3D cell culture
A 3D cell culture is an artificially-created environment in which biological cells are permitted to grow or interact with its surroundings in all three dimensions. This is an improvement over the previous method of growing cells in 2D (on a petri dish) because the 3D model more accurately models the in vivo cells. These three-dimensional cultures are usually grown in bioreactors, small capsules in which the cells can grow into spheroids, or 3D cell colonies. Approximately 300 spheroids are usually cultured per bioreactor.
Early studies in the 80’s, led by Mina Bissell from the Lawrence Berkeley National Laboratory, highlighted the importance of 3D techniques for creating accurate in vitro culturing models. This work focused on the importance of the extracellular matrix and the ability of cultures in artificial 3D matrices to produce physiologically relevant multicellular structures, such as acinar structures in healthy and cancerous breast tissue models. These techniques have proven to be very important for in vitro disease models with a wide range of applications, including evaluating cellular responses to pharmaceutical compounds in drug discovery applications.
3D cell cultures are an improvement over 2D cultures for many reasons. In living tissue cells exist in 3D microenvironments with intricate cell-cell and cell-matrix interactions and complex transport dynamics for nutrients and cells. Standard 2D, or monolayer, cell cultures are inadequate representations of this environment, which often makes them unreliable predictors of in vivo drug efficacy and toxicity. 3D spheroids more closely resemble in vivo tissue in terms of cellular communication and the development of extracellular matrices. These matrices help the cells to be able to move within their spheroid similar to the way cells would move in living tissue. The spheroids are thus improved models for cell migration, differentiation, survival, and growth. Furthermore, 3D cell cultures provide more accurate depiction of cell polarization, since in 2D, the cells can only be partially polarized. Moreover, cells grown in 3D exhibit different gene expression than those grown in 2D.
A real 3D environment is often necessary for cells in vitro to form important physiological structures and functions. The third dimension of cell growth provides more contact space for mechanical inputs and for cell adhesion, which is necessary for integrin ligation, cell contraction and even intracellular signalling. Normal solute diffusion and binding to effector proteins (like growth factors and enzymes) is also reliant on the 3D cellular matrix, so it is critical for the establishment of tissue scale solute concentration gradients 
For the purposes of drug toxicology screening, it is much more useful to test gene expression of in vitro cells grown in 3D than 2D, since the gene expression of the 3D spheroids will more closely resemble gene expression in vivo. Lastly, 3D cell cultures have greater stability and longer lifespans than cell cultures in 2D. This means that they are more suitable for long-term studies and for demonstrating long-term effects of the drug. Another reason for this is that the 3D environment allows the cells to grow undisturbed. In 2D, the cells must undergo regular trypsinization in order to provide them with sufficient nutrients for normal cell growth. 3D spheroids have been cultured in a lab setting for up to 302 days while still maintaining healthy, non-cancerous growth.
Today, there are a large number of commercially available culturing tools that claim to provide the advantages of 3D cell culture. The main categories are extracellular matrices or scaffolds, modified surfaces, rotating bioreactors, microcarriers, magnetic levitation, and hanging drop plates. The Bioreactors used for 3D cell cultures are small plastic cylindrical chambers that are specifically engineered for the purpose of growing cells in three dimensions. The bioreactor uses bioactive synthetic materials such as polyethylene terephthalate membranes to surround the spheroid cells in an environment that maintains high levels of nutrients. They are easy to open and close, so that cell spheroids can be removed for testing, yet the chamber is able to maintain 100% humidity throughout. This humidity is important to achieve maximum cell growth and function. The bioreactor chamber is part of a larger device that rotates to ensure equal cell growth in each direction across three dimensions.
MC2 Biotek has developed a bioreactor to incubate ProtoTissue that uses gas exchange to maintain high oxygen levels within the cell chamber. This is an improvement over previous bioreactors because the higher oxygen levels help the cell grow and undergo normal cell respiration.
The various cell structures in the human body must be vascularized to receive the nutrients and gas exchange help that they need to survive. Similarly, 3D cell cultures in vitro require certain levels of fluid circulation, which can be problematic for dense, 3D cultures where cells may not all have adequate exposure to nutrients. This is particularly important in hepatocyte cultures because the liver is a highly vascularized organ. One study cultured hepatocytes and vascular cells together on a collagen gel scaffold between microfluidic channels, and compared growth of cells in static and flowing environments, and showed the need for models with tissues and a microvascular network  
A primary purpose of growing 3D cell spheroids in vitro is to test pharmacokinetic and pharmacodynamic effects of drugs in preclinical trials. Toxicology studies have shown 3D cell cultures to be nearly on par with in vivo studies for the purposes of testing toxicity of drug compounds. When comparing LD50 values for 6 common drugs: acetaminophen, amiodarone, diclofenac, metformin, phenformin, and valproic acid, the 3D spheroid values correlated directly with those from in vivo studies. Although 2D cell cultures have previously been used to test for toxicity along with in vivo studies, the 3D spheroids are better at testing chronic exposure toxicity because of their longer life spans. The three-dimensional arrangement allows the cultures to provide a model that more accurately resembles human tissue in vivo without utilizing animal test subjects.
