Cell-based assays (CBAs) have been used for as long as cell culture has been around. But as hard as scientists tried, obtaining physiologically relevant data from CBAs was hit-and-miss. Some assays provided actionable results while others generated noise.

One problem was the cells themselves. Cultured cancer cells grow indefinitely but differ genetically and biologically from normal cells; primary cells work better, but their availability and expansion capacity is limited.

Advances in primary cell culture, and the emergence of induced pluripotent stem cells, have changed prospects for CBAs for the better. Modern cell biology has enabled creation of complex three-dimensional monocultures and co-cultures. An industry was thus born around cell clusters, spheroids, organoids, and variations where "physiologic relevance" became the operative term.

Initial considerations

3D CBA developers must consider several factors when designing 3D cell culture experiments—the first of which are the advantages or disadvantages of various cell types and models. Organoids and spheroids vary greatly in complexity and physiological relevance, from simple homogeneous 3D spheroids made from existing lines to tissue-derived multifunctional organoids. "Simpler models may be easier to establish and manipulate," says James Clinton, Lead Scientist at ATCC, "but they may not reproduce the relevant physiology or culture environment of more complex models."

As with more conventional models using cancer cell lines, 3D organoids from patient-derived primary cells can be maintained over time, expanded to increase yield, cryopreserved, and recovered back into culture. "However, they also pose additional challenges such as the need for extracellular matrix (ECM) as part of the culture system and specialized, complex media formulations that may require customization. "Proper preparation, storage, and handling of culture and ECM reagents can greatly improve your prospects for success in culturing organoids," Clinton comments.

Getting the right cells

"Cells cultured in 3D behave completely differently from 2D cultures," says Audrey Dubourg, Product Manager at CN Bio. Dubourg advocates for "rigorous testing" to identify cells that offer the greatest physiological relevance given research objectives. To achieve this she suggests acquiring cells from multiple sources, especially when working with primary cells with limited proliferative capacity and higher-than-average sensitivity to in vitro culture conditions.

"Some primary cells, such as hepatocytes, form stronger 3D structures when co-cultured with neighboring cells from the original organ,” Dubourg adds. “Another consideration is the performance of cells in static versus flow media conditions. Perfusion cultures, where media is constantly replenished, help to prolong culture longevity by preserving cultured cells' phenotypes and metabolic functions for up to one month. With static cultures, primary cells begin to de-differentiate after just seven days without access to fresh nutrients and oxygen."

Designers of 3D CBAs must also consider lot-to-lot or batch-to-batch variability when selecting cells from different sources. "Nominally identical cells from different suppliers can behave quite differently," Dubourg tells Biocompare. "Some work perfectly in 2D but when cultured in 3D they exhibit poor viability and inconsistent 3D structure formation."

Organ chips

Organ chips are living, micro-engineered environments that take 3D cell culture to a higher level of sophistication than spheroids and organoids. As with all 3D systems, maintaining an optimal cellular environment, particularly the ECM and cell culture media, is critical for success.

Most cells require ECM proteins to form consistent 3D cultures, but finding the right choice can be challenging, especially for co-cultures where ECM proteins often vary among cell types. A good starting point, says Dubourg, is to characterize the ECM surrounding cells in their natural niche. "You can find this information through published literature or experimentally, where the gold standard approach is to perform immunohistochemistry with ECM protein antibodies." Alternately, researchers can employ a high-throughput approach based on protein expression at the RNA level using single-cell RNA sequencing. "Once thus identified, your choice of 3D culture method can be tailored appropriately."

"For organ chips, users can start with an ECM that they have previously used with their cell types, or select a suitable ECM based on in vivo data," says Lorna Ewart, EVP, Scientific Liaison of Emulate. "ECM should be optimized to support cell viability, morphology, and function. Not doing this will lead to poor cell morphology, uneven coverage of channels, and could also result in cell loss when channel flow is introduced."

For its own organ-chip products Emulate uses multi-layered substrates to accommodate the multiple cell types comprising an organ. Their Intestine-Chip, for example, can be seeded with dissociated human organoids on the top channel and organ-specific endothelial cells on the bottom channel.

Since the Emulate organ chips comprise two separate culture compartments developers must optimize culture media for each. "Users can start with culture media that they know will work individually for each cell type in both top and bottom channels," Ewart adds. "If the different cells survive and function under these conditions, continue using these media. If not, you will need to optimize the best medium condition to support different cells in both top and bottom channels, as needed."

A question of throughput

Like many assays, CBAs may be run efficiently, and at high throughput, in microplates. One of the most useful, high-throughput readouts for complex cell models is automated digital microscopy (ADM). When combined with appropriate environmental control in automated workflows, ADM enables kinetic experiments to continue over weeks while maintaining the health of the cell model.

But it contributes to complexity as well, as experimental design must satisfy requirements for both cell growth and ADM analysis. Satisfying two criteria such as these often involves tradeoffs.

"Aspects to consider are the ease of model formation and the optical clarity of the transparent well bottom," says Peter Banks, Scientific Director at BioTek Instruments, an Agilent business unit. "Agilent has found that for 3D spheroids ultra-low-attachment coated microplates allow for the relatively simple progression from spheroid formation to assay readout in the same microplate, while allowing ADM to be used for verification and monitoring of cell aggregates, and for the readout once the experiment begins in earnest."

Banks notes that the U-shaped well bottoms, while not optimal for imaging, keep spheroids in the center of the well, which enables capturing images of the complete spheroid in one or a minimal number of fields of view without the need to search for a very small object (200–500 m ID) within a much larger-diameter well—more than 100 times larger for a 96-well microplate.

Simple brightfield microscopy using an inverted geometry is a very useful readout for both the verification of cell aggregation and monitoring spheroid proliferation. "A useful starting point for spheroid aggregation is seeding with between 1,000–5,000 cells per well," Banks explains. This cell density is optimal for achieving satisfactory aggregation, long-term cell model viability, and ease of imaging. "Spheroid area" measurements quantify spheroid proliferation.

For information from within the core of the spheroid, investigators use confocal fluorescence microscopy to collect fluorescence from out-of-focus planes, which tend to obfuscate quantitative measurements such as cell counts within the spheroid.