Dynamic Cell Cultures: Organoids and Spheroids in Suspension

Compared to conventional 2D cell culture, organoids and spheroids represent complex 3D structures that have revolutionized in vitro investigations of human diseases, human development, and precision medicine. Research is moving forward at a breathtaking pace—with many groups focused on potential therapeutic applications—and there's an increasing need to generate large numbers of high-quality spheroids and organoids. This has led to advances in dynamic cell cultures, which allow for organoids and spheroid aggregate cultures generated in suspension.

Growing a huge quantity of cells would typically involve a bioreactor, which can provide continuous control of pH, nutrients, metabolites, and dissolved oxygen to promote cell growth. However, bioreactors can be cost prohibitive and require larger volumes than necessary for many purposes, so numerous protocols for dynamic cell cultures use ErlenmeyersCorning® disposable spinner flasksCorning Elplasia® 12k flasks, and Corning Elplasia® plates.

Advantages of Aggregate Cultures

Organoids are three-dimensional cell culture models that self-organize into complex organ-like tissues. They are commonly generated from pluripotent stem cells or adult stem cells. Spheroids are aggregates of one or more cell types and are commonly used to model multicellular tumors. When cultured on non-adherent surfaces, cells are able to interact with each other in ways that more closely mimic in vivo conditions.

Elizabeth Abraham, Market Manager for the Corning Life Sciences 3D Cell Culture division, explains the advantages of organoids and aggregate cultures generated in suspension: "The reason it's advantageous to culture these 3D structures in suspension is that it simplifies harvest since you're not removing from a scaffold. In addition, if you're growing 3D structures in a matrix or a tissue culture plate, they can reach a size that's too big for the plate or the vessel. In a suspension format, that size constraint is reduced."

Abraham continues: "Another advantage is that there's nutrient access from all sides. Recent literature indicates that cells in 3D suspension cultures are more able to mimic the mesenchyme, which is not easily mimicked in attached 3D cultures, so there's a functional advantage, as well."

Challenges of Suspension Culture

According to Abraham, some of the challenges associated with dynamic cell cultures include sheer stress. "This happens when the force to prevent cells settling is harsh enough to cause the cells to actually break apart or sheer them." It's a balance between preserving cell health and using enough force to keep the cells moving or suspended.

To address that challenge on a small scale, Abraham says, "You can use ultra-low attachment coated or non-adherent vessels that keep the cells in suspension or non-adherent. You can also use micro-organospheres," which are miniature droplet-encapsulated tissue or cell models. This would allow for culture in a Petri dish on an orbital shaker, for example.

At a larger scale, Abraham suggests scientists should look toward Erlenmeyers, disposable spinner flasks, or bioreactors with low shear stress.

Control of Spheroid Size

"The Erlenmeyers or the spinner flasks allow for spheroid generation, but they are often heterogenous in size," says Abraham. To allow for consistent spheroid generation, Corning developed the microcavity technology used in Elplasia 12K flasks and plates. When combined with an ultra-low attachment surface, the microcavities allow a high density of uniformly sized spheroids to self-assemble in a scaffold-free environment. "The sphere size with Elplasia flasks will typically be 50 microns and under. With the Erlenmeyer, sphere sizes could vary between 100 or 200 microns, or grow to larger spheres when cultured for a longer time."

Elplasia technology is available in multi-well formats and in the Corning Elplasia 12k flask, which allows for the culture of about 12,000 spheroids in a T-75 format. Abraham explains, "You have better control of culture conditions with Elplasia technology. It's not a dynamic culture per se, because you have to transfer the spheres formed in the Elplasia platform into something like a spinner flask to keep it in suspension. However, it can act as an important stepping stone in the process of moving to dynamic culture."

The Elplasia 12k flask makes it easier to generate the large number of spheroids required for suspension culture and potential downstream applications.

Extracellular Matrix for Organoids

According to Abraham, a general rule is: "If the organoids are grown from a tissue-derived stem cell—and to get that stem cell to multiply and then differentiate—you definitely need Corning Matrigel® Matrix." To grow organoids in suspension, researchers have developed some innovative solutions. "They're adding Matrigel matrix to the medium in a very dilute form," or using micro-organospheres to encapsulate the Matrigel matrix droplet in another droplet before adding it to suspension.

Growing organoids in suspension would typically be a multistep process. For example, dynamic cultures are "a tried-and-true method for growing brain organoids," explains Abraham.

For this application, researchers first grow pluripotent stem cells and then "differentiate to a neural lineage by using growth factors, which produces neural rosettes" in 2D culture. The rosettes "are mixed with Matrigel matrix and then a droplet is dispensed onto a parafilm" that prevents the droplets from attaching. Once the organoids grow to a certain size, they can be transferred to a Petri dish kept on an orbital shaker, where they typically develop for several months.

"Corning has recently launched a synthetic hydrogel called Synthegel™, which allows you to grow induced pluripotent stem cells (iPSCs) as 3D cultures in an embedded or suspension format," says Abraham. The innovative Corning Synthegel 3D Matrix is a defined, self-healing hydrogel that supports 3D cultures of human iPSCs. Thus allowing researchers to stay within the 3D format to both grow and differentiate iPSCs and alleviate the need to transfer from 2D to 3D formats.

Liver and Kidney Organoids

By using disposable spinner flasks, researchers have developed protocols to generate billions of healthy liver organoids with relative ease. When liver organoids were cultured in disposable 125-mL spinner flasks with 10 percent (vol/vol) Matrigel matrix, they rapidly proliferated to reach 40-fold cell expansion after 2 weeks, compared with 6-fold expansion in static cultures.

The spinner flask organoids were also larger and had better expression of mature hepatocyte markers, meaning they more closely mimic the function of the native organ. Huge numbers of functional cells will be necessary for potential applications in tissue engineering or transplantation.

Kidney organoids are also routinely cultured from iPSCs in 125-mL spinner flasks. One advantage of using spinner flasks is that the approach is easily scalable.

Perfusion Cultures

For some applications, producing a large volume of cellular material is less important than having tightly controlled conditions for each organoid or spheroid. Microfluidic devices are miniaturized liquid-handling systems that incorporate fluidic channels and chambers, which can be designed for and seeded with cells. These systems allow for the continuous infusion of nutrients and growth factors, tight control of spheroid or organoid size, and precise replication of cell-cell contacts.

As a result, cellular structures can reach more advanced stages of development. For example, brain organoids cultured on microfluidic chips exhibit neural differentiation and a wrinkly cortical structure. Microfluidic devices can include multiple cell types to produce an organ-on-a-chip.

Exploring the Potential

Abraham cautions, "Some cell types may respond better to dynamic culture than others. Some are far more sensitive. It would be a matter of trying to see which works. I would definitely recommend starting with static culture and then moving into dynamic perfusion because, for perfusion, the threshold is a bit higher."

Learn more about scaling up bulk spheroid production and dynamic cultures and how to get started with Corning Elplasia Technology. Corning Scientific support and Field Application Scientists can help you choose the right solution to achieve your goals—from basic cell culture to 3D models and cell therapy production.