The Need for Mass Produced Spheroids | Corning

Spheroids have proven to be simple but versatile 3D models that can better recapitulate physiological conditions than 2D models. They also provide a convenient starting point for more complex 3D models, including embryoid bodies and organoids.

With so many applications, it's no surprise that researchers now need reliable ways to mass produce spheroids and other 3D objects.

Bulk Spheroid Tools

According to Hilary Sherman, Senior Applications Scientist at Corning Life Sciences, there weren't a lot of great tools for scaling up spheroid production until recently.

"One tool that customers have used is spinner flasks, but the problem is that you get non-uniform aggregates. If you're trying to do any kind of drug screening, and your spheroids are different sizes, then the drug penetration and drug response can be different. If you're working with stem cells—perhaps creating embryoid bodies—the same concept applies to differentiation. The growth factors penetrate at different rates, so you might get good differentiation in your small organoids but not so much in your bigger organoids. Thus, being able to control size is really important."

Corning® Elplasia® plates provide this control by combining the Corning Ultra-Low Attachment Surface with microcavity technology. Each U-shaped microcavity is designed to hold a single spheroid as it's generated, cultured, and analyzed. This allows hundreds to thousands of spheroids to be cultured in each well of a microplate for up to 21 days or more.

The technology is available in open well plates with 6 to 384 wells. For large-scale production of a single type of spheroid, the Corning Elplasia® 12K flask enables approximately 12,000 spheroids to form in a vessel footprint similar to that of a T-75 flask.

"These products work well and are easy to use," says Sherman. "Cell seeding is the same as for any traditional vessel. It's just that instead of cells laying flat on the surface, they settle into these cavities by gravity to form a spheroid. Customers are having a lot of success, too. Many are already using our Elplasia products for the purpose of creating embryoid bodies from induced pluripotent stem cells (iPSCs) to further differentiate them into their organoids."

Technical Considerations

When working with Elplasia microcavity technology, Sherman emphasizes that what's really most important is "starting with a uniform, single cell suspension." If you have a cell suspension with clumps of different sizes, "you're going to end up with spheroids of all different sizes in your cavities."

Many labs work with cancer cell lines or iPSCs, "which, in general, form beautiful single cells." This simplifies the process of distributing cells uniformly across the plate, "but if you do have small clumps and clusters of cells, hopefully all the clumps and clusters are about the same size." One option that may help is to use a cell strainer.

Spheroid culture is straightforward in Elplasia plates but does require some optimization depending on cell type, seeding density, and desired culture time. The Guidelines for Use recommend between 100 and 1,000 cells per microcavity depending on the application and cell type.

Care should be taken to avoid dislodging spheroids out of the microcavities—for example, when transporting the plates from one location to another. Care needs to be taken when performing media exchanges, if possible. When possible, half media changes are recommended to reduce the risk of disturbing the spheroids. The Elplasia 12K flask features an internal diverter to minimize disruption of spheroids during liquid handling steps.

Synthegel 3D Matrix Kits for Mass Production

Corning innovation has produced yet another option for mass producing spheroids: Corning Synthegel™ 3D matrix kits, which provide chemically defined synthetic hydrogels for 3D cell culture.

As Sherman explains, "The microcavity substrates, like Elplasia plates, are very easy to work with because there's no hydrogel involved and no sticky viscous material to play with. But the cells only grow in one plane. This means if you need more three-dimensional structures, you need more surface area."

Sherman also notes, "With the hydrogels, you're working in a three-dimensional space. The structures are forming throughout the gel, which means you can generate more three-dimensional structures in a volumetric space."

Synthegel 3D matrix is available in two formulations for iPSC culture. "One is a very viscous hydrogel, so you can only get a certain thickness before nutrient penetration becomes a problem. The other is a bit looser, which allows you to make a thicker layer without worrying about diffusion gradients. In a given volumetric space, you can actually put in more hydrogel, so you can get more spheroids." It's designed more for scaling up and can be used in any standard vessel, like a standard T-flask or Corning CellSTACK®.

The Utility of Spheroids

When it comes to current applications for spheroids, according to Sherman, "For me, the most exciting is probably the organoid work. As research in the organoid area develops, there are more established protocols for how to create different organoid models from iPSCs." By growing uniform spheroids to produce uniform embryoid bodies, "customers are now able to mass produce more uniform organoids—like brain organoids, kidney organoids, and liver organoids."

Cancer spheroids are widely used as disease models and for the screening of potential anti-cancer therapeutics. Compared to 2D cell culture, 3D cell culture more accurately mimics in vivo cellular conditions, including cell-cell and cell-matrix communications, nutrient status, and physiological/biochemical properties. Proteomic analysis has revealed significant differences in cancer cells cultured in 2D versus 3D. Compared with 2D culture models, 3D culture models are also more likely to show resistance to anti-cancer drugs. This difference is possibly caused by reduced drug penetration into the core cells of the 3D spheroid and increased hypoxia-induced drug resistance, which mimic in vivo conditions.

Sherman is also seeing a lot of interest in bulk producing 3D structures to generate more cellular material for RNA, DNA, or protein analysis. For example, "One well of a 96-well plate (with cells in 2D) might not give researchers enough material to work with, but a spheroid in that same footprint can give them a lot more material." Extracellular vesicle (EV) production "is also an emerging and very hot area of research right now. There is literature to suggest that you can get higher EV production when cells are cultured in three dimensions compared to two dimensions."

"In the last year or so, there's been a lot more interest in 3D bioprinting because of the ability to spatially determine where your cells are going to grow and how they're going to align," Sherman says. "The addition of spheroids to bioprinting is very new. Instead of seeding single cells to create a structure in a hydrogel, with the cells then slowly doubling and growing together, you start with spheroids, and thus, everything merges together much faster to generate an actual tissue. This is potentially a more efficient way to generate a tissue."

With the expanding applications of spheroids, mass production can impact how these spheroids are utilized and increase their use across experimental and clinical applications.

Learn more about mass spheroid production tools and request samples.