
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.
Read more about the development and future of organoids for research.
Organoid Recipes
Gasteroids
Stomach epithelial organoids
Base Media Components | |
---|---|
N-2 MAX Supplement | Similar to N-2 |
N21-MAX Supplement | Similar to B27 Supplement |
N-Acetylcysteine | |
Penicillin/Streptomycin | |
DMEM/F-12 | |
Glutamine | |
Organoid Harvesting Solution |
Additives for long-term growth | |
---|---|
A 83-01 | See all TGF-beta/ALK inhibitors |
Nicotinamide | |
SB 202190 | See all p38 MAPK inhibitors |
Recommended Markers | |
---|---|
Pepsinogen C | Chief cells |
Somatostatin | Enteroendocrine cells |
TFF1 | Gastric cells |
TFF2 | Gastric cells |
MUC5AC | Gastric pit mucous cells |
Periodic acid-Schiff (PAS) staining | Gastric pit mucous cells |
MUC6 | Gland mucous cells |
LGR5 |
Gastric stem cell marker |
E-Cadherin |
Gastric epithelium |
Enteroids
Small intestine epithelial organoids
Base Media Components | |
---|---|
N-2 MAX Supplement | Similar to N-2 |
N21-MAX Supplement | Similar to B27 Supplement |
N-Acetylcysteine | |
Penicillin/Streptomycin | |
DMEM/F-12 | |
Glutamine | |
Organoid Harvesting Solution |
Additives for long-term growth | |
---|---|
A 83-01 | See all TGF-beta/ALK inhibitors |
Nicotinamide | |
SB 202190 | See all p38 MAPK inhibitors |
Recommended Markers | |
---|---|
Alkaline Phosphatase | Enterocytes |
Villin | Enterocytes |
Chromogranin A | Enteroendocrine cells |
Synaptophysin | Enteroendocrine cells |
MUC2 | Goblet cells |
Periodic acid-Schiff staining | Goblet cells |
Lysozyme | Paneth cells |
LGR5 | Intestinal stem cell marker |
E-Cadherin | Intestinal epithelium |
ASCL2/Mash | Crypt cell marker |
Colonoids
Colon epithelial organoids
Base Media Components | |
---|---|
N-2 MAX Supplement | Similar to N-2 |
N21-MAX Supplement | Similar to B27 Supplement |
N-Acetylcysteine | |
Penicillin/Streptomycin | |
DMEM/F-12 | |
Glutamine | |
Organoid Harvesting Solution |
Additives for long-term growth | |
---|---|
A 83-01 | See all TGF-beta/ALK inhibitors |
Nicotinamide | |
SB 202190 | See all p38 MAPK inhibitors |
Recommended Markers | |
---|---|
Ki-67/MKI67 | Cell proliferation marker |
EphB2 | Colon crypt cell marker |
Chromogranin A | Enteroendocrine cells |
EpCAM/TROP-1 | Epithelial cell marker |
Periodic acid-Schiff staining | Mucous cells |
LGR5 | Intestinal stem cell marker |
E-Cadherin | Intestinal epithelium |
ASCL2/Mash | Crypt cell marker |
Liver Organoids
Base Media Components | |
---|---|
N-2 MAX Supplement | Similar to N-2 |
N21-MAX Supplement | Similar to B27 Supplement |
N-Acetylcysteine | |
Penicillin/Streptomycin | |
DMEM/F-12 | |
Glutamine | |
Organoid Harvesting Solution |
Recommended Markers | |
---|---|
Cytokeratin 7 | Centrilobular hepatocytes |
EpCAM/TROP-1 | Epithelial cells |
CD24 | Hepatocyte progenitors |
CD44 | Hepatocyte progenitors |
Glutamine Synthetase | Hepatocyte progenitors |
SP5 | Hepatocyte transcription factor |
Cytokeratin 19 | Hepatocytes |
HNF-1 alpha | Hepatocytes |
HNF-4 alpha/NR2A1 | Hepatocytes |
Integrin beta 1/CD29 | Hepatocytes |
Serum Albumin | Hepatocytes |
Transthyretin/Prealbumin | Hepatocytes |
Lgr5/GPR49 | Liver stem cells |
PROM1 | Liver stem cells |
Pancreatic Organoids
Base Media Components | |
---|---|
N-2 MAX Supplement | Similar to N-2 |
N21-MAX Supplement | Similar to B27 Supplement |
N-Acetylcysteine | |
Penicillin/Streptomycin | |
DMEM/F-12 | |
Glutamine | |
Organoid Harvesting Solution |
