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What Are Organoids?

Organoids are 3D 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.1 

Mini-brain organoids generated from human tissues in a dish.
Brain organoids, also called cerebral organoids or mini-brains, are stem cell-derived self-organizing structures that emulate neurodevelopment and 3D brain composition.
Courtesy of Muotri Lab/UC San Diego

Organoids versus Spheroids

A spheroid is a round cluster of primary or immortalized cells that scientists commonly grow with 3D culture techniques for tumor research.2 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.1

How Are Organoids Made?

Organoids allow researchers to study matrix-adhered cells and learn about organ development. A process that could take years using live model organisms now only takes months with organoids grown in culture. Combined with CRISPR genome editing technology, researchers also culture organoids to model genetic diseases in tissues that are otherwise unobtainable, such as the brain. Researchers can produce numerous organoids from tumors or patient-derived induced-pluripotent stem cells (iPSCs), making them ideal models for drug screening and personalized cancer therapies.2 Organoid culture protocols are continuously improving, giving researchers a chance to focus on novel and targeted treatments while avoiding the risks posed to human subjects.

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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 such as Matrigel, 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 3D organoid structures that behave similarly to a specific tissue. 3,4

Alysson Muotri, professor at UC San Diego School of Medicine, holding a tray of stem cell-derived human brain organoids.
Researchers generate spheroids and organoids using standard laboratory and tissue culture equipment.
Courtesy of Muotri Lab, photo by Erik Jepsen UC San Diego

Researchers can adapt most protocols using a typical tissue culture room and standard equipment. When generating organoids 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 Matrigel droplets and either cryopreserve or continuously culture them for several months using tissue-specific media.5 There are now also a number of commercial resources that provide pre-generated cryopreserved organoids for purchase.

Schematic of how different organoids are made.
Scientists grow stems cells in the presence of different substrates to mimic development and intercellular interactions. They typically harvest and induce stem cell aggregates to create cell types found in one of the three germ layers. During normal development, these germ layers give rise to specific tissue types, and scientists emulate this process to grow organoids that recapitulate structural and functional features of different tissues in vitro.
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Examples of Human Organoids1

  • Brain organoids derived from hPSCs
  • Lung organoids derived from iPSCs
  • Intestinal organoids derived from hPSCs
  • Stomach organoids derived from adult stem cells
  • Liver organoids derived from adult stem cells or iPSCs
  • Kidney organoids derived from hPSCs
  • Cardiac organoids derived from hESCs
  • Skeletal muscle organoids derived from myoblast progenitors
  • Bone spheroids derived from osteogenic cells
  • Retinal organoids derived for hPSCs

How Do Researchers Use Organoids?

Investigating neurodevelopment and neurovascular connections

Brain organoids, also called cerebral organoids, are stem cell-derived self-organizing structures that emulate neurodevelopment and 3D brain composition.6 The brain is a highly complex organ with elements that are unique to humans, so using animal models to study brain development is rather ineffective. Scientists use mainly hPSCs or mouse embryonic stem cells along with neural stimulatory factors to grow brain organoids in 3D cell culture protocols. Researchers have designed these protocols to promote cell differentiation into organoids representing specific brain regions. As these develop and form multiple tissue layers, they can represent the actual progression of the human brain during gestation. Researchers are continuously improving their methods, pushing the development of these 3D brain regions further in gestational age to discover unknown brain development mechanisms. For many regions of the brain, however, inducing in vitro development past early gestational ages remains a challenge. Nevertheless, brain organoids provide significant insights into neurodevelopmental, psychiatric, and genetic diseases as well as neural tumors.6

See also "Human Brain Organoids Transplanted Into Rats Respond to Visual Stimuli"

Another challenge that researchers face when creating brain organoids is incorporating multiple cell types that influence brain biology. In living organisms, vascular cells, immune cells, and other non-neural cells are responsible for brain function. For instance, neurovascular cells deliver oxygen and nutrients to the brain, influencing neural growth and development. To replicate these intertwined dynamics, scientists create vascularized brain organoids. They develop strategies such as co-culturing brain organoids with blood vessel cells or genetically engineering stem cells to express transcription factors that drive blood vessel cell fate.7

A microscopy image of a fluorescently stained brain organoid, demonstrating complex cellular heterogeneity with cells stained using different colored markers.
A cross-section through a fluorescently stained brain organoid showing the cellular heterogeneity that can be achieved in organoid culture.
Courtesy of Muotri Lab/UC San Diego

Regenerating the airway

The lung is another complex tissue that is difficult to study. With its many cell types, highly vascularized structure, and function in oxygen exchange, 2D in vitro experiments are limiting and in vivo studies are costly. Lung organoids representing alveolar tissues were first developed in 1980, but were not truly successful until 30 years later when Darrell Kotton from Boston University purified lung precursor cells from mouse embryonic fibroblasts and stimulated BMP/FGF signaling to induce lung cell differentiation.8 Techniques have since improved as researchers use hPSCs and iPSCs to model airway epithelial cell function, track early lung bud development, and perform lung cancer screens. The lung is also unique in its ability to regenerate following damage. Therefore, researchers can derive lung organoids from adult stem cells.9 This makes it possible to generate lung organoids from adult patients with genetic or environmental diseases. More recently, lung organoids have become an invaluable tool for studying covid’s effect on respiratory function and development.10

