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Three-dimensional cell culture has played a steadily growing role in drug discovery since the technology’s inception more than two decades ago. Since cells in the human body, including those in cancer tumors, live in three dimensions, 3-D cell models in the lab better mimic in vivo physiology, often providing better predictive ability in drug discovery than 2-D cell models. Predictive ability is crucial, since 90% of drugs ultimately fail to meet required efficacy or safety margins in clinical trials. More biologically relevant models can lead to higher success rates for drug-compound testing, a faster path to market, and reduced development costs.
For most of the last century, 2-D culture has been the preferred cellgrowing method mainly due to the ease with which cells can proliferate in single layers on flat and rigid glass or polystyrene surfaces. Unfortunately, these cells can become flat and distended in their physiology, and resist the formation of cell assemblies and intercellular communication seen in vivo. Thus, their response to drugs and other stimuli can differ markedly from an in vivo response.
Tools and technologies for 3-D cell culture have rapidly evolved in the past three years or so, elevating its value in drug and toxicity screening as well as stem cell culture and differentiation, cancer biology, and tissue engineering.
Advantages of 3-D models
At a fundamental level, 3-D cellular models have a minimum depth of 50 μm, and possess both stroma and structure. When these features are absent, as in 2-D culture, the result is poor recapitulation of in vivo physiology, including tissue-specific architecture, morphology, polarity, cell-to-cell communication, cell microenvironment, proliferation rates, gene and protein expression, as well as sensitivity to drug molecules and their metabolism. These shortcomings, together with the emergence of stem cells as powerful research tools, have spurred the development of 3-D culture models. Among them are 3-D cell tumor models existing as multicellular spheroids composed of tumor cells growing in a 3-D structure simulating in vivo growth and microenvironment.
Cellular spheroids provide researchers with several favorable attributes, including a defined geometry; optimal physiological cell-cell and cell-to-extracellular-matrix (ECM) interactions; and better gradients of nutrients, growth factors, and oxygen, allowing transport to occur for several hours or even days. These attributes facilitate screening assays for a wide variety of compounds, including those that can modulate tumor growth, invasion, and blood-vessel development. Cellular spheroids can be generated from many types of cells, becoming not only tumor spheroids, but embryoid bodies, mammospheres, hepatospheres, and neurospheres.
3-D culture gels, scaffolds, and bioprinting
Instead of letting cells proliferate in a single layer, 3-D culture often involves embedding cells in either ECM gels or solid scaffolds. To date, more than 100 types of matrices and scaffolds have been developed, most of which are optimized for the growth of the specific cells under investigation. Naturally derived ECMs are widely used in 3-D cell culture, providing the appropriate microenvironment needed for morphogenesis and organogenesis of cells. Immortalized cell lines and tissue fragments form structures that recapitulate key tissue features when embedded in ECM gels and exposed to appropriate growth factors. Naturally derived hydrogels for 3-D culture comprise proteins and other ECM components, including collagen, laminin, and fibrin. The Corning Matrigel matrix (Corning Life Sciences, Tewksbury, MA) has been widely used for over 40 years because it contains many of the common ECM components found in basement membranes.
Hanging-drop and low-attachment methods are commonly used approaches for spheroid development due to their compatibility with automated screening instrumentation and detection systems. Spheroid microplates with ultralow-attachment (ULA) surfaces and innovative well geometries are ideal for generating, culturing, and assaying 3-D multicellular spheroids in the same microplate. The ULA coating attached to the interior surface of the spheroid microplate well bottom enables highly reproducible growth of 3-D cell spheroid cultures. Automation-friendly 96-well and 384-well formats support high-throughput screening (HTS) platforms.
Polymeric scaffolds use synthetic hydrogels or other biocompatible polymeric materials to generate the physical supports for 3-D cell cultures. Use of synthetic materials can minimize the relatively poor reproducibility of biological ECMs between batches and the resulting inconsistency between cultures. The scaffold can also be biodegradable, which is critical to applications in which cell utilization is required, such as tissue engineering and regenerative medicine. Scaffolds can be made using a variety of techniques, such as 3-D printing, particulate leaching, and electrospinning.
