Getting Started with 3D Cell Cultures

Our abilities to grow cells in three dimensions, or 3D cell culture, is advancing steadily, offering options to scientists with different research aims. For example, spheroids are well-suited for tumor biology studies and high-throughput drug screening assays. On the other hand, organoids offer greater cellular complexity and are more suitable for disease models and therapeutic applications. “Organoids are patient-relevant in vitro models representing the original tissue’s genetic, phenotypic, and disease characteristics,” says Sylvia Boj, CSO at HUB Organoids. “If you are trying to study a human disease or biological function, use patient-derived organoids suitable for high-throughput screens using luminescence or fluorescence readouts.” 

Choosing a 3D system

Options for 3D cell culture types mainly fall into 3 categories: spheroids, organoids, and bioprinted tissues. These have advantages and disadvantages depending upon your research goals. Spheroids are simpler, self-assembling cell clusters that are cost-effective and relatively easier to set up, but may be less standardized in size. Organoids contain more cell types and can approximate organs, while bioprinted tissues allow the precise positioning of cell types for bioengineering, such as creating layers or lines of cells.

“Organoids and bioprinted tissues can provide more standardized and reproducible experimental models since they can be expanded and propagated from a renewable source, such as pluripotent stem cells,” says Carolina Lucchesi, Principal Scientist of the Microphysiological System Program at ATCC. “[But they also] require more extensive culture protocols, specialized equipment, and higher costs.” Choosing a 3D model system necessitates considering such trade-offs.

When considering 3D model systems, Boj recommends thinking about aspects of both biology and technology. “Depending on the question we try to answer, we can choose either spheroid when we want to address a simple mechanistical question, or organoids if we’re going to address a disease or patient-relevant question,” she says. “If we need a level of complexity where different cell types need to be combined or additional physical properties are required, then microfluidics is an option.”

Consider complexity only when warranted—simpler might be better. “Sometimes very complex systems are needed to answer complex questions, but often a simpler model can be enough and can save time and money,” says Hilary Sherman, Senior Scientist at Corning Life Sciences. “Choosing the right model is always a balance between complexity, cost, throughput, and the ability to answer the desired questions.”

The type of research question often guides the choice of model system. “For example, if we are trying to screen toxic compounds, spheroids might be the simplest first pass test,” says Sherman. “If we are trying to understand how a population of individuals with pancreatic cancer might respond to a particular drug, then it might make more sense to use patient-derived organoids, since they better recapitulate patient diversity than cell lines.” Questions involving cell-cell interactions, on the other hand, might be better served with tissue bioprinting.

Choosing protocols and reagents

As in 2D cell culture, using the right reagents can make or break your culturing success. High-quality reagents are key, but the types needed will depend on your experiment—you may require animal-free extracellular matrix, or particular media for expanding your cultures. Check out current published protocols for working with 3D cultures before trying your own. “If someone has already proved that something works, why reinvent the wheel? Use it!” says Boj. “If you are developing something new, test different extracellular matrix and media components to identify the ones that better represent the in vivo tissue or model you aim to reproduce in vitro.”

Scaffolding or extracellular matrix (ECM) provides structure for cell growth and tissue engineering. “Most labs use an ECM derived from Engelbreth-Holm-Swarm (EHS) murine sarcoma because it has been rigorously validated,” says Lucchesi, but ultimately the choice of ECM should be guided by what’s appropriate for your cells and experimental aims. The same is true in choosing media, when considerations should include cell type, culture system, the desired cellular behaviors, nutrient requirements, the presence or absence of serum and other factors, and regulatory or clinical implications.

Choose scaffolding that best mimics the in vivo environment of your cells or tissue. The choice between animal-derived or xeno-free ECM materials depends upon the nature of your research and future directions. “Using xeno-free ECM formulations may be the preferred option if your research involves clinical translation or therapeutics,” says Lucchesi. “However, if you are working on basic research or evaluating cell behavior, animal-derived ECMs could still be a viable choice.”

Measuring organoids

Various methods are available to measure the size and morphology of organoids over time, including microscopy, image analysis software, flow cytometry, and biochemistry. Measuring organoids is important because their size is related to their structure, function, and useability. “If the size of organoids varies from batch to batch, it can lead to inconsistencies in experimental results, thus affecting reproducibility,” says Lucchesi.

“Optimizing the organoid size can help maintain cell viability, and ensure the accuracy and reliability of results.” Organoid size also affects timelines for passaging. “When routinely culturing organoids, the size when passaged can be important since some organoids do not recover well if passaged when they are too small or immature,” says Sherman. “Some cell counters can assess organoid size, but most analysis is currently done with high-content imagers or using imaging analysis software applied to photomicrographs.”

Handle with care

Monitor the growth and conditions of 3D cultures carefully, as they can be more fragile than their 2D counterparts. Sherman finds the biggest challenge in working with 3D cultures is the need to handle them differently compared to 2D cultures. “Since 2D cultures are attached to the growth surface, media exchanges can be performed very easily,” she notes. “With 3D culture, cells could be accidentally disturbed during media exchanges if not done carefully.” Assays for 3D cultures may need further optimization, because reagents don’t penetrate 3D tissue structures as readily as more accessible 2D cell cultures.

With these guidelines as a jumping off point, remember to handle your cultured cells with kid gloves by learning as much as possible before diving in. “3D cell cultures, especially organoids derived from patients, are closer to primary cell cultures than conventional 2D cell lines, and sensitive to wrong handling or reagents,” says Boj. “Therefore, pay attention to all the details available to take the best care of them.”