The past decades have witnessed significant efforts toward the development of three-dimensional (3D) cell cultures. Today, 3D cell cultures are emerging not only as a new tool in early drug discovery, but also as potential therapeutics to treat disease.
Cell culture is a fundamental technique in both medical research and drug discovery, and for decades, two-dimensional (2D) culture has been the preferred method, due to the ease with which cell monolayers can be induced to proliferate on planar surfaces.
However, early pioneering work in cell culture utilised tissue explants grown in three-dimensions (3D). It was evident that such 3D cultures maintained many attributes that resembled cell growth and differentiation in vivo (1). Today, the limitations of 2D culture, the development of advanced laboratory products and sophisticated detection instrumentation and analytic software, as well as the emergence of stems cells as powerful research tools, has led to the growing adoption of 3D culture models in many phases of drug discovery, including target validation, lead identification and preclinical optimisation.
In the past, 3D cell culture models were mostly developed for oncology research, given that tumours exist as 3D entities in vivo and, therefore, should be better approximations of the tumour microenvironment (2). Consequently, several 3D cell tumour models now exist, from multicellular layers on porous membranes coated with collagen, to matrix-embedded cultures, hollow fibre bioreactors and multicellular spheroids. A more recent approach, yet to have a major impact on drug discovery, has been the growing use of dish-based organogenesis, using technologies from stem cell research and mixed cell culture techniques (3). This approach has a high degree of physiological relevance and results in the development of organoids with realistic micro-anatomy.
Taken together, 3D cell culture techniques are no longer confined to the research space, but are emerging as a powerful new tool in preclinical drug discovery. Recent advances in cell biology, microfabrication techniques and tissue engineering have enabled the development of a wide range of 3D cell culture technologies. These include multicellular spheroids, organoids, scaffolds, hydrogels, organson-chips, and 3D bioprinting, each with its own advantages and disadvantages, since patient-specific cells can also be grown in 3D, the exciting possibility exists for drug discovery to be undertaken on cells with very precise pathophysiologies. Finally, 3D cell culture is growing in the area of bioproduction, notably as a means to scale-up abundant and reproducible numbers of cells as potential therapeutics.
3D cell culture in drug discovery
Cellular assays are routinely used in compound screening and optimisation, with growing evidence showing that compound library hits and optimised leads translate into better candidates for clinical evaluation (4,5). The majority of cell-based screening is performed using 2D culture technologies, due in part to the demands of the automation and detection instrumentation in use. However, cells grown in 3D better reflect drug-target interactions in vivo (6,7). Additionally, drug sensitivity in 3D culture models differs markedly from that obtained using 2D culture (Figure 1). In particular, cell-based screening technology has been pivotal in the area of imaging technologies as used in high content screening (HCS) assays. 3D culture technologies, therefore, coupled with confocal optical imaging, allow for drug screening in protocols that are unfeasible with 2D cultures. Further, many of the current technologies that enable 3D cell culture also support co-culture conditions, allowing for multiple cell types to be integrated into a 3D model that more closely mimics the in vivo microenvironment. These co-culture models are important for drug discovery, as the presence of certain cell types within tumour spheroids can greatly shift drug responsiveness.
Advances in hydrogels and scaffolds for creation of spheroids and organoids
3D culture often involves embedding cells in either extracellular matrix (ECM) gels or solid scaffolds. To date, more than 100 types of matrices and scaffolds have been developed, most of which are optimised to the growth of the specific cells under investigation.
Naturally-derived ECMs are widely used in 3D cell culture. These basement membrane hydrogels can provide the appropriate microenvironment needed for morphogenesis and organogenesis of cells possessing intrinsic developmental programmes. Immortalised cell lines and tissue fragments form structures that recapitulate key tissue features when embedded in ECM gels and exposed to appropriate growth factors. Stem cell-derived organoids have been developed from embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs) or from primary stem cells purified from organs3.
The possibility now exists for patient-derived organoids that potentially enable personalised approaches to identify the mechanisms underlying human diseases8-10, and to evaluate the efficacy and predict toxic potential of drugs prior to administration. This transformative approach may help identify the best therapies for individual cancer patients over the course of their disease.
Cellular spheroids embedded in ECMs present several features, including a defined geometry, optimal physiological cell-cell and cell-ECM interactions, and better gradients of nutrients, growth factors and oxygen, upon which transport occurs for several hours or even days (7). These attributes facilitate screening assays for compounds to modulate tumour growth, invasion and angiogenesis. Cellular spheroids can be generated from many types of cells; those formed include embryoid bodies, mammospheres, tumour spheroids, hepatospheres and neurospheres.
Naturally-derived hydrogels for 3D culture comprise proteins and other ECM components, including collagen, laminin and fibrin. A major advantage of 3D culture using a naturally-derived hydrogel is that the protocols are robust and simple. However, they have certain disadvantages due to their origin as undefined, complex material of variable compositions. Furthermore, naturally-derived hydrogels may lack the mechanical properties provided by endogenous ECMs. Finally, their nonhuman origin can preclude their use in human regenerative or transplantation therapies.
