Testing in the third dimension

Experiments with a new three-dimensional model of liver tissue find that the toxic effects of nanoparticles are reduced when compared with tests that use two-dimensional models.

Molly M. Stevens

Cells reside in a three-dimensional (3D) environment in the body and they are sensitive to nanoscale topographical and chemical alterations. The varied landscape of ridges, posts and grooves in the extracellular matrix influences whether a cell will grow or die1. Synthetic nanomaterials, which also present a nanoscale landscape, hold enormous promise in biomedical applications, but assessing and predicting their potential toxicity towards cells remains a challenge.

Now in Small, Nicholas Kotov and colleagues2 of the University of Michigan and Nico Technologies report a new 3D model of liver tissue for assessing the toxicity of various nanoparticles. The new culture model aims to bridge the gap between in vitro and in vivo testing of nanoparticles, and to improve the predictive power of in vitro screening procedures.

In vitro tests of the toxicity of nanoparticles on 2D cell cultures have helped describe fundamental mechanisms of how cells interact with materials. This method involves growing the cells of interest on a flat substrate and measuring their response to the test material using various colorimetric, fluorescence, protein and gene expression assays. The problem with these cultures is that they do not reproduce many of the complex cell-cell and cell-matrix interactions found in the natural 3D environment of tissues and organs, and this limitation frequently means that the results from these studies cannot accurately predict outcomes in animal experiments.

Three-dimensional cell cultures in the form of tissue 'spheroids' are routinely used in cancer and pharmaceutical testing and are expected to be effective models for toxicity studies because they could potentially approximate the in vivo tissue structure and cell behaviour more closely than 2D cultures3. Liver tissue spheroid models are popular because the liver is the main organ where drugs are metabolized and nanoparticles accumulate. However, the development of simple and reproducible in vitro liver toxicity screening models has been hampered by the lack of control over the dimensions and organization of liver spheroids. Because the functional bioactivity of a spheroid is closely related to its diameter, reproducibility is necessary if they are to be used to screen for nanoparticle toxicity.

As a scaffold for growing the 3D liver spheroids, Kotov and co-workers prepared a cell-repulsive transparent polyacrylamide hydrogel4,5 consisting of highly organized and uniformly sized spherical pores with small openings on the top side, and sub-cell-sized porosity throughout the walls of the scaffold (Fig. 1). Liver cells are delivered through the small openings and after a few days of culture the single cells grow into balls of cells called spheroids that are eventually trapped in the pores. Obtaining quantitative data from such 3D cultures remains challenging, but the well-confined spheroids in this case means that the total number of cells is kept constant and so the effects of different nanoparticles on liver tissue could potentially be characterized. By controlling the size of the pores, it is possible to reproducibly form spheroids that are 100 |im in diameter and still maintain good transport of gas and nutrients throughout the scaffold. Hypoxic conditions, which cause cell death, are a common problem in other poorly controlled spheroid models.

Liver acinus

Septal branch Kupffer

Hepatocyte ce^

Hepatic artery branch

Liver acinus

Septal branch Kupffer

Hepatocyte ce^

Hepatic artery branch

Central vein

Bile canaliculus

Bile duct

Figure 1 | Architectures of the liver tissue in vitro and in vivo. a, Schematic of the scaffold of the 3D spheroid model. Pores (light grey circles) in the hydrogel are made using a colloidal crystal template. Liver spheroids (dark grey circles), composed of liver cells, grow to 100 ^m in diameter and are trapped in the pores. b, Confocal image of a 3D spheroid culture after 12 h of exposure to CdTe quantum dots. Live cells are green and dead cells are red. c, The liver tissue is complex with arteries (red) bringing blood in and veins (blue) taking it out, bile ducts (green) for the excretion of waste products, and various types of cells, such as Kupffer cells — specialist macrophage cells that remove foreign material. In the liver, nanoparticles are immediately taken up by Kupffer cells, so future 3D liver models should also include macrophage cells. Panels a and b reprinted with permission from ref. 2 (© 2007 Wiley ); panel c reprinted from ref. 9 (© 2006 NPG).

