While the primary benefit of 3D cell cultures—greater resemblance to in vivo conditions than their 2D counterparts—is undoubtedly valuable in physiological research, 3D cultures are still more difficult to use compared to 2D cultures. But increasingly, innovations are streamlining 3D cultures workflows.
Today, a variety of 3D culture models—e.g. scaffolds, ultra low attachment microplates, hanging drops, and spheroids—make it easier to choose a type that better satisfies particular research goals and throughput levels. “3D cell models are as diverse and imaginative as the scientists that create them,” says Andrew Niles, senior research scientist at Promega. This article looks at tools that are helping scientists streamline workflows using 3D cell cultures to derive greater physiological information with greater efficiency.
More tailored extracellular matrix
Using 3D cell culture materials that are more physiologically relevant can accelerate workflows. Xylyx Bio offers TissueSpec ECM hydrogels and scaffolds for 3D cell culture; both are derived from specific organs (human or porcine) and correspond to a specific cell type, e.g. scaffold or hydrogel for a 3D cell culture of cardiomyocytes is derived from heart tissue. “We see that cells cultured on these tissue-specific substrates behave more like they would behave in vivo,” says Tanya Yankelevich, director of product management. “Our tissue-specific extracellular matrix products provide highly physiologically relevant microenvironments with predictive values, specifically important in drug testing and cancer research.”
Hydrogel is the most frequently used format in 3D cell culture, says Yankelevich, because it's easier to use and it's easier to image than the scaffold. However, the scaffold provides greater physical structure, which is very important to 3D cell growth. “The scaffold retains the natural 3D structure, biomechanics, and topography of native tissues, which is very important when studying the effects of native tissue mechanics and structure on the differentiation, migration, and function of cells,” explains Yankelevich. She believes that using more physiological 3D culture environments earlier in the drug development process will prove to be beneficial in the long run. “The closer the cellular environment is to actual human environments early on in the drug development process, the higher the chances that the drug failure rate will improve,” she adds.
Ready-to-use microorgans
StemoniX offers assay-ready microOrgan® platforms containing human iPSC-derived tissue cells along with AnalytiX™, their advanced data analysis package. MicroOrgans are 3D spheroids constructed from neural or cardiac tissues, termed microBrain® and microHeart®. “Our microOrgan technology significantly shortens screening workflows at the bench and first pass data analysis,” says Blake Anson, senior director of marketing and strategic alliances at StemoniX.
Because 3D cell cultures are believed to recreate the cellular microenvironment better than 2D cultures, microOrgan and AnalytiX technology can help scientists uncover more physiologically relevant results. “This provides time and resource savings by avoiding experimental dead-ends and having to repeat work due to incomplete, or even incorrect, biology and/or data analysis,” notes Anson.
Many of StemoniX’s customers are biopharmaceutical companies using “our microOrgan platforms across multiple aspects of drug discovery, including early and late-stage toxicity testing, disease modeling, and high-throughput screening,” adds Anson.
Cell-based assays optimized for 3D cultures
Promega’s efforts to streamline the 3D cell culture workflow include simple and reliable plate-based assays for some types of 3D cultures. “This enables our assay users a means to quickly understand the effects of their treatment on multiple parameters associated with cell health,” says Niles. “Our preferred format follows the ‘add-mix-measure’ homogeneous configuration that requires no washes or other processing steps.” Promega offers 3D endpoint assays, as well as 3D real-time assays that allow data collection during the experiment.
One of the challenges presented by 3D cultures is their physical structure, which restricts access to solubilized compounds or to microscopic investigation. “The density and tenacity of 3D structures often impede the permeability of plate-based assay reagents from fully penetrating larger masses to measure biomarkers,” says Niles. “Similarly, microscopic methods often reach practical size limitations due to laser efficiency and other optical considerations.”
Analysis software for 3D imaging
Brendan Brinkman, life science new business strategy for Global Olympus Corporation, agrees that the quantitative imaging and analysis of 3D cell cultures is challenging. Olympus’ NoviSight software aims to streamline the microscopic study of 3D cultures, including a range of rapid 3D object recognition algorithms, which is the basis of their True 3D segmentation analysis. “The analysis is rapid and statistically robust for multiple samples simultaneously across a multiwell microplate,” Brinkman explains.
NoviSight allows “cross linking of data modes, from initial raw images, to 3D volume views, scatterplot display of population data, and segmentation mask overlays resulting in rapid object identification galleries, statistical data tables, and microplate heat maps,” says Brinkman. The NoviSight software can be used with Olympus’ confocal and multiphoton imaging systems.
Olympus also recently released the Provi CM20 (currently available only in the U.S. and Japan), a cell culture monitoring system that uses artificial intelligence for automated measurements of cell counts and confluency, and identification of iPSC colonies. While Provi is used to monitor the growth of 2D cell cultures, these cells are often used to construct 3D cultures. In such workflows, the quality of 2D cultures is critical to the reproducibility of 3D cell culture assays. “The consistency of cell-based assays can be dramatically affected by the passage timing of cells before moving on to the microplate-based assay step,” notes Brinkman.
Even though 3D culture models are not a standard drug discovery tool as yet, more and more biopharmaceutical companies are interested in incorporating them. “We have customers in drug discovery using [multiple 3D cell culture] models, and most of the drug discovery scientists we have spoken to are either actively using such models or planning to adopt them due to their greater physiological relevance,” says Brinkman. With the increasing use of 3D cultures, as well as more tools available to expedite their workflows, it will be interesting to see how research and drug discovery changes in the near future.