Primary vs. Stem-Like Cells in 3D Cell Cultures

Biocompare readers are familiar with the rationale for three-dimensional (3D) cell culture: biological, and therefore clinical, relevance. 3D cultures range from simple cellular suspensions to spheroids and organoids, plus they can be 3D-printed, scaffolded, or substrate-based “organ chips,” system-chips, or hybrids of any of the above plus microfluidics. Cell sources are also quite varied, to include adult and embryonic stem cells, induced pluripotent stem cells, immortalized cells, and primary cells.

The literature on 3D cultures leaves the impression that the more complex the system, the more likely it will require immature cells that have not undergone final differentiation. In other words, primary cells for spheroids, simple suspension cultures, and some bioprinted applications and stem-like cells for everything else.

The reality is much more complicated.

Inherent limitations

Non-immortalized primary cells are the “real deal,” as they represent the cells under study in their original, pristine state. But as noted by Thomas Hartung, M.D., Ph.D., at the Center for Alternatives to Animal Testing, Johns Hopkins University, “Primary cells are rarely available from healthy humans, and each donation is unique. You can get blood, sometimes skin, but that’s it.” Hartung studies the brain, for example. “Try getting a donation from somebody not deceased or very ill undergoing surgery.”

By contrast stem cells are a reproducible, renewable source of human cells, and differentiation protocols are improving all the time. “But this technology is fairly young—the ethically non-problematic induced pluripotent stem cells were only developed in 2006, and have been broadly available for only about 10 years,” Hartung tells Biocompare.

In addition to sourcing woes, primary cells have a reputation for being difficult to work with, and having very limited expansion capability—a factor limiting their commercialization in commodity cell-based assay platforms. CN Bio, which specializes in complex 3D cultures (including organ-on-a-chip), has noted that the challenges of primary cells exist even for very simple 3D cultures (with the given that not all primary cells that plate nicely in 2D are capable of forming 3D structures predictably and reproducibly).

The secret to greater success with primary cells may involve conditioning such cells, which are normally attachment-dependent, to thrive in standard 3D environments and formats, including those structures at the high end of the complexity scale. Remember that most cells used in mammalian cell cultures to produce biopharmaceuticals, including Chinese hamster ovary (CHO) cells, had to be adapted through years of effort to thrive in suspension culture.

Sometimes less (complex) is more

Arguments regarding the superiority of primary vs.“stem” cells cultured in 3D arise in part from the assumption that more complex cultures, e.g., organoids and organ/system chips, are more advanced and therefore are always preferred to spheroids or even 2D or suspension cultures.

But often, as noted by Hilary Sherman, Senior Applications Scientist at Corning Life Sciences, the simpler platform provides all the biological relevance needed—despite its greater simplicity in terms of construction and a response set to toxicology or pharmacology. “This is the big question! It's always a balancing act when trying to get the most relevant data for the question being asked in the simplest and most cost-effective manner possible. Sometimes a complex model is required to obtain your answer, but other times a relatively simple, inexpensive model will do the job."

For example, investigators create 3D hepatocyte (liver) spheroid models from both primary cells and stem-like cells, but the former are easier to work with than cultures derived from primary cells and, since they often serve admirably for simple tox/pharm assays, are more commonly used, according to Dr. Ovidiu Novac, Senior Scientist, CN Bio.

“Spheroids generated from primary cells or differentiated stem cells provide improved physiological relevance compared with immortalized cells and are relatively simple to adopt into screening regimes. But, to gain an extra dimension of physiological relevance, we have shown that co-culturing human-relevant ratios of primary liver cell types under perfusion creates advanced 3D microtissues that even more accurately recapitulate the phenotype and function of their human organ counterparts in health and disease. These more advanced liver-on-a-chip models deliver greater metabolic 'horsepower' and clinically relevant biomarker detection. They perfectly complement primary hepatocyte spheroid assays by providing a test environment to ask more in-depth questions such as drug mechanism of action or toxicity.”

The question, therefore, is not whether primary, immortalized, or stem-like cells are best, but which assay platform is superior.

“For us, the organ and application dictate our choice of cell type,” Novac explains. “In our hands, for example, primary human hepatocytes function more highly than iPSC-derived hepatocytes, for example in determining cytochrome p activity. Primary cells remain in a differentiated state for a month in culture when perfused to replicate blood circulation. This allows us to run chronic dosing studies, to profile human drug metabolism, and to identify metabolite-driven toxicity in both healthy and diseased liver models. However, in jurisdictions in which the use of human tissues is restricted, stem cells represent the obvious alternative.”

Spheroids are generally simpler, easier to make, and less expensive to culture than organoids, says Corning’s Hilary Sherman. “Organoids typically require more specialized, expensive media formulations and may require longer culture periods depending on the source of the organoid culture.” iPSC-derived organoids typically take the longest to make since the cells must undergo differentiation. Those derived from adult stem cells have shorter culture times since some degree of tissue-specificity already exists. Note as well that organoids and spheroids require an extracellular matrix, which adds to cost and complexity.

The key to succeeding at 3D cell culture is therefore not to overengineer your assay platform. Keep things as simple as possible, and let the assay and desired readout dictate the type of culture you use and the cells comprising it.