Three-Dimensional Cell Culture: A Rapidly Emerging Approach to Cellular Science and Drug Discovery

Physiologically, cells grow in three dimensions (3D) to form discrete tissue and organ structures. The specific architectures of these structures have evolved to provide optimal conditions for cellular proliferation, differentiation, and function. By contrast, the majority of cell culture studies are conducted by growing cells on two-dimensional (2D) flat, glass, or plastic planar surfaces, and chemically modified to promote growth, differentiation, and adherence. Such planar surfaces, while suitable for many detection and imaging instrumentation protocols, clearly do not fully mimic the 3D cell environment in vivo. Consequently, 3D cell culture is seen as an emerging area of high interest, as it provides better predictive in vitro models to study fundamental cell biology, disease pathophysiology, and the identification of novel therapeutic agents. ¹

Historically, the limitations of 2D cell culture are well known, and several attempts have been made to work with cells in a 3D environment. In oncology research, notably, 3D culturing techniques were developed using multicellular spheroid cultures derived from tumor cells, where the spheroid was used to recapitulate the functional tumor phenotype and, consequently, responsiveness to antioncologic agents. ² Many researchers wish to work with a broader range of cells (and organoid structures with heterogeneous cell populations) grown in environments closely resembling those found in vivo. The growing adoption of 3D cell culture is reflective of this need, and the techniques now being developed allow cells to develop such that tissue-specific architectures, the morphology, the polarity, cell–cell communication, the cell microenvironment, proliferation rates, and gene and protein expression, collectively, resemble the situation in vivo more precisely than 2D cell culture. ³ Furthermore, 3D cell culture techniques have now evolved such that many cell types, beyond those involved in cancer, can be evaluated, including hepatocytes, myocytes, and neurons.

Human primary cells (including those genetically modified or isolated directly from the patient) are widely used in drug discovery and screening. As occurs in basic research, many of these are cultured and used in assays with traditional 2D culture techniques. However, for the reasons discussed above, a growing body of literature obtained in 3D cell culture shows data that are more similar to those found in vivo. Consequently, 3D cell culture methods, when used in compound screening and lead optimization studies, could allow more predictive lead identification, as well as optimization in toxicity and metabolic liability testing. ⁴ Related to this is that 3D culture technologies also provide the means by which patient-specific cells can be better studied, thereby defining the pharmacology of novel compounds at targets expressed in cells with relevant disease phenotypes. The approach also provides a simple means to create cocultures, including immune cells from the same donor, or cells from diseased donors, which are difficult to harvest, isolate, and expand. Potentially more extensive use of 3D culture-based assays may mitigate the high attrition rate of compounds entering clinical evaluation.

Many 3D culture techniques employ either spheroids or organoids. In practice, most 3D cellular models have a minimum depth of 50 µm, and possess both stroma and structure. A widely used approach in 3D cell culture is the formation of multicellular spheroids, which comprise tumor cells growing in a 3D structure closely mirroring the growth patterns and microenvironment of the tumor in vivo. ¹ More complex is an organoid, which is a 3D miniature organ bud, cultured in vitro, that displays realistic microanatomy. Organoids are classified into tissue and stem cell organoids, with the former possessing stromal cell-free (or mesenchyme-free) culture. Human stem cell organoids, alternatively, are generated from stem cells, where the 3D cellular environment can markedly influence cell differentiation and organoid formation. ⁵

Recent advances in tissue and instrumentation engineering have driven development of a wide range of 3D cell culture technologies. Numerous 3D cell tumor models are currently available, ranging from multicellular layers on porous membranes coated with collagen to matrix-embedded cultures, hydrogels, spheroid microtiter plates, and hollow fiber bioreactors. In parallel, several detection technologies have converged that are accelerating the adoption of 3D cell culture with robust experimental protocols.^6,7 For example, the use of high-content imaging, and associated improvements in analytic software, has provided for detailed analysis of cell function in 3D culture, both in end point assays ⁸ and in kinetic assays (in which cellular responsiveness is assessed over time, sometimes termed 4D cell culture). The availability of microtiter plate formats, of varying well density configurations (including 96, 384, and 1536 wells), enables the growth of abundant, reproducible spheroids in formats and amounts suitable for high-throughput screening automation and instrumentation.

