Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents
Introduction
EU legislation currently states that all chemicals that come into contact with man must be tested. There is also a drive to reduce the number of animal experiments that are being conducted particularly for dermatotoxicity studies. Consequently, more and more cell-based testing methods have been employed especially early in the discovery process of drug research (Stratowa et al., 1999, Taylor et al., 2001). It is believed that the use of cell-based systems that are specifically engineered to mimic in vivo behavior can reduce costs, add efficiencies and, most importantly, increase predictive accuracy of the drug discovery process (Bhadriraju and Chen, 2002).
The time-honoured conventional 2-dimensional (2D) cell culture has proven to be a valuable method to quickly identify toxic compounds. The low cost and high speed of testing have made 2D cell culture a key component of drug discovery programs (Giese et al., 2002). However, the limitations of 2D cell culture have become increasingly recognised. Due to the highly unnatural geometric and mechanical constraints imposed on cells, 2D cell cultures only approximate properties of normal tissues, and this approximation is always limited to single cell types and does not take into account the impact of the other cells and the environment. Consequently, most current drug testing methods using 2D cell culture continue to give unsatisfactorily misleading and non-predictive data for in vivo responses (Weaver et al., 1997, Bhadriraju and Chen, 2002, Birgersdotter et al., 2005).
In situ environment of a cell in a living organism is that almost all the cells are surrounded by other cells and extracellular matrix (ECM). It has been shown that 3-dimensional (3D) ECM and its receptors can promote normal epithelial polarity and differentiation (Roskelley and Bissell, 1995, Ingber et al., 1995). ECM was also reported to dictate the phenotype of mammary epithelial cells, and the tissue phenotype is dominant over the cellular genotype (Weaver et al., 1997). Several laboratories, including this one, have shown that the culture of skin cells in 3D results in a more physiologically relevant morphology of both fibroblasts and keratinocytes (including differentiation) compared with cells cultured as monolayers in 2D cultures (Ralston et al., 1997, Birgersdotter et al., 2005, Wittea and Kao, 2005). Interestingly this seems to hold true whether cells are cultured in a physiologically relevant natural dermis or in a simple 3D polystyrene matrix (Sun et al., 2005).
In our laboratory, a simple 3D cell mono-culture and/or co-culture system for skin tissue has been developed recently using a biologically inert non-hydrolysable scaffold (Azowipe®). Here, cells can undertake more complex morphologies and can also interact with different cell types in the immediate environment. Human dermal fibroblasts and keratinocytes co-cultured in Azowipe® scaffold were found to self-organise to a reasonable degree even under serum-free conditions (Sun et al., 2006). Apart from immediate clinical importance, this tissue engineered 3D skin model may also offer a physiologically relevant improvement for dermatotoxicity studies compared to conventional 2D models. To investigate this possibility, the 3D cell culture model was used to assess how the response of skin cells to xenobiotic agents compared to responses in 2D culture.
Hydrogen peroxide and silver nitrate were selected as two physiologically relevant but unrelated agents known to induce cell damage. Hydrogen peroxide is usually used to generate reactive oxygen species when investigating the influence of UV on skin cells (Lima-Bessa and Menck, 2005) and silver is a commonly used antimicrobial, which is also cytotoxic to skin cells (Hidalgo and Dominguez, 1998).
In this study, human dermal fibroblasts, keratinocytes, and small vessel endothelial cells were cultured in 3D Azowipe® scaffold. The abilities of the cells to respond to xenobiotic stress were then investigated and compared with the same cells cultured in 2D. The difference between monoculture and co-culture and the presence and absence of serum were also studied in both 2D and 3D cultures.
Section snippets
Cell culture
Keratinocytes and fibroblasts were isolated and cultured in Greens media and DMEM media (Sigma–Aldrich, Poole, Dorset, UK), respectively, as described previously (Sun et al., 2004). The HaCaT human keratinocyte cell line was kindly supplied by Professor N.E. Fusenig (Institute of Biochemistry, German Cancer Research Centre, Heildelberg, Germany). Cells were cultured in DMEM medium with 5% (v/v) foetal calf serum (FCS) (Labtech, East Sussex, UK), 2 mM l-glutamine, penicillin/streptomycin (100
Effects of differentiation on the response of keratinocytes to hydrogen peroxide in 2D cell culture
Primary keratinocytes over a range of densities (5.0 × 105, 2.0 × 105, 1.0 × 105, 5.0 × 104 cells per well of 24 well plate) were cultured initially for 3 days on standard 24 well tissue culture plates. This was followed by 24 h exposure to a range of hydrogen peroxide concentrations. The viability of cells was assessed using the MTT-ESTA assay. As shown in Fig. 1A, the effect of hydrogen peroxide on cell viability was very dependant on the initial seeding density of the cells. For example, the IC50 for
Discussion
It becomes more and more apparent that the development of a tissue can only be explained once we understand the contributions of and interactions with the microenvironment. A growing body of research indicates that the extracellular matrix (ECM) is not only a support structure that gives the tissues their mechanical properties and helps to organize communication between cells embedded in the matrix but it also contains information that helps organise cells (Mac Neil, 1994, Roskelley and
Acknowledgements
We gratefully acknowledge financial support from EPSRC and BBSRC for this research.
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