Although the use of 2D cellular assays for drug discovery predominates, the adoption of 3D assays is steadily growing. Three-dimensional assays (also known as 3D microtissues) are self-generated clusters of cell colonies. Such assays provide greater physiological and morphological relevance compared to 2D assays, aiming to reduce drug candidate failures and the use of animal testing. Participants in the 3D assay market include consumables, matrix and media vendors, as well as assay providers. Technical advances, standardization and participation in the market of major vendors signal adoption of 3D assays for drug discovery applications. However, multiple scientific and market challenges remain, with the full potential of the technology only beginning to be realized.

Although 3D assays’ advantages for drug discovery are evident, especially for high-throughput screening (HTS), implementation and acceptance are an issue. “It is clear that spheroids experience gradients of oxygen, growth factors and other nutrients that more closely resemble the in vivo microenvironment,” explained Mark Rothenberg, PhD, manager, Scientific Training and Education at Corning Life Sciences. “Furthermore, the cells in spheroids engage in cell-cell and cell-ECM [extracellular matrix] interactions that are not seen in 2D adherent cultures.” However, he added, “It remains to be demonstrated that these factors will translate into more effective hits from compound screens.”

Corning Life Sciences provides a wide range of 2D and 3D cell-based assay consumables, including a variety of matrixes, media and formats. Spheroid formats can be classified as scaffold based, including natural or synthetic matrixes, and nonscaffold. Among the most popular matrix materials are hydrogels, such as Corning Life Sciences’ Matrigel. In nonscaffold systems, spheroids grow suspended in liquid. Nonscaffold formats include hanging drop plates, in which 3D assays are formed in a drop; nonadherent surfaces; and micropatterned surfaces. Providers of nonscaffold systems include Corning Life Sciences and InSphero.

Regarding the use of 3D assays in drug discovery, Randy Strube, director of Global Marketing at InSphero, told IBO, “The uptake of 3D assays is definitely still in the early stages, as users sort through different platforms for 3D cell culture out there and discover their compatibility (or often lack thereof) with existing cell-based assays.” He emphasized the advantages of 3D assays compared to 2D assays for drug discovery. “With 3D, we are talking about more complex model systems, often co-cultures, with a more dense tissue-like structure. The advantage is greater biological relevance, and there is much support in the literature as to the differences in gene and protein expression profiles of the same cells grown in 2D versus 3D.”

The use of 3D assays has progressed from later to earlier stages of drug discovery, according to InSphero CSO Jens M. Kelm, PhD. “Even though multi-cellular tumor spheroids have been used primarily in hit identification and lead optimization, current technologies and assays enable the use of spheroids also in primary screening campaigns as shown, e.g., by Bayer.” Discussing use in primary and secondary screening, Terry Riss, PhD, Global Strategic Marketing Manager at Promega, a provider of 3D assays, noted, “Most new screening use of spheroids is for primary screening. Secondary screening will likely use the same 3D culture model but change to an orthogonal assay of similar endpoint.”

The greater usage in earlier stages of drug discovery is due to improvement of 3D microplate formats, which enabled adaption to a standard high-throughput format and allowed automation of preparation and testing. “In terms of current approaches to 3D spheroid assays, one key factor has been the development of microplate-based formats that facilitate uniform cell aggregation into single spheroids per well,” explained Dr. Rothenberg. Corning Life Sciences’ spheroid microplates utilize an ultra-low attachment surface and round-well geometry so each well contains a uniform-sized, single tumor spheroid. “The most commonly employed formats are hanging drop or low-attachment methods that are compatible with automated screening instrumentation and detection systems,” he explained. “These methods have been validated using multiple cell lines derived from a wide array of tumor types, including patient-derived cells to enable personalized approaches to drug discovery.”

InSphero provides the GravityPLUS Hanging Drop System, which utilizes the GravityTRAP Ultra-Low Attachment Plates. Describing the system, Dr. Strube said, “Once the microtissues form in the hanging drop, they are transferred to a companion assay plate that is coated to prevent attachment of the spheroid and designed to simplify medium exchange, compound dosing and imaging.” Last year, PerkinElmer announced an agreement to exclusively distribute InSphero’s GravityPLUS Hanging Drop System, which can be used with PerkinElmer’s HCS platforms.

As Dr. Riss told IBO, “Probably the most widely used technologies for 3D HTS are ultra-low binding plates and the hanging drop approaches, with some adoption of the modified surfaces.” Utilization of 2D assay features have also been instrumental in increasing the use of 3D assays for drug discovery. “The ultra-low binding plates and modified surfaces that enable generating spheroids using a homogeneous (i.e., no wash or transfer) approach are contributing significantly to adoption. Another contributor to adoption of 3D is the promise to reduce or replace the use of animals.”

