Increasingly complex three-dimensional cultures, which include spheroids, organoids, and organ-on-a-chip, bridge the space between traditional cellular models, like 2D cultures, and organs, according to Oksana Sirenko, Ph.D., Senior Manager, Assay Development, at Molecular Devices. These culture systems are being used for drug discovery and development, including personalized models, especially in cancer therapeutics. Further, Sreethu Sankar, Product Manager at Proteintech, adds, “As more countries adopt laws to reduce the use of animals in drug discovery and toxicity studies, the relevance of 3D models is growing rapidly.”

As this system becomes more mainstream and its capabilities expand, labs will need to decipher the best way to culture, stain, and image so as to derive the most data in an efficient way. When imaging under microscopy, challenges include adapting staining protocols to best elucidate subcellular structures and markers, especially near the core, and microscopy limitations related to the sample thickness. For example, “monitoring 3D cell cultures for longer duration is difficult due to the hypoxic core region and high autofluorescence from extracellular matrices secreted by the cells,” explains Sankar. Hilary Sherman, Senior Scientist at Corning Life Sciences, adds “Excessive light scattering results in poor image quality…additionally, more traditional widefield microscopy isn’t as effective for imaging 3D structures compared to confocal.”

Here we’ll explore common obstacles in 3D cell culture imaging, and show you how to best visualize your samples, whether fixed or alive.

3D cultures are different from 2D cultures

It may seem obvious, but 3D cultures require more planning and ingenuity when it comes to staining and subsequent microscopy. The best images begin before the cells hit the culture plates. Sherman recommends beginning with appropriate cultureware, such as Corning® Spheroid Plates and Corning Elplasia® products, fashioned to produce uniform structures in every well. “Using microplates designed for imaging that have little well-to-well variability will significantly reduce scan times, which could get long when imaging 3D structures.”

Sirenko seconds the use of U-bottom plates to center organoids and adds, “Some 3D culture formats are less suitable for accurate imaging, for example floating organoids in the flat low attachment plates. During imaging those objects can shift. Researchers may intentionally place organoids onto some sort of matrix (Matrigel for example) to enable immobilization during imaging.”

Once the plates are chosen, “the most important aspect is to start with a single cell suspension,” says Sherman. “Uniform spheroids will only form if a uniform single cell suspension is used. If cells are in non-uniform clumps or clusters, they will not settle uniformly into cavities and the result will be non-uniform spheroids.” And if you have a choice, keep spheroids on the smaller side: “Staining and imaging smaller 3D structures is easier than larger ones,” suggests Sherman.

Optimize, optimize, optimize—Reconfiguring staining protocols

Fixing, slicing, and mounting spheroids on slides is always a possibility, but it’s labor-intensive and time-consuming. To stain intact cultures, tailoring conditions may take some troubleshooting, but it will be well worth it in the long run.

“Optimizing every step of sample processing is extremely important when imaging 3D cell cultures,” emphasizes Sankar. Expect to increase fixation and permeabilization times, and concentrations of reagents, like antibodies, to compensate for the thicker structures. Sirenko reports that small molecule stains such as Hoechst, MitoTracker, and Calcein AM, are able to easily penetrate the 3D structures and matrix, but still require longer times, a two- to threefold increase in concentration (compared to traditional cultures), and if possible, should be used in the incubator.

Sankar thinks nanobodies are a great tool for organoids, as they are significantly smaller than conventional ones; “Nanobodies could penetrate deep into tightly packed 3D structures and [thereby] improve [imaging] resolution.” He is also excited about Proteintech’s FlexAble, the novel antibody labeling kit conjugates as little as 0.5 ug of antibody to a fluorochrome in under ten minutes, further cutting down on prep time required to image spheroids.

Making samples transparent

In recent years, a range of clearing agents have come to market, able to make fixed three-dimensional structures such as organoids, thick slabs of tissue, and even entire animals transparent. Biological samples are mixtures of protein, lipids, and water, which all have different refractive indices (RI), a measure of how quickly light can travel through the material. A high RI, for example, means light will travel more slowly through the substance. Clearing agents work by equalizing the RIs of a specimen through removing and changing the constituents of the tissue.

“Clearing solutions such as Corning® 3D Clear Tissue Clearing Reagent can significantly improve image capture by increasing the refractive index of cytosolic and intercellular space to match that of cell-membranes and proteins,” says Sherman. “This allows better light penetration and less light scattering.” Sirenko notes she prefers clearing protocols where the reagents are simply added to the wells, such as Visikol. She also points out that strong clearing agents require more sophisticated protocols, which can damage the plastic in cell culture plates.