The microenvironment is clearly critical to complete organ development; but this feature has, nonetheless, been difficult to reconstitute completely in many 3D cultures. The development of synthetic structures or scaffolds using naturally-derived ECM, synthetic hydrogels or other biocompatible materials may address this issue, as they are designed to either replace or complement naturallyderived ECMs with clinical-grade materials. Tissue organoids in 3D ECMs have been developed for mammary, stomach, intestinal, liver, brain, salivary, kidney, lung and pancreatic ductal epithelium (3,15). However, the thick ECM gel can limit optical imaging and cell recovery is more complex.
To overcome some of these limitations, several lab consumables and methods have been developed to take advantage of spontaneous cell-cell interactions, which occur when cells are in an environment that promotes greater attraction towards each other than to any available surface. Of the techniques developed to take advantage of the phenomenon of cell aggregation, the hanging drop (13) and low-attachment methods (14) are widely used due to their compatibility with automated screening instrumentation and detection systems. Additionally, technologies to circumvent the issues of vascularisation have been reported with microfluidic systems and/or organ-on-chip platforms.
A novel approach to cell therapy
Cell therapy and tissue engineering not only offer new hope for patients with injuries, end-stage organ failure, or other clinical issues, but will eventually transform our lives. However, it is becoming clear that realising the full potential of cell therapy and tissue engineering requires advances in cell culture technologies to meet the demand in quantity, quality and process robustness for commercialisation and clinical trials. 3D cell cultures offer not only a solution for cell scale-up production, but also a new form of therapeutics for treating many different diseases.
Stem cells are widely used as a cell source for regenerative medicine and cell therapy applications. However, conventional 2D culture techniques, in combination with the current best practice, may be ineffective in the expansion of stem cells for clinical applications. This is reflected by the fact that 2D cultures are inadequate to reproduce the in vivo microenvironment of stem cells (16). In addition, clinical observations show that the beneficial effects of stem cell-based therapeutics seen in initial small-scale clinical studies are often not validated by large, randomised clinical trials (17,18). In fact, mesenchymal stem cells (MSCs) often decrease their replicative ability, colony forming efficiency and differentiation capabilities over time when culturing and passaging in 2D adherent monolayer (19,20).
In contrast, MSCs cultured in spheroids display a morphology that is significantly different from 2D culture (21). Furthermore, compared with 2D culture, MSCs cultured in spheroids have different gene expression patterns, with up-regulation of many genes that are associated with hypoxia, angiogenesis, inflammation, stress response and redox signalling (22).
Spheroid cultures have been reported to improve the efficacy of MSC-based therapeutics. Compared with 2D cultures, MSC spheroid cultures were also found to have additional benefits, such as enhanced anti-inflammatory and tissue regenerative and reparative effects, as well as better post-transplant survival of MSCs (22). Furthermore, compared with 2D cultured cells, spheroids of human adiposederived MSCs produced higher levels of ECM proteins, exhibited stronger antiapoptotic and antioxidative capacities and increased the paracrine secretion of cytokines.
When considering transplantation, organoids could provide a source of autologous tissue, as organoid research advances rapidly. For instance, renal organoids derived from pluripotent stem cells were successfully transplanted under the renal capsules of adult mice (24). Here, the organoid reconstituted the 3D structures of the kidney in vivo, including glomeruli with podocytes and renal tubules with proximal and distal regions and clear lumina. Furthermore, the glomeruli were efficiently vascularised upon transplantation, which is a promising step toward kidney replacement strategy.
Although early in development, organoid-based replacement may find applications in other diseases, such as retinal organoids obtained from human ESCs for treating certain types of retinal degeneration and blindness, intestinal organoids for replacement of damaged colon after injury or following removal of diseased tissue and genecorrected organoids for replacement of damaged organs with gene defect(s).
In addition to the advantages of 3D culture techniques for expanding stem cells for therapeutic use and transplantation, there is also promise for stronger in vitro screening models applicable to immune cell therapy. While the use of immune cells in cancer treatment has shown great success, especially for B cell malignancies, with best case clinical outcome of complete remission, targeting solid tumours has added complications. Evaluating immune cell homing, efficacy and selectivity against tumour spheroids may be advantageous in helping to overcome these challenges, as the invasion and cytotoxic activity of immune cells against tumour cells cultured in 3D is quite different than that of a 2D adherent monoculture.
What the future may hold: bioprinting and organ-on-chip
As we move into the future of cell culture, 3D bioprinting is an emerging technology currently utilised to create complex 3D tissue models for biomedical research (27). Bioprinting technologies may enable biofabrication of organ structures accompanied by defined positioning of a range of cell types and with optimal ECM components. This may provide a more optimal cell/organoid architecture, topology and functionality that are highly representative of the in vivo organ. Furthermore, automated systems may provide layer-by-layer deposition of biological materials that can ensure good reproducibility of the bioprinted structures.