As with microplate formats, validated assays have also enabled greater adoption of 3D assays for drug discovery, particularly for cell viability testing. “Much of the early adoption for 3D HTS is for detecting cell viability (i.e., a standard cytotoxicity assay),” stated Dr. Riss. “The toxicology area of research is probably leading the adoption of 3D for screening and searching for methods to replace animal use.” He said Promega adapted its CellTiter-Glo Cell Viability Assay. “Promega has reformulated an ATP cell viability assay to improve the ability to lyse all cells in 3D cell culture model systems. The CellTiter-Glo 3D Cell Viability Assay is becoming widely adopted as the go-to method for measuring viability in individual spheroid cultures.” And the company continues to adapt other 2D assay protocols. “Promega is also developing modified protocol applications examples for many other assays to detect mechanisms leading to cytotoxicity such as the Caspase-Glo 3/7 Assay, RealTime-Glo MT Cell Viability Assay, RNA extraction methods, various luciferase reporter assays, etc.”

Despite progress, challenges for the greater use of 3D assays for drug discovery remain. “The somewhat higher cost of assay plates is among the barriers slowing adoption of 3D culture model systems for screening,” noted Dr. Riss. But, he added, “The labor of the homogeneous methods to create spheroids is insignificant compared to the advantage of getting more physiologically relevant data. There has not yet been (nor will there likely ever be) a single standardized method researchers will use.”

Comparison to 2D assays is also a hurdle. “The major barrier is still the status quo. There are a litany of established 2D cell lines, model systems, cell-based assays and ‘big data’ generated using them, against which 3D models need to be compared,” stated Dr. Strube. “Current barriers to a more widespread adoption of 3D screening assays include the need to optimize and standardize assay procedures, particularly enabling compound access to the cells throughout the spheroid complex. 3D cultures are more heterogeneous than 2D cultures, and therefore, interpretation of data can be more challenging,” explained Dr. Rothenberg. He said, “3D assays typically exhibit lower sensitivity to drug compounds than those grown in 2D culture, and the full physiological relevance of this observation remains to be determined. Another reason for the slow acceptance of 3D cultures in HTS screening are methods to test functionality.”

However, as Dr. Rothenberg added, ”As new and more advanced assay systems are developed, 3D HTS screens should become more prevalent.” Dr. Strube also believes that spheroid assays for drug discovery will become more complex. “The disadvantage is that existing 2D assays may require reformulation or protocol modification to work with a 3D microtissue. So 3D assays at the moment are more simple, but will certainly gain complexity with time as assay providers and imaging systems adapt.” As Dr. Kelm told IBO, “[C]lassical monolayer cultures are highly data rich, and compound classification schemes have access to data generated over decades. Standardized in vitro model–validation schemes are required to compare and assess strength and weakness of the individual model systems for efficacy and safety testing.”

Progress in 3D assay technologies include new types of assays and new formats. “Assuming that this approach becomes more widely adopted in the next few years, the next advance may be the development of microplate-based organoid assays that more fully recapitulate the complex, multicellular tissue structure of organs,” said Dr. Rothenberg. “Development of these assays will be facilitated by advances in other technologies, such as 3D bioprinting to create complex 3D tissue models and microfluidics to provide organoid vascularization.”

Although Dr. Riss believes that formats will evolve, he thinks these new formats may not be suitable for HTS. “The HTS market will continue its rapid pace of adopting the use of the simplified methods to generate 3D spheroids. While the more complex model systems (e.g., microfluidics, organs on a chip, 3D printing of ‘organs’) can provide advantages in some cases, and may be useful for secondary screening of small numbers of compounds, the throughput of systems are prohibitive for HTS.”

For InSphero, whose products also include preplated 3D assays, formats continue to evolve. “Naturally, the next phase is interconnecting tissues via microfluidics, such as body-on-a-chip and organ-on-a-chip systems,” noted Dr. Strube. “To date, these mostly have consisted of 2D cultures interconnected by microfluidics to create flow and mimic more complex multi-organ physiological systems.” Describing InSphero’s development of new systems, he said, “[we] have shown proof of concept with bioactivation of an anti-cancer drug by liver microtissues, which resulted in killing of tumor microtissues cultured in the same microfluidic device.” He added, “Such devices can help predict the effect of metabolites on other organs to better predict toxicity and efficacy.”