Connecting the gut and the microbiome in culture

The gastrointestinal tract has many functions and consists of multiple germ layers that must work together. Researchers used hPSCs to develop the first gastric and intestinal organoids that contained multiple tissue types.11,12 However, the gastrointestinal system is closely tied to neural and immune cells, which made many protocols relatively limited in their ability to recapitulate what goes on inside the body. To make things more complicated, the stomach and intestines have their own microbiomes, which influence multiple processes ranging from the immune response to pharmacokinetics. With this in mind, researchers recently developed protocols that co-culture intestinal commensal bacteria with organoids, which can model gastric diseases and allow researchers to study microbes’ influence on cancer cells.13

Modeling patients with cancer organoids

Patient-derived cancer organoids are invaluable tools for studying cancer, as well as designing and implementing precision therapy. Cancer organoids accurately model patient tumors and scientists use them for basic research and drug screens. Organoids developed from tumors also have a surprising ability to maintain genetic and molecular signatures even when extensively passaged.14 Recently, researchers developed methods to generate and screen patient-derived organoids to determine patient-specific therapies within a matter of days.15

Assembloids and organ on a chip models: the future of 3D cultures

Researchers have now gone a step further in organ modeling by developing cultured assembloids, which are the next generation of organoids that combine multiple tissues. In organs such as the brain where several different regions and tissue types interact, researchers developed protocols that combine multiple tissues in culture, allowing them to observe and model the tissue interactions that affect cellular function.16 Recently, laboratories adapted these protocols to additional tissues that are increasingly complex, quickly making assembloids an important tool in modeling disease.

In addition, researchers are beginning to combine organoids with revolutionary bioengineering tools such as organ on a chip systems.17 These are microfluidic devices that contain and fuel mini tissues or organs. Scientists use these systems to biochemically mimic the 3D in vivo environment on a microscale.18 For instance, by pairing organoids and organ on a chip devices, researchers have investigated how immune cells respond to tumor cells, and created multi-organoid metastasis and drug response models. Combining organoids and engineering technologies generates opportunities for next-generation models of complex human physiology and pathology.17

Replacing animal models with organoids

In the past two decades, scientists and governing bodies have begun introducing new regulations to replace or eliminate animal testing in pharmaceutical research. Because organoids can recapitulate complex physiological functions, they may be viable alternatives to animal research. In contrast to conventional 2D cell cultures and animal models, organoids enable researchers to model patient-specific genetics, cell organization, and tissue-like structures in vitro. Organoids are also more financially accessible than animal models for in-depth biological studies. As such, organoids hold promise for a wide variety of applications, including drug discovery, diagnostics, and cellular therapy testing.19

References

  1. Lancaster MA, Knoblich JA. Organogenesis in a dish: modeling development and disease using organoid technologies. Science. 2014;345(6194):1247125.
  2. Hirschhaeuser F, et al. Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol. 2010;148(1):3-15.
  3. Huch M, Koo BK. Modeling mouse and human development using organoid cultures. Development. 2015;142(18):3113-25.
  4. Kretzschmar K, Clevers H. Organoids: Modeling development and the stem cell niche in a dish. Dev Cell. 2016;38(6):590-600.
  5. Lancaster MA, Knoblich JA. Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014;9(10):2329-40.
  6. Sidhaye J, Knoblich JA. Brain organoids: an ensemble of bioassays to investigate human neurodevelopment and disease. Cell Death Differ. 2021;28(1):52-67.
  7. Cakir B, Park IH. Getting the right cells. Elife. 2022;11:e80373.
  8. Longmire TA, et al. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell. 2012;10(4):398-411.
  9. Archer F, et al. State of the art on lung organoids in mammals. Vet Res. 2021;52(1):77.
  10. Han Y, et al. Identification of SARS-CoV-2 inhibitors using lung and colonic organoids. Nature. 2021;589(7841):270-5.
  11. McCracken KW, et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature. 2014;516(7531):400-4.
  12. Spence JR, et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature. 2011;470(7332):105-9.
  13. Puschhof J, et al. Intestinal organoid cocultures with microbes. Nat Protoc. 2021;16(10):4633-49.
  14. Sachs N, et al. A living biobank of breast cancer organoids captures disease heterogeneity. Cell. 2018;172(1-2):373-386.e10.
  15. Ding S, et al. Patient-derived micro-organospheres enable clinical precision oncology. Cell Stem Cell. 2022;29(6):905-917.e6.
  16. Birey F, et al. Assembly of functionally integrated human forebrain spheroids. Nature. 2017;545(7652):54-9.
  17. Corrò C, et al. A brief history of organoids. Am J Physiol Cell Physiol. 2020;319(1):C151-C165.
  18. Leung CM, et al. A guide to the organ-on-a-chip. Nat Rev Methods Primers. 2022;2(33).
  19. Zhao Z, et al. Organoids. Nat Rev Methods Primers. 2022;2:94.

This article was updated on October 3, 2023

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