Recent examples of bioprinted tissues include muscle fibres, printed with human myoblasts and tenocytes that exhibit contractile movement in multiwell devices containing physical posts for attachment (28). These devices offer the possibility of identifying compounds that modulate fibre contractility. In a different model, human lung endothelial and epithelial cells, bioprinted in a 3D air-blood barrier on multiwell permeable support system (29), offer the potential to screen for aerosolised toxic compounds.
Organs-on-chips represent a family of miniature models of human organs on plastic chips. These micro-engineered models leverage novel technologies including microfabrication, microfluidics, tissue architecture engineering and sensors.
Microfabrication techniques (such as soft lithography, photolithography and contact printing) enable the creation of well-defined structures, patterns and scaffolds to control the position, shape, function and physical microenvironment of the cells in culture (30). Microfluidics permits the precise control of spatiotemporal gradients of chemical and physical cues to the cells (31). Tissue architecture engineering allows for designing practical and simple tissue models to recapitulate the tissue-tissue interfaces, as well as the chemical and physical microenvironments found in vivo for drug testing (32). Furthermore, sensors can also be incorporated and provide in-process controls for pH, O2, temperature, flow velocity, metabolic status and organ viability and functionality.
Currently, organs-on-chips are designed to reconstitute the structural and functional complexity of human organs and clinically-relevant disease phenotypes and pharmacological responses. The past decade has witnessed dramatic expansion of different types of organs-on-chips, including lung, heart, brain, liver, kidney, intestine, fat, muscle and bone marrow.
Human organs-on-chips hold promise in replacing animal models for assessing drug safety, efficacy and pharmacokinetics in human, as results from animal models often fail to predict human responses.
Limitations and commercial implications
There are, of course, some limitations to using 3D cell culture in drug screening. One of these lies in the technical aspects that relate to assay protocols. These include the need to optimise and standardise procedures for cell harvesting, cell lysis, production scale up, as well as control of pH and temperature to reduce well-to-well and lot-to-lot variations.
Since 3D culture can be more heterogeneous than 2D culture, interpretation of data is more challenging. In addition, the potential for compound non-specificity may be increased due to the more complex culture conditions used, as well as physicochemical issues, including compound access to the cells within the 3D complex.
Nonetheless, as occurred in the adoption of 2D cell culture procedures in HTS/HCS, defined protocols – and novel instrumentation – are now being developed for 3D culture that could circumvent these limitations.
3D cell cultures have a clear role in drug discovery and development. There is enormous potential to model development and disease, as advanced cell models under development may fully capture the in vivo functions of organs and tissues. Furthermore, the development of screening-compatible 3D cell cultures would transform the drug discovery process, as it becomes possible to obtain early the physiologically-relevant efficacy and toxicity data. In addition, the optimisation of 3D cell cultures for scaling-up cell production would improve quality, quantity and efficacy, thus making cells as therapeutics a reality.
Today, the market is rapidly adopting 3D cell culture technologies, and the commercial implications for the industry are starting to become clear. According to a recent HTStec global benchmarking survey, 70% of respondents are currently performing 3D cell cultures, and the median annual budget allocated to spheroid culture consumables is growing – from US$1,000-$2,500 per laboratory in 2015 to an estimated US$5,000-$10,000 per laboratory in 2018. In addition, 3D spheroids are expected to be the customary 3D platform for drug research within the next three to five years. HTStec has assessed the 2016 market for 3D spheroid culture/generation consumables at approximately US$33 million (39). If the predicted growth numbers are accurate, advances in 3D cell culture technology will have a significant impact on laboratory spend in the next few years.
The growing interest in using cells in an environment that authentically replicates their function in vivo, yet is amenable to manipulation and experimentation, has driven the adoption of 3D culture in both medical research and drug discovery. Technologies are rapidly advancing in the area, with many protocols being reduced to routine practice, as well as for use in the automation systems employed in compound screening and drug evaluation. The use of cultured primary human cells can help doctors choose the most appropriate medicine for specific patients, as well as provide compound screening systems to develop novel therapeutics.
Indeed, as the industry continues to adopt new 3D cell culture technologies, the future of discovery looks bright.
The authors appreciate Elizabeth Fryman’s writing and editorial support.
Hannah Gitschier is an Applications Lab Manager for Corning Life Sciences. Since joining Corning in 2011, Hannah has focused on developing protocols and new applications using lab consumables for cell culture, cell-based and biochemical assays. Hannah received her MS degree in Biochemistry and Molecular Biology from the University of New Hampshire (Durham, NH), and her undergraduate degree in Biochemistry from Colgate University (Hamilton, NY).
Dr Ye Fang is the Research Director and Research Fellow of biochemical technologies in the science and technology division of Corning Incorporated. He received his BSc in Chemistry from the Hubei University, MSc in Physical Chemistry from the Wuhan University and PhD in Physical Chemistry from the Institute of Chemistry, Chinese Academy of Sciences, China.
Dr Richard M. Eglen is Vice-President and General Manager of Corning Life Sciences. Eglen joined Corning in 2011 with more than 35 years’ experience in the Life Science industry. He has authored more than 320 publications, book chapters and patents, and serves on numerous industry, academic advisory and journal editorial boards.
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