Recommended Markers | |
---|---|
Insulin C-Peptide | Beta cells |
NeuroD1 | Beta cells |
NKX6.1 | Endocrine cells |
HNF-6 | Pancreatic progenitors |
Neurogenin-3 | Pancreatic progenitors |
PDX-1 | Pancreatic progenitors |
SOX9 | Pancreatic progenitors |
Prostate Organoids
Base Media Components | |
---|---|
N-2 MAX Supplement | Similar to N-2 |
N21-MAX Supplement | Similar to B27 Supplement |
N-Acetylcysteine | |
Penicillin/Streptomycin | |
Glutamine | |
Organoid Harvesting Solution |
Recommended Markers | |
---|---|
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 |
TMPRSS2 | Prostate cells |
NKX3.1 | Prostate-specific transcription factor |
Laminin gamma 1 | Stromal cells |
Vimentin | Stromal cells |
References: | |
---|---|
Karthaus W.R. et al. (2014) Cell. 159:163 |
Kidney Organoids
Base Media Components | |
---|---|
N-2 MAX Supplement | Similar to N-2 |
N21-MAX Supplement | Similar to B27 Supplement |
N-Acetylcysteine | |
Penicillin/Streptomycin | |
DMEM/F-12 | |
Glutamine | |
Holo-Transferrin | |
Organoid Harvesting Solution |
Recommended Markers | |
---|---|
Brachyury | Mesoderm |
LHX-1/LIM1 | Mesoderm |
Cytokeratin 8 | Uteric bud |
GATA-3 | Uteric bud |
GFR alpha-1/GDNFR alpha-1 | Uteric bud |
HOXB7 | Uteric bud |
Pax2 | Uteric bud |
Pax8 | Uteric bud |
Ret | Uteric bud |
Brain Organoids
Base Media Components | |
---|---|
N-2 MAX Supplement | Similar to N-2 |
N21-MAX Supplement | Similar to B27 Supplement |
N21-MAX Vitamin A Free Supplement | Similar to B27 Vitamin Free Supplement |
Penicillin/Streptomycin | |
DMEM/F-12 | |
GlutaMAX | |
Insulin | |
2-mercaptoethanol | |
Organoid Harvesting Solution |
Recommended Markers | |
---|---|
EMX1 | Dorsal cortex marker |
FOXG1 | Forebrain marker |
Frizzled-9 | Hippocampus marker |
Neuropilin-2 | Hippocampus marker |
Prox1 | Hippocampus marker |
EOMES | Intermediate progenitor marker |
Pax6 | Neural induction marker/ventral cortex marker |
SOX1 | Neural inductions |
beta-III Tubulin (clone TuJ-1) | Neuronal marker |
MAP2 | Neuronal marker |
Vimentin | Neuronal marker |
Lung Organoids
Base Media Components | |
---|---|
N-2 MAX Supplement | Similar to N-2 |
N21-MAX Supplement | Similar to B27 Supplement |
N-Acetylcysteine | |
Penicillin/Streptomycin | |
DMEM/F-12 | |
Glutamine | |
Organoid Harvesting Solution |
Recommended Markers | |
---|---|
p63 | Basal airway cells |
ACTTUB | Ciliated cells |
FoxJ1 | Ciliated cells |
Uteroglobin/SCGB1A1 | Club 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 |
References: | |
---|---|
Dye B.R. et al. (2015) eLIFE. 4:e05098 |
Inner Ear Organoids
LIF-2: Base Media Components | |
---|---|
N-2 MAX Supplement | Similar to N-2 |
N21-MAX Supplement | Similar to B27 Supplement |
Leukemia Inhibitor Factor (LIF) | |
CHIR 99021 | See all GSK-3 inhibitors |
PD 0325901 | See all MEK inhibitors |
DMEM/F-12 | |
Penicillin/Streptomycin | |
Glutamine | |
Organoid Harvesting Solution |
Cancer 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.
References:
Fang, Y. et al. (2017) SLAS Discov. 22:456.
Kelm, J.M. et al. (2003) Biotechnol. Bioeng. 83:173.
Fatehullah, A. et al. (2016) Nat. Cell Biol. 18:246.
Shamir, E.R. et al. (2014) Nat. Rev. Mol. Cell Biol. 15:647.
Takasato, M. et al. (2015) Nature 526:564.
Zhu, R. et al. (2014) Stem Cell Res. Ther. 5:117.
Li, Y. et al. (2014) Organogenesis 10:159.
Birgersdotter, A. et al. (2005) Semin. Cancer Biol. 15:405.
Wilson, S.S. et al. (2015) Mucosal Immunol. 8:352.
Lancaster, M. et al. (2013) Nature 501:373.