Central vein

Bile canaliculus

Bile duct

Figure 1 | Architectures of the liver tissue in vitro and in vivo. a, Schematic of the scaffold of the 3D spheroid model. Pores (light grey circles) in the hydrogel are made using a colloidal crystal template. Liver spheroids (dark grey circles), composed of liver cells, grow to 100 ^m in diameter and are trapped in the pores. b, Confocal image of a 3D spheroid culture after 12 h of exposure to CdTe quantum dots. Live cells are green and dead cells are red. c, The liver tissue is complex with arteries (red) bringing blood in and veins (blue) taking it out, bile ducts (green) for the excretion of waste products, and various types of cells, such as Kupffer cells — specialist macrophage cells that remove foreign material. In the liver, nanoparticles are immediately taken up by Kupffer cells, so future 3D liver models should also include macrophage cells. Panels a and b reprinted with permission from ref. 2 (© 2007 Wiley ); panel c reprinted from ref. 9 (© 2006 NPG).

At present there is no clear consensus on the toxicity profile of quantum dots and many other nanoparticles of biomedical interest because of the variability in test methods, materials and cellular systems6. The Michigan-Nico team evaluated the toxicity of cadmium telluride quantum dots and gold nanoparticles in the 3D liver spheroid and compared the results with a conventional 2D cell culture. Significantly b b

lower cell death and fewer morphological alterations in cells were seen in the 3D spheroid cultures.

The results are similar to drug screening studies that show higher drug resistance to anti-cancer drugs in 3D tumour spheroids than 2D cultures and is probably due in part to the spheroids, like natural tissues, being covered with an outer layer of extracellular matrix that can reduce the penetration of certain toxic agents into the inner layers of the cells. The cellular proliferation rate and metabolic activity is also much lower inside the spheroid.

Nonetheless, the absence of cell death does not necessarily mean that the cells are not undergoing any changes in function or signalling, so it remains necessary to determine the biochemical, genomic and proteomic output of cells within this 3D spheroid model. Although such 3D models bring us yet another step closer to recreating the environment in our organs, we are still a long way from reproducing the complex vasculature systems, immune responses and nanoparticle clearance systems in our body. Concurrent advances in tissue engineering may, however, one day allow us to introduce engineered vasculature and bile ducts into larger organ-specific constructs in vitro to help mimic in vivo processes of clearance and bioaccumulation.

Reproducible 3D tissue models provide an important extension of current testing strategies. However, for widespread use, they should be standardized, simple and adaptable to high-throughput screening. Because of the enormous diversity of nanomaterials7, such a precise and reproducible screening tool will be useful for quickly assessing structure-activity profiles of toxicity and for identifying coatings that will minimize toxicity. For example, in industries where workers are exposed to quartz, inhalation of small particles of quartz leads to progressive lung disease, yet the same particles with a thin coating of clay are less harmful8.

Similarly, in cancer therapeutics, screening tools can help identify the necessary surface modifications for enhancing the specific toxicity of nanoparticles. These developments are an essential step forward for engineering safer nanoparticles and for producing physiologically relevant toxicological information. □

Molly M. Stevens is in the Department of Materials and the Institute of Biomedical Engineering at Imperial College London, London SW7 2AZ, UK. e-mail: [email protected]

References

1. Stevens, M. M. & George, J. H. Science 310, 1135-1138 (2005).

2. Lee, J., Lilly, G. D., Doty, C., Podsiadlo, P. & Kotov, N. A. Small 5, 1213-1221 (2009).

3. Cukierman, E., Pankov, R., Stevens, D. R. & Yamada, K. M. Science 294, 1708-1712 (2001).

4. Lee, J., Shanbhag, S. & Kotov, N. A. J. Mater. Chem. 16, 3558-3564 (2006).

5. Kotov, N. A. et al. Langmuir 20, 7887-7892 (2004).

6. Hardman, R. Environ. Health Perspect. 114, 165-172 (2006).

7. Maynard, A. D. et al. Nature 444, 267-269 (2006).

8. Donaldson, K. & Borm, P. J. A. Ann. Occup. Hyg. 42, 287-294 (1998).

9. Adams, D. H. & Eksteen, B. Nat. Rev. Immunol. 6, 244-251 (2006).

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