the same way as traditional 2-D cultures. Despite these challenges, it is evident that organoids have great potential to revolutionize the way we approach disease modeling, drug discovery, and toxicology. Their limitations are rapidly being overcome as emerging technologies (such as organ-on-a-chip, microfluidics, or bioprinting) open new avenues for more accurate in vitro testing.
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