Three-Dimensional (3D) Multicellular Structures Are Thought to Be More Representative of the In Vivo Environment
Over the last century, there has been a steady progression in the diversity and complexity of tissue culture methodologies used in biomedical research, the earliest reported examples of in vitro cell culture being that conducted by Ross G. Harrison who observed neuronal sprouting from frog embryo spinal cords on a microscope slide in 1907.1 At present, cell-based research is often performed on a variety of planar surfaces that have been modified to promote the growth of two-dimensional (2D) cellular monolayers. These monolayers are utilized for majority of in vitro evaluations in research and have proven very effective. However, it is evident that while these approaches provide a convenient means of treating and analyzing cells, they do not reliably permit the formation of multicellular structures, which in turn form microenvironments similar to that found in vivo.2–8 Hence the interest in generating more biologically relevant in vitro models, such as three-dimensional (3D) culture systems.
Limitations associated with 2D models have been identified; such as the loss of tissue-specific architecture, mechanical and biochemical cues, and cell-to-cell interactions.9–12 Conversely, the microenvironment generated by 3D cell culture appears more representative of that observed in vivo, resulting in relevant cell-to-cell and cell-to-extracellular matrix (ECM) signalling.13–16 Such signalling cascades are deemed essential for a multitude of cellular processes, including differentiation and proliferation.9,17,18
In contrast to conventional 2D methods, cells cultured in a 3D format may exhibit unique biochemical and morphological features similar to their corresponding tissues in vivo19 (summarized in Table 1). It should be noted that the cell type, as well as the 3D culture method, impacts on cell organization and formation of the 3D structure. However, the concentric arrangement of heterogeneous cell populations in 3D cultures, as well as their growth pattern, mimics the initial (i) avascular stages of solid tumors in vivo, (ii) not-yet-vascularized micrometastatic foci, (iii) intercapillary tumor microregions with a high proliferative activity close to the capillaries, (iv) quiescent cells as intermediates, and (v) necrotic areas at larger distances.20,21
Two-dimensional cell-based assays are well established in the drug discovery process, particularly in cancer.22 However, their value in predicting clinical responses to new agents is limited. This unpredictability is attributable to the fact that such systems do not accurately mimic the response of cells in the 3D microenvironment present in vivo.23
Billions are spent every year on developing targets identified from in vitro systems through to Phase III trials in patients. The vast majority of these compounds fail due to either unacceptable toxicities or limited efficacy in humans.13 This in itself demonstrates that the more traditional 2D cell systems are ineffective in predicating clinical responses. Indeed, 3D models tend to have better drug predicative value compared with 2D.13,24,25 Furthermore, issues are associated with the ECM component in 2D culture, which appears to be overcome in a number of 3D systems.25–29
Incorporating 3D cell culture with in vitro screening processes such as high-throughput screening (HTS) and high-content screening (HCS) is necessary to identify clinically relevant compounds. Drug discovery is heavily reliant on HTS; the process of identifying hits by testing a large number of diverse chemical structures against disease targets and is characterized by its simplicity, efficacy, low cost per assay, and high efficiency.30 In addition, HCS-facilitated phenotypic screens yield more complex biologically relevant information and increased data generation relative to conventional in vitro assays, such as protein enzyme assays, binding assays, and endpoint assays.31,32 Highly sensitive fluorescence-based HCS assays are important for and complement HTS, therefore contributing to the industry-wide initiative to simplify, miniaturize, and speed up assays.30 Active compound hits, identified by HTS and HCS screens, act as templates for further drug development.31 The features of both HTS and HCS, and the potential impact of combining these technologies, are summarized below (Figure 1).
HTS and HCS technologies are, for the most part, conducted and optimized with cells cultured in 2D monolayers. However, creating the means to facilitate screening of 3D models using the same technologies is essential. Unfortunately, not many 3D cell culture technologies are compliant with both HTS and HCS. We believe that this is perhaps a consequence of technological development being promoted above design principles.33 Very few commercially available products are readily available technologies designed to improve the accuracy of in vitro 3D cell culture analyses in a routine and cost-effective manner.34 In this study, we discuss the merits of 3D cell culture models and the technologies that are currently attempting to mainstream their utilization in HTS. The need to identify the optimal method to facilitate their generation and use in translational research in the most effective and efficient way possible, is essential.
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* For reference pelase see the original article "Drug Discovery Approaches Utilizing Three-Dimensional Cell Culture".
ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. GEN presents here one article titled "Drug Discovery Approaches Utilizing Three-Dimensional Cell Culture". Authors are Sarah-Louise Ryan, Anne-Marie Baird, Gisela Vaz, Aaron J. Urquhart, Mathias Senge, Derek J. Richard, Kenneth J. O'Byrne, and Anthony M. Davies.