Spheroid vs. Organoid: What's the Difference?

The terms "spheroid" and "organoid" are kind of like jam or jelly.

Sure, they mean similar things, and they're often used interchangeably — most of the time, you'll get by just fine using either. But there are distinct differences between the two in how they're made and what they do.

If you're just looking to make a sandwich, either jam or jelly will do the job. But if you want to get started in complex 3D cell culture, you need to know the difference — and pick the right one.

Spheroid vs. Organoid Definitions: Learning the Basics of Cell Clusters

Spheroids and organoids are 3D structures composed of multiple cells. Each can be useful in 3D cell research — but in different ways because they're made differently.
  • Spheroids are simple clusters of broad-ranging cells, such as from tumor tissue, embryoid bodies, hepatocytes, nervous tissue, or mammary glands. They don't require scaffolding to form 3D cultures; they do so by simply sticking to each other. However, they can't self-assemble or regenerate and, thus, aren't as advanced as organoids.
  • Organoids are complex clusters of organ-specific cells, such as those from the stomach, liver, or bladder. They're made of stem cells or progenitor cells and self-assemble when given a scaffolding extracellular environment, such as Corning® Matrigel® matrix or collagen. When that happens, they grow into microscopic versions of parent organs viable for 3D study.

Scientific Applications of Organoids and Spheroids

How do spheroid vs. organoid cultures compare? Each has unique applications, and different lab scenarios might call for different multicellular structures.

Organoid Applications

Organoid technology has been used to great success in personalized medicine — in disease modeling as well as optimizing drug discovery and regenerative medicine. The applications of organoids in CRISPR research could similarly help scientists better study organ development within the context of gene editing.

Specific to cancer research, 3D organoids can provide insight into the mutational signatures of selected cancers because they can mimic the pathophysiology of human tumors.

Organoids can also function as a self-assembling miniature manifestation of a parent organ, which can be of particular benefit to researchers. For example, neural organoids are bringing us closer to understanding diseases in the brain.

Spheroid Applications

Perhaps most notably, tumor spheroids can help scientists understand the in vivo microenvironments of tumors, which can help researchers predict drug efficacy in cancer research.

One research team recently screened a natural product library against non-small cell lung cancer spheroids in 1536-well plates and identified several compounds active against the cancer cells. Another team used spheroids with macrophage infiltrates to model how macrophages interact with tumors and anti-cancer drugs in the human body.

Spheroids can also be used in stem cell research to develop embryoid bodies from induced pluripotent stem cells, which can then be turned into high-purity neural stem cells for studying neural diseases and their related treatments.

Spheroid Culture Techniques

In scaffold methods, researchers use an artificial hydrogel, an extracellular matrix, or a solid scaffold to shape cell growth and encourage spheroid formation.

Anti-adherence methods rely on preventing cell adherence to a surface so cells are forced to clump together. Growing cells in a bioreactor with constantly flowing media, thus preventing them from adhering to the vessel, is one such method. The use of low-attachment coatings in plates so cells clump and form spheroids is another.

In the hanging-drop culture method, cells are seeded in media, which is used to form hanging droplets below a surface. With no surface to attach to, cells clump together by gravity, forming one spheroid inside each drop.

Spheroid Analysis Techniques

To analyze spheroid experiments, researchers use plate readers, various microscopy techniques, or flow cytometry.

Transwell migration and invasion assays with tumor spheroids can be analyzed using microscopy with image capture, microscopy with direct counting, a plate reader, or flow cytometry.

Viability assays rely on differences between dead or dying and viable cells, such as the presence of apoptotic markers or the ability of live cells to metabolize certain molecules into a fluorescent or colored product. The cells in spheroids may need to be disaggregated into single cells before analysis.

To evaluate the penetration of drug-carrying nanoparticles or other therapeutic products into tumor spheroids, researchers have used flow cytometry, confocal microscopy, and various other microscopy techniques. For example, a research team used two-photon microscopy to analyze the ability of drug-delivery nanoparticles with different properties to penetrate deeply into tumor spheroids.

Another group of researchers first treated colorectal cancer spheroids with drug-carrying nanoparticles, then stained the spheroids with Hoechst 33342 dye, which stains cells closer to the exterior of the spheroids more strongly. After staining, the team disaggregated the spheroids and analyzed their nanoparticle content and staining strength via flow cytometry, thus determining each cell's location in the spheroid and the depth of penetration of the nanoparticles.

Challenges and Future Directions in 3D Culture

Compared to 2D cultures, spheroids and organoids do come with greater complexity in analysis. It can be difficult to image the interior of spheroids or organoids without disaggregation or sectioning, and making histology sections is too labor-intensive for many applications. Another limitation is that certain cancer cell types are unable to form spheroids or have a tendency to form non-spherical aggregations.

Another challenge is that nutrient and oxygen diffusion limitations for cells in the spheroid interior increase as the size of spheroids increase, so larger spheroids develop a necrotic core.

Future directions and emerging trends in spheroid research include the use of spheroids as building blocks to create more complex tissue constructs, and the development of new biosensing methods (e.g., optical coherence tomography and electrochemical biosensing) that can analyze whole spheroids without damage.