Noteworthy in this field is the emergence of organ-on-a-chip technologies. In this case, microfluidic approaches provide systems that function in 3D and can thus mimic basic organ—or multiple organ—physiology. ⁹ Manufactured with high-precision microfabrication techniques, the chip comprises well-defined structures, patterns, and scaffolds, in which the cellular position, shape, function, and chemical and physical microenvironments in culture are controlled with precise spatiotemporal precision via microfluidics. Several cellular systems have been evaluated in this manner, some of which allow for “on-chip” assessment of compound toxicity and metabolic liability. A second, also rapidly emerging area is 3D bioprinting, in which a biocompatible matrix containing cells is printed into complex 3D tissue architectures, with the desired cell/organoid architecture, topology, and functionality. ¹⁰

This special issue of SLAS Discovery contains several papers that describe novel data in this rapidly advancing field. The area, in general, is extensively reviewed by Fang and Eglen, ¹¹ which covers the historical development of the area, but also emphasizes novel technologies now emerging for drug discovery, including novel surfaces, organ-on-a-chip methods. and bioprinting.

As discussed above, the early applications of 3D cell culture were applied in the areas of cancer research. This special issue has several reports in this area, including the report by Selby and coworkers ¹² using NCI160 cells for compound screening, Escobar and coworkers with WM115 cells, ¹³ and Lal-Nag and colleagues, who have developed a tumor model of ovarian cancer for high-throughput screening. ¹⁴ Multicellular spheroids are growing as important 3D cell models, as several papers are also included in this area, including the use of spheroids in screening by Thakuri and colleagues, ¹⁵ 1536-well-based screening by Madoux and colleagues, ¹⁶ high-throughput RNAi screening by Fu and colleagues, ¹⁷ and the use of real-time imaging with spheroids by Lal-Nag and colleagues. ¹⁸ The use of imaging technologies with spheroids is also covered in papers by Cribbes and colleagues ¹⁹ and by Pampaloni and coworkers using live fluorescence microscopy. ²⁰

The area of 3D culture with organoids is addressed by Ley et al., ²¹ focusing on intestinal organoids, while 3D culture and other cell phenotypes are covered by Watson and coworkers ²² with glial cells and Unterleuthner and colleagues ²³ with coculture of cells in the screening for antiangiogenic drugs. In addition, Ott and colleagues compare 2D and 3D culture when testing compounds with HepaRG liver cells. ²⁴

The growing interest in hydrogel-based surfaces is illustrated in two papers, one from Zhu and colleagues, ²⁵ assessing the impact of viral infection on fibroblasts in a hydrogel matrix, and the other from Zhang and colleagues, ²⁶ who describe the use of hydrogels in providing surface density gradients and their application to 3D cell culture. A different, microfluidic approach is described by Lee and colleagues, with the use of micropillars and microchip design in the analysis of patient-specific cells. ²⁷

In summary, cultured primary human cells, and those derived from induced pluripotent stem cells, have been used in basic research and drug discovery for many years. Yet it is only comparatively recently that such cells, when cultured in 3D, have been routinely prepared and employed in robust experimental protocols, some of which are now amenable to high-throughput automation and detection. The emerging data are providing new insights not only into cell fundamental biology, but also in terms of better screening systems for novel agents entering clinical evaluation.

Guest EditorsRichard M. Eglen, PhDCorning Life SciencesCorning Inc.Tewksbury, MA USA Jean-Louis Klein, PhDGlaxoSmithKlinePhiladelphia, PA USA