Cells grown in monolayer fail to accurately model endogenous biochemical processes and disease phenotypes, according to Ha Nam Nguyen, a postdoctoral fellow at John Hopkins and a founder of 3Dnamics. This is important because, as he points out, “This discrepancy could cause the majority of preclinical drug candidates to fail in downstream clinical trials.”
Mark Rothenberg, manager of scientific training and education at Corning, offers a similar point of view, saying, “More biologically relevant models can lead to higher success rates for drug compound testing, a faster path to market, and reduced development costs.”
Unlike traditional cell culture, grown on plastic in two-dimensions, three-dimensional (3D) culture recapitulates conditions cells experience in vivo. Even the simple spheroid model compensates for many deficiencies seen in monolayer culture. These structures can develop gradients of oxygen, nutrients, metabolites, and soluble signals, creating a diverse cell population. They also experience physiological cell-cell and cell-extracellular matrix (ECM) interactions.
3D culture is likely to revolutionize the way scientists do research. The capability of crafting models that more closely mimic human anatomy, physiology, and disease means paradigms with less caveats attached to them. Nguyen cites the mouse, a model used near ubiquitously, to understand biology: “A human brain is so much different from a mouse brain, which has been used to understand human brain development and function. The human brain is so much bigger and evolutionarily, has gained new structures (expanded neocortex) and functions (diseases) that are not observed in mice.”
3D culture is likely to revolutionize the way scientists do research.
He says that, when done correctly, 3D cultures could be a valuable alternative to live animals and human testing. He has already seen a number of studies that use 3D mini brains to effectively study the Zika virus. “Having a new model that can recapitulate human brain structure and physiology has been a boon to the field.”
But scientists need to exercise caution: Nguyen warns that “some 3D systems are just aggregates of cells that do not have any resemblance to human tissues at all. How useful are they?”
Choosing the right model for the job
East River Bio TissueSpec Heart Matrix: Human embryonic stem cell derived cardiomyocytes differentiated in TissueSpec™ Heart Matrix Hydrogel and immunostained for cardiac troponin T (green), show differentiation and functional gene expression. Embryonic stem cells cultured in heart matrix hydrogels demonstrated significantly higher expression of markers cardiac troponin by immunostaining and PCR, which corresponded to significant increases in embryoid body size and function, specifically beating area and contraction amplitude. Image courtesy of East River Bio.
The term “3D culture” runs the gamut from mini organs (organoids) to spheroids to new organ-on-a-chip technology and growing human organs inside of pigs. Tanya Yankelevich, director of product management and business development at East River BioSolutions, says that the models can be broadly divided into scaffold-based or scaffold-free models that use hang-drop, low-adherence plates, microfluidic chambers, or bioreactors.
While most research fields stand to benefit from the advancing of 3D cell culture, Thomas Villani, CSO and co-founder of Visikol, believes that personalized medicine and drug toxicity/efficacy research have the most to gain. “The end goal for 3D cultures in personalized medicine is to be able to grow a plate of a patient’s tumors, based on their own cells, screen an array of drugs in vitro, and then make determinations about what therapeutic strategy [should be used] informed by responses of tumor microtissues.”
According to Villani, a lab undertaking 3D culture for the first time could be daunted by the sheer availability of different methods and products on the market. On the bright side, he says that lots of reagents have now already been validated by companies.“ I think we just crossed a major inflection point in the usage of 3D culture models in the past few years and major strides have been made in the commercial availability of kits, plates, scaffolds, etc. that are consistent and high quality.”
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For example, many physiologically relevant ECM substrates are now available commercially. Yankelevich says that ECMs are moving away from a one-size-fits-all approach. “The extracellular matrix varies drastically throughout the body and appears to be highly specific to each organ—particularly with respect to the structure and biochemical composition of the extracellular environment.” With this in mind, the company currently offers TissueSpec™ ECM products from 12 organs as a hydrogel for 3D culture (scaffold and sponge ECM constructs can also be custom ordered).
She says that TissueSpec is different from what is found in commonly used products, which contain many tumor-associated growth factors, or from collagen, which only contains one ECM component and no tissue-specific factors. “Our products contain the suite of natural adhesion and signaling factors present in the native tissue matrix.”
As systems grow in complexity, so too, do obstacles
“Several challenges remain for the widespread adoption of 3D cell culture technologies, especially in the drug discovery process,” says Rothenberg.
He says that these include figuring out how to best assess complex morphology and function seen in 3D cultures. “Assays using 3D cell models are far less developed with respect to imaging, analysis, quantification, and automation, compared to established 2D models.” What’s more, Rothenberg says that use of 3D culture for drug efficacy and toxicity studies needs further validation. “Only a small set of these data confirmed that the efficacy and toxicity of drugs in 3D models are close to the clinical data.”
But while any new system must be put through its paces and validated, Villani says that 3D technology is rapidly evolving. “The space is changing so frequently that by next year, the answers to all of these questions will be different. The field is growing super quickly and I’m sure there will be even more exciting developments to come.”
One of the major advantages of 3d cell culture is that the majority of assays can be converted to fluorescent endpoints for use in high content image screening. Depicted here are a few common examples of endpoints of use to drug screening. Image courtesy of Visikol.
And as the technique becomes more mainstream, so too do solutions for the most common problems that plague researchers. One major issue, with respect to analysis, is visualization of cells within a 3D structure. “Until recently, the only way to observe cells in the interior of the 3D culture was to section and stain it, because they are too thick for direct imaging,” explains Villani. Viskol is currently collaborating with InSphero and Corning to develop ways to use the company’s HISTO tissue clearing agent in confocal imaging of 3D cultures. “Applying a clearing agent allows for imaging of the entire microtissue with high content imaging.”
Yankelevich concurs that imaging remains an issue for most scientists, but says that there are innovations in the field. “[There’s an] emergence of companies that are developing new technologies to improve the imaging of the thick 3D cell constructs to improve the number of cells detected in organoids.”
The size of structures in 3D culture can also become an impediment to the experiment. “As they grow bigger, the cells on the inside will starve of oxygen and nutrients and will die,” says Nguyen. He cautions that consistency issues can also arise since not all tissues will have the same size and structure.
As such, Villani says that the most critical question a scientists can ask is how the data will be extracted. “It is essential to consider the various parameters and options and how they may affect the data you are trying to acquire, as there are benefits and drawbacks to each technique.”
Bénédicte Gagny, product manager for 3D cell culture technologies at Millipore Sigma/Merck, advises that before doing anything, “You first need to ask yourself why you are considering 3D studies. The answer should not be ‘because others are doing it in my area of interest,’ but for a very clear purpose.”
Image: Corning’s Transwell® permeable supports are used by Organovo to print a full plate of tissue, such as these human liver tissues. Image courtesy of Organovo.