Organoids
Recently, 3-D cell culture has seen the increased practice of dish-based organoid growth using technologies from stem cell research and mixed-cell culture techniques. In organogenesis, cells undergo spontaneous self-organization into properly differentiated functional cell types and progenitors. Although it has generally been difficult to foster complete organ development in 3-D cell culture microenvironments, synthetic structures or scaffolds may help address this issue.
The potential also now exists for the growth of patient-derived organoids that enable personalized approaches for addressing cancer and other diseases. Organoids representing the embryonic kidney have also been developed as potential models for evaluating the toxicity of clinical drug candidates. Similar approaches are feasible in miniature hearts, guts, and livers. Small-intestinal organoids are the first in vitro model system to enable concurrent investigations of nutrient and drug transport, sensing incretin hormone secretion as well as allowing fluorescent live cell imaging of intracellular signaling processes.
Bioprinting
Three-dimensional bioprinting is an emerging technology that can create complex 3-D tissue models for biomedical research. Bioprinting technologies employ automated systems to deposit biological materials layer by layer to fabricate organ structures that display cell/ organoid architecture, topology, and functionality highly representative of the in vivo organ. Although printing an intact organ still remains elusive, 3-D-bioprinted bladders, tracheal grafts, bone, and cartilage have proven to be functional, sometimes in humans.
One variety of bioprinted organs are miniature models of human organs on plastic chips. These microengineered organs on chips leverage novel technologies, including microfabrication, microfluidics, tissue architecture engineering, and sensors. 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. Lung, heart, brain, liver, kidney, intestine, fat, muscle, and bone marrow have all been incorporated into chip form.
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. For instance, bone marrow-on-a-chip was formed by implanting a disk-shaped bone structure under the skin on a mouse’s back.1
Human organs-on-chips may eventually replace animal models for assessing drug safety, efficacy, and pharmacokinetics, as results from animal models often fail to predict human responses.
Cell therapy and tissue engineering
Three-dimensional cell culture holds tremendous potential in cell therapy and tissue engineering, medical approaches that offer new hope for patients with grievous injuries, end-stage organ failure, and other issues. Conventional 2-D culture techniques may not be effective to expand stem cells (the source for cell therapy and regenerative medicine) for clinical applications. Spheroid cultures, however, have been reported to improve the efficacy of stem cell-based therapeutics, including enhanced anti-inflammatory, tissue regenerative and reparative effects, as well as better post-transplant survival of stem cells. Spheroid cultures have also been used to scale up stem cell products for use in clinical trials.
Research is promising. When injected into the kidney of model rats with a form of acute kidney injury, stem cell spheroids in one experiment were more effective than 2-D cultured cells for protecting the kidney against certain types of cell death, reducing tissue damage, promoting vascularization, and ameliorating renal function.
Spheroid cultures have also been used to enrich patient-specific stem cells for disease treatment in animals. For instance, researchers applied spheroid culture to enrich adult lung stem cells for use in treating idiopathic pulmonary fibrosis in mice.2 Here, in a suspension culture, the outgrowth cells from healthy lung tissue explants were self-aggregated into spheroids, which recapitulated stem cell niche and acquired mature lung epithelial phenotypes. Mice that received these spheroids showed decreases in inflammation and fibrosis.
Conclusion
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 3-D culture in both medical research and drug discovery. Technologies are rapidly advancing in this area, with many protocols being used in routine practice, as well as in the automation systems used 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. As synthetic systems increase in complexity, other attributes such as real-time responses and modulation of differentiation and growth become highly possible, and may well refocus research toward cell cultures in which organ function can be studied, as opposed to simple cellular assemblies. Future research will no doubt bring 3-D cell culture closer to its potential.
References
- Torisawa, Y.-S.; Spina, C.S. et al. Bone marrow-on-a-chip replicates hematopoietic niche physiology in vitro. Nat. Methods 2014, 11(6), 663–9.
- Henry, E.; Cores, J. et al. Adult lung spheroid cells contain progenitor cells and mediate regeneration in rodents with bleomycin-induced pulmonary fibrosis. Stem Cells Transl. Med. 2015, 4, 1265–74.
Richard M. Eglen, Ph.D., is vice president and general manager, Corning Life Sciences, 836 North St., Bldg. 300, Ste. 3401, Tewksbury, MA 01876, U.S.A.; tel.: 978-442-2200; e-mail: [email protected]; www.corning.com