3D Cell Imaging | Best Practices in 3D Cell Culture Imaging | Corning

3D cell culture, including the increased use of spheroids and organoids, presents unique cell imaging problems, especially when creating a digital model is necessary for experimental analysis. When thinking in terms of three dimensions rather than traditional cell culture monolayers, researchers need to consider the steps required to maximize the benefits of 3D. According to Ann Rossi Bilodeau, a senior bioprocess applications scientist with Corning, preparation and optimization before imaging is key.

"Start simple," she advises. "Do a lot of optimization and aim for uniform cultures, especially where high throughput is the goal."

3D Cell Culture in Clinical Research

Going beyond the cell monolayer is driving deeper and faster research into personalized medicine and rapid drug screening. Spheroids and organoids are replacing standard cell culture in many traditional cell-based assays. For example, cytotoxicity studies are faster and provide more comprehensive data with higher throughput.

Furthermore, as part of the Reduce, Refine, Replace approach to in vivo research (described here in the Journal of the American Association for Laboratory Animal Science), 3D cell culture offers a highly valuable in vitro alternative. Spheroids and organoids also offer a faster way to screen drug responses and study pathology compared to equivalent studies in lab species.

Data Collection in 3D Cell Imaging

Advances in cell culture consumables mean that spheroid and organoid culture is now highly amenable to high content screening. For example, U-bottom spheroid culture plates not only optimize growth conditions but also create uniform cell growth that sits centrally in each well.

However, to maximize the cost savings and time efficiency, scientists must be aware of the factors that influence whether or not cell imaging returns high quality data, for both multiwell plate assays and for creating a digital model from z-stack layers. As noted by In Vitro Cellular & Developmental Biology — Animal, you may not want to rely on assays developed for 2D when moving into 3D data gathering. You can't just move from the monolayer to the spheroid without optimizing for 3D.

But how?

Considering 3D in Cell Imaging

Light penetration is the number one issue to address when imaging 3D cultures, according to Rossi Bilodeau.

"The sample thickness creates an opacity barrier," she says. "You must be able to image the whole structure. Without good quality images, sampling bias can creep in."

The risk of missing entire subsets of cells is a problem. The architecture of 3D cultures relies on the diffusion gradients creating different microenvironments inside the bundle of cells. Cells on the outside behave differently compared to cells in the center. If light cannot penetrate, interior resolution is poor and imaging will miss important details.

For this reason, in addition to thoroughly optimizing for cell growth characteristics and staining protocols, Rossi states it is important to prepare the 3D cultures before imaging.

The traditional route when imaging solid samples is to prepare tissue sections prior to histopathology. Although this is possible with 3D cultures, it's time-consuming and disruptive. Rossi Bilodeau suggests using a tissue clearing reagent, such as the Corning® 3D Clear Tissue Clearing Reagent, to replace this approach for 3D cultures. An advantage of the Corning 3D Clear Tissue Clearing Reagent is that it is completely compatible with high content processing; there's no need to transfer cultures and you can complete all steps in the microplate. Not only is the clearing process reversible, making 3D cultures available for further analysis, but the reagent does not alter cell morphology.

For spheroids, tissue clearing is the only step. Rossi Bilodeau notes that when dealing with organoids, there's an additional step to clear the culture matrix they're embedded in.

Quality Control

As Rossi Bilodeau describes, any imaging study requires optimization with a return to the basics for each cell type. She also advises adding in quality control steps that ensure intra- and interassay consistency.

Along with a well-optimized staining protocol with good stain penetration, predicting cell behavior helps pinpoint cell responses.

"Hypothesize cell location and thus activity within your spheroids to predict ahead where to focus on for results," she suggests. She also advises adding a nuclear stain; this will not only show penetration but also indicate cell viability and spheroid structure.

Finally, she says it's also a good idea to check in with your imaging experts as they may have specific advice on imaging modalities you can tap into for your study.