Spheroid 3D Cell Culture Protocols
Sherman also explained that no single 3D cell culture protocol will fit the needs of every laboratory, and there's often a need to balance different considerations. Laboratories should consider the cell type they're working with, their analysis method, how much uniformity they need in spheroid size and shape, and how much signal they need.
One traditional option is embedding cells within a hydrogel, such as Corning® Matrigel® matrix. "Think of fruit inside of jello. The matrix keeps the cells entrapped and encapsulated," Sherman said.
More recently, some laboratories have moved toward growing spheroids on low-attachment surfaces, such as Corning's Ultra-Low Attachment (ULA) surface. Because cells cannot attach to these surfaces, the cells must aggregate together, forming spheroids.
Organoid 3D Cell Culture Protocols
Organoids tend to have more specific requirements than spheroids in terms of growth factors and proteins, and the majority of organoid culture is done by embedding organoids in an extracellular matrix (ECM). This is partly because organoids have polarity, with different cell types on the inside versus the outside.
"Most folks who are growing organoids want them in the appropriate orientation, and that's where ULA is just not enough. You need ECM and ULA, or you just need ECM," Sherman said.
The simplest way to achieve this is to mix cells with Matrigel matrix and place them in a plate so they distribute throughout the hydrogel. Sherman said that, while this is a good way to bulk-produce organoids, the result is more difficult to image because the organoids are in different focal planes.
Instead, some researchers coat a plate with Matrigel matrix first, creating a flat, thick bed. Then, organoid cells are added on top in a much more diluted mixture with Matrigel matrix. This technique is called "sandwich culture." While it requires multiple steps for setup, it simplifies imaging because the organoids are in a single focal plane.
Another option is a dome assay or a droplet assay, a technique in which small droplets (5–10 uL) of hydrogel mixed with cells are placed on a surface. The small volume forces organoids into a narrow field of view for imaging but only requires a simple, one-step process for seeding. Sherman said that, because only a small number of cells are needed to seed the tiny droplets, this technique is especially beneficial when researchers are working with precious organoids, such as those derived from a patient's cells.
A newer technique, which has been demonstrated with several types of organoids, is to use a ULA product plus Matrigel as a supplement in the media. The ULA keeps the cells from attaching, and the Matrigel signals the organoids to develop the correct orientation. This can reduce costs because only a dilute Matrigel solution is used, and it can help balance labs' imaging and simplicity needs.
Troubleshooting Spheroid and Organoid Cultures
Because of the specific requirements of 3D cultures, procedures for spheroid and organoid culture may need more optimization compared to 2D cultures. Here are some troubleshooting considerations.
Uniformity of Cell Suspensions
Sherman explained how failure to keep cell suspensions mixed properly throughout the process of seeding plates can lead to non-uniform spheroids. "If you have a heterogeneous cell suspension, the cells are going to land where they land. So, if you get a clump of cells that lands in one well versus a single cell that lands in another well, you could get different-sized spheroids. For all spheroid protocols, it's really important to start with a uniform suspension."
Organoid Optimization Required
Sherman also noted that, when organoids are grown from patient-derived cells, each cell line needs to be tested for the optimal density and the optimal size to break the organoids into for passaging, because these factors greatly impact viability. "Every organoid line from a patient is going to behave slightly differently, even if it is the same organ," she said.
For spheroids, as well, researchers should optimize seeding density to control the final size of spheroids. "A larger spheroid is going to have greater nutrient needs, so media changes will need to be more frequent, and that could start to impact the viability," Sherman explained. "Making sure your cells get the appropriate nutrients and aren't building up waste is important."
Lysis and Imaging Considerations
When researchers move from 2D to 3D culture, they should be aware of lysis and imaging considerations. For example, it is harder to lyse a 3D than a 2D structure.
Commercially available cell lysis kits that have stronger lytic compounds are available for 3D cells so the entire spheroid can be lysed before analysis. For cell imaging, scientists should consider that it may take longer for dyes to penetrate and for fixation to occur in 3D versus 2D.
Recent Advancements and Real-world Applications of 3D Culture
Recent advances are leading to new applications in the exciting field of 3D cell culture. Here are a couple areas of active research.
Inside-out Organoids
An interesting development in organoid culture techniques involves growing organoids with inside-out polarity. Sherman explained, "With an airway organoid, if it's grown using Matrigel so it has the same orientation as it would have in your body, you'll have no access to the cilia because they'll be on the inside of the organoid. But when you grow them on ULA, they actually flip so the cilia are on the outside." Gut organoids can also be grown in a flipped orientation for easier access to cells of interest.
In an article published in the journal Viruses, scientists created an inside-out airway organoid model and used it to model SARS-CoV-2 infection of the lungs. The technique could also be applied to study other respiratory illnesses.
Spheroids for Toxicology
Spheroids are not only used in cancer research; they are also being employed in toxicology studies. "Primary liver cells can be cultured as spheroids, and they do show more sensitivity than traditional 2D cultures (in toxicology studies)," Sherman said. "You can also co-culture multiple primary cell types that would be found in the liver to make a more complex model. It's not an organoid, but it's a much more complex spheroid."
Some laboratories are now using liver spheroids to test drug candidates for possible hepatotoxic effects and investigate mechanisms of toxicity that are difficult to study in 2D.
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