Sankar admits that there are a variety of clearing methods available, but says researchers may want to consider expansion microscopy, instead. “Expanded samples exhibit high transparency facilitating detailed imaging across large volumes by minimizing light scatter and absorption.” He adds, “The hydrogel-like properties of expansion resins allow for uniform antibody binding of buried epitopes throughout the sample. This enhanced labeling and image clarity unveil intricate details within the organoid's core, previously visible only through serial sectioning.” Not all antibodies work with this method, although Proteintech antibodies perform well with this approach.

There are caveats to expansion microscopy. According to Samantha Fore, Ph.D., Product Marketing Manager at ZEISS, the reagents used in the expansion process will alter the RI, and the enlargement of the sample can dilute fluorescent signals.

Which brings us back to the first rule: optimize everything. “Spend the time optimizing your staining protocol to make sure you have as good of a signal-to-background ratio as possible. The better the staining, the easier time the imager will have separating signal from noise,” advises Sherman.

The main event: Imaging

The best place to start is with uniform 3D cultures in appropriate cell culture plates that are healthy and adequately stained with minimal background. If samples are fixed, clearing reagents or expansion microscopy have hopefully helped to achieve a clear view of stained and antibody-labeled structures down to the very core. This should make imaging samples less cumbersome, especially when combined with the right microscopy system (which doesn’t necessarily include the confocal).

“While the confocal is the go-to for imaging of thicker, denser samples, there are many other viable alternatives,” says Fore, such as a quality widefield microscope with deconvolution capabilities, which can generate images good enough for downstream quantitative analysis. ZEISS’s Axio Observer is one option, which she believes is an ideal inverted microscope for imagining 3D cultures. “The key is the ability to achieve high-quality optical sections without introducing artifacts or rendering the images non-quantitative.” The Apotome 3 add-on tool provides “beautifully optically sectioned 3D images using a version of structured illumination methods.” Another option is the ZEISS Celldiscoverer 7, which is available as widefield, confocal, and Airyscan configurations. This microscope is also ideal for live cell imaging, including high throughput, and includes full incubation control.

Image of rat cortical primary culture using 40x/0.95 autocorr objective. Antibody-staining of bIII-tubulin (Cy2) and DCX (Cy3). Nuclei Dapi-stained, mounted on standard slides with coverslip. Images show a projection (extended depth of focus) of a 4 channel z-stack. Acquired on ZEISS Cell Discoverer 7 using Widefield acquisition and subsequent GPU based deconvolution processing. Sample courtesy of H. Braun, LSM Bioanalytik GmbH, Magdeburg, Germany.

There are, of course, a variety of confocal options available, including the LSM 9 family and the recently launched Lattice SIM family. The Lattice SIM 3 and SIM 5 are “especially focused on easy live cell super resolution imaging, including 3D imaging, by leveraging the versatility and optical sectioning of structured illumination microscopy,” explains Fore.

Often photobleaching can be an obstacle to 3D imaging. When speed and gentle imaging are critical, Fore says the Lattice Lightsheet 7, which offers minimal phototoxicity alongside fast imaging speeds “making it suitable for long-term imaging of viral infection of cells, small organoids (up to 150 um), and [organisms such as] C. elegans.”

Continue to be aware of RI mismatch, as Fore cautions this is the critical factor that degrades image quality. “The one parameter that can almost always be improved upon is RI matching.” This means selecting the correct objective, and thinking beyond just the magnification. “Not all 40X objectives are alike,” she remarks—the immersion media is also key and should match the RI of the sample. As such, ZEISS’s multi-immersion objectives are available over a wide range of magnifications. “A new objective is not needed to switch from water versus silicone for your immersion media.”

Finally, if expansion microscopy is the protocol of choice, or if researchers are working with larger spheroids and model systems, Fore again proposes the ZEISS Lightsheet 7 since “its light-sheet principle and large offering of samples to accommodate large organisms” makes it a fitting choice.

Final considerations

This type of work can generate enormous quantities of data, which labs will need to figure out how to store and analyze. Fore says scientists should have the software infrastructure to accommodate it, along with AI tools “to improve repeatability in analysis.” To that end, ZEISS has options including ZEISS arivis Cloud, a web-based application that lets users train custom AI deep learning models that “can evolve with your science over time and requires no coding.” Similarly, Sankar thinks AI-based image segmentation and thresholding methods to analyze 3D are going to continue to drive the field forward. “It becomes absolutely necessary to have advanced algorithms for high-throughput analysis of vast amounts of data generated from 3D models.”

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