All of the different methods of 3D culture claim relative ease of use and produce 3D structures with improved in vivo similarity compared to 2D methods. However, none of the 3D methods has yet replaced 2D culturing on a large scale, including in the drug development process. However, existing 3D methods are not without limitations, including scalability, reproducibility, sensitivity, and compatibility with high-throughput screening (HTS) instruments. Cell-based HTS relies on rapid determination of cellular response to drug interaction, such as dose dependent cell viability, cell-cell/cell-matrix interaction, and/or cell migration, but the available assays are not optimized for 3D cell culturing. The next challenge faced by 3D cell culturing is the limited amount of data/publications that address mechanisms of drug interaction, cell differentiation, and cell-signalling in in vitro 3D environments and correlate results with in vivo drug response. Although the number of 3D cell culturing publications is increasing rapidly, the current limited biochemical characterization of 3D tissue diminishes the confidence necessary to drive adoption of new methods. The rate at which these challenges are met will determine the pace at which 3D cell culturing is adopted as a routine tool.
There are also problems using spheroids as a model for cancerous tissue. Although beneficial for 3D tissue culture, tumor spheroids have been criticized for being challenging or impossible to “manipulate gradients of soluble molecules in [3D spheroid] constructs, and to characterize cells in these complex gradients”, unlike the paper-supported 3D cell culture for tissue-based bioassays explored by Ratmir et al.
- Fey, Stephen; Wrzesinski, Krzysztof (2013). "5". In Boucher, Alexis. Valproic Acid. Nova Science Publishers, Inc. pp. 141–165. ISBN 978-1-62417-952-5.
- Marx, Vivien (11 April 2013). "A Better Brew". Nature. Retrieved 7/9/2013.
- Souza, Glauco (14). "Three-dimensional tissue culture based on magnetic cell levitation". Nature Nanotechnology: 291–296. doi:10.1038/nnano.2010.23. Retrieved 7/9/2013.
- Pampaloni, Francesco (October 2007). "The third dimension bridges the gap between cell culture and live tissue". Nature Reviews 8: 839–845. doi:10.1038/nrm2236. Retrieved 7/9/2013.
- Boudreau, Nancy (5). "A pericellular collagenase directs the 3-dimensional development of white adipose tissue". Cell. PMID 16678100. Retrieved 7/9/2013.
- Yamada, KM (24). Modeling tissue morphogenesis and cancer in 3D. PMID 17719539. Retrieved 7/9/2013.
- Friedrich, Seidel (12). "Spheroid-based drug screen: considerations and practical approach". Nature Protocols 4: 309–324. doi:10.1038/nprot.2008.226. Retrieved 7/9/2013.
- Prestwich, Glenn (15). Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: a pragmatic approach.. PMID 17492655. Retrieved 7/9/2013.
- Griffith, Linda; Melody A. Swartz (March 2006). "Capturing complex 3D tissue physiology in vitro". Nature Reviews 7: 211–224. doi:10.1038/nrm1858. Retrieved 7/9/2013.
- Lee, J; Cuddihy MJ, Kotov NA. (14). Three-dimensional cell culture matrices: state of the art.. doi:10.1089/teb.2007.0150. PMID 18454635.
- Haycock JW. (2011). "3D cell culture: a review of current approaches and techniques.". Methods Mol Biol. 695: 1–15. doi:10.1007/978-1-60761-984-0_1.
- Prestwich, G.D. (15). Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: a pragmatic approach.. PMID 17492655. Retrieved 7/9/2013.
- Suuronen, E. J.; Sheardown, H., Newman, K.D., McLaughlin, C.R. & Griffith, M. (2005). "Building in vitro Models of Organs". Building in vitro Models of Organs 244: 137–173.
- Louekari, K (2004). "Status and prospects of in vitro tests and risk assessment". Altern. Lab. Anim 32: 431–435.
- Knight, B.; et al. (2000). "Visualizing Muscle Cell Migration in situ". Curr. Biol. 10: 576–585.
- Roskelley, CD; Desprez PY, Bissell MJ (1994). "Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction.". Proc. Natl. Acad. Sci. USA 91: 12378–12382.
- Krzysztof Wrzesinski et al. (2013). "HepG2/C3A spheroids exhibit stable physiological functionality for at least 24 days after recovering from trypsinisation". Toxicol. Res. 2: 163–172. doi:10.1039/C3TX20086H.
- "After trypsinisation, 3D spheroids of C3A hepatocytes need 18 days to re-establish similar levels of key physiological functions to those seen in the liver".
- Du, Yanan (January 2008). "Synthetic sandwich culture of 3D hepatocyte monolayer". Biomaterials 29 (3): 290. doi:10.1016/j.biomaterials.2007.09.016. PMID 17964646.
- Derda, Ratmir; et al. (2009). "Paper-Supported 3D Cell Culture for Tissue-Based Bioassays". Proceedings of the National Academy of Sciences of the United States of America 106 (44). doi:10.1073/pnas.0910666106.
- Fey, Stephen J. "WO2012022351". European Patent Register.
- Chung, Seok; Kamm, Roger D., Sudo, Ryo, et al. (July 2009). "Transport-Mediated Angiogenesis in 3D Epithelial Coculture". The Journal of the Federation of American Societies for Experimental Biology (FASEB) 23 (7): 2155–2164.
- Fey, Stephen J. (June 2012). "Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line". Toxicological Sciences 127 (2): 403. doi:10.1093/toxsci/kfs122. PMID 22454432.
- Messner, S; Agarkova, I., Moritz, W., & Kelm, J. M. (August 2012). "Multi-cell type human liver microtissues for hepatotoxicity testing". Archives of Toxicology 87 (1): 209–213. doi:10.1007/s00204-012-0968-2.
- Ratmir, D; et al. (2009). "Paper-Supported 3D Cell Culture for Tissue-Based Bioassays". Proceedings of the National Academy of Sciences of the United States of America 106 (44). doi:10.1073/pnas.0910666106.