Cell culture in the third dimension

Sustaining life outside the body through the use of cell culture for research and discovery purposes has always been challenging. However, despite these challenges, cell culture has resulted in the discovery of most of today’s knowledge about disease processes and their cures. There has always been the desire to increase the relevance of cell culture by reproducing the 3D context of the body and thus the rate of peer reviewed publications employing 3D cell culture has increased dramatically over the past several years [1]. Furthermore, many new 3D cell culture products are emerging in the bioscience market that are focused on making the process simpler and more affordable.

The papers highlighted in this newsletter demonstrate the biologic relevance of 3D cell culture in several unique ways.   The paper by Justice et al. reviews 3D cell culture techniques that have the benefit of not only the biological relevance of cell growth in 3D, but also lend themselves to scalability and ultimately automation [2].   The future success of high-content screening, high-throughput ADME Tox, and stem cell production for therapeutics will have to adopt 3D cell formats that contain costs while yielding increasing numbers of commercial successes.   Justice et al. reviews the three principle formats for 3D cell culture, including filter supports, sponges and gels, and microcarriers. He concludes that microcarriers possess the necessary increased cost-performance ratio for commercial success with the added benefit that microcarrier culture lends itself better to automation because it maintains cells in a pipettable liquid during the entire cell culture process. A step by step guide to 3D microcarrier culture will be published in Methods in Molecular Biology [3].

Physical stresses can also be incorporated purposefully and reproducibly in 3D cell culture. In the paper by Asthana and Kisaalita they review various approaches that have been used to constrain cell growth to specific shapes, sizes, or configurations [4]. For example, the size of growth area may be constrained so that there is a reproducible deprivation of oxygen to the growing tissue. Lack of oxygen is a characteristic that stimulates cancer progression and thus useful for high-throughput screening of anticancer agents. Markers of hypoxia can be even included in the growth system so that it may be monitored directly. The incorporation of microenvironmental markers of various types is gaining in popularity because a continuous and direct measurement can be made to assure the continued relevance of the culture system during the entire growth and testing cycle.
 3D cell culture challenges current measurement and data reduction technologies because it can yield additional data in terms of cell–cell interaction. Scott and Peters in this publication describes how label-free measurements are providing better insight into the integration of individual biochemical pathways into total cell physiology or redistribution of cellular biomass [5]. Using either impedance or optical methods can measure subtle changes cell shape and adhesion (to their growth surfaces or each other) in response to pharmacologic stimuli. These integrated cell behaviors can then be correlated with the activity of a specific stimulus such as the ability of a G-protein-coupled receptor stimulation of adenylyl cyclase activity. The measurement of the redistribution of cellular biomass can be measured more sensitively than current fluorescent assays for selected biochemical changes. However, it isn’t clear yet how these sensitive methods will translate into data that will provide useful information for drug discovery. 
How sure are we that 3D cell culture actually provides data that is more biological relevant and actionable than convention flat-surface cell culture? Lai et al, in this issue, review papers that directly demonstrate improved biological relevance as evidenced by specific cell behavior or biomarker expression [6].   In particular, many cancer cells exhibit in vivo behaviors in 3D cell culture that they don’t exhibit in 2D.   Toxicology studies of new drug candidates are often performed in 2D cell culture. However, many potential drug candidates don’t demonstrate toxic effects until human phase III trials. There are a few reports of more sensitive toxic effects being demonstrated in 3D. Lai et al describe how 3D achieves its increased relevance, namely through a combination of chemical and physical cues. 2D cell culture media components try to mimic in vivo microenvironments, but 3D adds the relevance of providing chemicals that are attached to the growth surface and available through body fluids as well as temporal chemical gradients. 3D can also try and mimic the physical nature of the growth surfaces found in the body by providing biomimetic shapes and molecular components. Ultimately, measuring cellular responses to 3D will provide the data that will convince the cell culturist that they are indeed providing an in vivo-like experience for the cell. Apparently, the expression of various cytokines is yielding data that provides good correlation with the quality of the 3D environment.
In summary and conclusion, 3D cell culture will inevitably become the principal approach by which cell biologists explore and capitalize on in vitro life.  When a wide range of unique 3D cell culture products become available cell biologists will be able to customize their cell culture process to provide them the most actionable biologically relevant data.   
Robin A. Felder, Ph.D., Professor of Pathology, Associate Director of Clinical Chemistry, The University of Virginia, Charlottesville, Virginia
2.     Justice, B.A. (2009) 3D cell culture opens new dimensions in cell-based assays. Drug Discov. Today 14, 104–107
3.     Gildea JG, Van Sciver R, Felder, RA. Epithelial Cell Culture Protocols. In Methods in Molecular Biology. ISBN: 978-1-62703-124-0, Chapter -20, Isolation, Growth, and Characterization of Human Renal Epithelial Cells Using Traditional and 3D Methods, 2012.
4.     Asthana, A. and Kisaalita, W.S. (2012) Microtissue size and hypoxia in HTS with 3D cultures. Drug Discov. Today 17, 810–817
5.     Scott, C.W. and Peters, M.F. (2010) Label-free whole-cell assays: expanding the scope of GPCR screening.  Drug Discov. Today 15, 704–716
6.     Lai, Y. (2011) Biomarkers for simplifying HTS 3D cell culture platforms for drug discovery: the case for cytokines. Drug Discov. Today 16, 293–297

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