Selecting and Generating 3D Cell-Based Models

Three-dimensional (3D) cell cultures are rapidly replacing 2D cell monolayers (conventional tissue cultures), and even some animal test models, in many industries but predominantly in biology and medicine. Applications of 3D cultures include “live” sensors, microfluidic devices such as organ-chips, and stand-alone test platforms in drug discovery and toxicology. Organoids and spheroids are two significant 3D cell culture models that don't require microfluidics or chip-like substrates to yield results that are unachievable through standard 2D cell culture. In selecting between the two, scientists consider the application or specific readout required, and of course cost and availability.

“Organoids possess additional complexity compared with spheroids,” explains Steve Budd, Scientist and Product Line Business Specialist at ATCC. “Organoids are composed of organ-specific cells such as stomach, colon, etc., while spheroids are made of less diverse cell types.” Organoids also require a scaffolding matrix on which the cells self-assemble. “Spheroids do not, but they do require a specialized liquid medium.”

Another variant, organotypic cell culture, involves culturing different cell types to form a more differentiated 3D structure. An example would be culturing airway epithelial cells with fibroblasts to form differentiated ciliated and goblet cells that mimic lung tissue.

According to Budd, deciding which model to employ depends on the end-user's ability to generate the models. “While brain organoids can be derived from induced pluripotent stem cells (iPSCs), obtaining these models via primary tissue is extremely hard, if not impossible.”

The requirement that users “roll their own” organoids has been a limiting factor in their widespread use, even as organizations like ATCC offer organoid models commercially.

Organoids can be made in different ways: from iPSCs, from normal primary cells, or from cancer cells. “In all cases a specialty medium is required, along with a scaffolding matrix. “The medium is quite complex and varies based on the model type,” Budd says. Most of ATCC's models require a base medium, conditioned medium containing specific growth factors, and other chemical reagents and recombinant proteins.

Recapitulating cardiac function

With cardiovascular disease the perennial number-one cause of death, and cardiac toxicity a leading cause of drug failures, interest in cardiac organoids is robust and growing.

Proper heart function depends on coordinated interaction of, and among, many cells that comprise heart tissue. “The challenge in 3D models has been to create an environment where human-derived cells can form functional tissue,” says Misti Ushio, CEO of TARA Biosystems. “Two areas must be addressed to achieve this goal: sourcing cardiac cells and achieving functional maturity.”

Obtaining human cardiac tissue is itself challenging. Historically cardiomyocytes, the cells responsible for cardiac contraction, are isolated from (non-human) animals. The isolation procedure can result in high batch-to-batch variability, a major issue for model development.

“Unlike many other cell types, cardiomyocytes, once isolated, can only be maintained in the lab for short periods of time,” Ushio explains. “Beyond the technical issues, there are fundamental differences in cardiac physiology between animal and human hearts. A human-derived cell would be ideal. Groundbreaking advances in stem cell technology have yielded the ability to generate cardiomyocytes derived from induced pluripotent stem cells.”

iPSCs have been a game-changer in terms of sourcing cells, as human heart tissue is no longer required. This approach holds great promise for 3D cell-based models, but it highlights a second area to be addressed, i.e., functional maturity.

“While iPSC-derived cardiomyocytes possess several desirable features of human cardiomyocytes they lack critical physiologic and drug responses,” Ushio says. “Researchers address this issue in two ways. They can first create ‘chips’ in which cells are in a physical microenvironment, or niche, that supports a tissue-like structure that allows interactions similar to those occurring in the human heart. The second is they provide relevant cues, such as electrical and mechanical stimulation, that promotes physiologic maturation.”

3D cell-based models come in several varieties, including spheroids, organoids, organ-chips, disease-chips, etc. How does a laboratory select among them?

“Organoids, spheroids, and other 3D models have all shown great potential in many applications, including disease modeling and regenerative medicine. All these formats bring value to the research ecosystem,” Ushio explains. “The choice depends on multiple factors, including the mix of cell types under study, desired endpoints, assay throughput, and cost.”

Organoids, for example, replicate microanatomy quite accurately. TARA Biosystems generates cardiac tissue using iPSC-derived cardiomyocytes, which replicate human organs and relevant biology, and generate rich datasets which, according to Ushio, “generate accurate information that increases the probability of success for drug discovery and development. This is the missing piece for cardiac drug discovery and development, cardiotoxicity testing, and more.” By contrast spheroids are best suited for cancer research and drug screening. “There has been significant progress in the application of in vitro tumor cell aggregates as models for in vivo tissue environments,” Ushio adds.

3D models are useful in modeling diseases where the cellular microenvironment plays an essential or vital role. The microenvironment influences a range of features including cell signaling, proliferation, viability, and drug response. 3D models recapitulate in vivo cellular responses, including responses to drug treatments, by incorporating original tissue architecture.

Barriers to industrialization

What will be required to “industrialize” cardiac—or other—organoids to allow researchers to order them for specific applications from catalogs, the way they do for cell lines? In contrast to many biologics, shelf life and storage of organoids is relatively straightforward. “Organoids are stored and shipped in liquid nitrogen, just as with standard cells, and may be kept under those conditions indefinitely,” Budd tells Biocompare, “so shelf life is not an issue, and they have no expiration date.” Cell viability is also comparable to standard cell lines cultured in 2D.

There is one thing to note regarding the initial expansion of organoids. When first plated after thawing, organoids are dissociated and exist as cells and cell clumps. They then reassemble into typical organoid morphology.

Organoids also have a rather long expansion time. “Slower-growing organoids could take several weeks to months to expand to quantities required for an experiment,” Budd adds. “Also with manufacturing, the medium used is somewhat complicated. It usually requires a base medium, common components like L-glutamine, conditioned media obtained from medium exposed to R-Spondin1-Fc, and/or Wnt-3A expressing cells. Medium complexity, of course, adds preparation time to the process.”