Within the pharmaceutical industry, business firms and organizations need to test the toxicity of the drugs they create. The common method used is in vitro testing employing Primary Human Hepatocytes (PHHs). According to Andria, Bracco, Cirino, and Chamuleau (2010), there are 5 different types of two-dimensional culture configurations and 3 different types of three-dimensional cell culture configurations, and there is a big difference between each approach.

Many studies have been conducted about the effectiveness and differences between 2D and 3D cultures. The comparative study by Bell et al (2018) and the review by Godoy et al (2013) assessed the effectiveness of 2D and 3D cultures in detecting toxicity. According to Bell et al (2018) PHHs serve different functions, however, when cultured as 2D monolayers, PHHs lose crucial hepatic functions within hours and leads them to be inaccurate measures of in vivo toxicity. This dedifferentiation can be ameliorated when PHHs are cultured in sandwich configuration (2Dsw), where cultures are regularly re-overlaid with extracellular matrix, or as 3D spheroids. The first difference recognized is that 2D cultures are less complex and this makes the cells more susceptible to losing many of their functions, whereas 3D culture models reproduce intrinsic properties of the cells' in vivo environment and promote cellular interaction, essential to perform their functions.

The first difference recognized is that 2D cultures are less complex and this makes the cells more susceptible to losing many of their functions, whereas 3D culture models reproduce intrinsic properties of the cells' in vivo environment and promote cellular interaction, essential to perform their functions.

This is because liver cells contain many receptors and proteins that are important in maintaining proper function. The levels of proteins in the cells contribute to the function control drug absorption, distribution, metabolism, and excretion, as well as catalytic activities of 5 different CYPs (CYP1A2, CYP3A4, CYP2D6, CYP2C8, and CYP2C9) (Godoy et al., 2013). These CYP enzymes are important for the metabolism and clearance of many compounds from the liver, and can be downregulated consistently in 2D monolayer cultures and upregulated in 3D spheroid cultures (Vorrink et al., 2017). Overall, the Vorrink et al (2017) study showed that in 2D monolayer culture various metabolites were affected and key hepatic functions were lost (urea cycle and bile acid biosynthesis) compared to 3D culture where these functions and metabolites were stable up to 3 weeks in culture.

Analyzing in depth the differences between culturing hepatic cells in 2D sandwich (2Dsw) vs 3D spheroid cultures, the results of the Bell et al (2018) study showed that PHHs in 3D spheroid cultures were overall more sensitive and detected toxicity of all the hepatotoxins used at clinically relevant concentrations after repeated exposures. In contrast, cryopreserved PHH in 2Dsw configuration exhibited substantially lower sensitivities to most of them after long-term exposure when using the chosen treatment regimen. Furthermore, the ratio between concentrations at which toxicity of some of the hepatotoxins were observed is significantly higher in 3D culture compared with 2Dsw, and indicative of elevated specificity of PHH in spheroid configuration.

Analyzing in depth the differences between culturing hepatic cells in 2D sandwich (2Dsw) vs 3D spheroid cultures, the results of the Bell et al (2018) study showed that PHHs in 3D spheroid cultures were overall more sensitive and detected toxicity of all the hepatotoxins used at clinically relevant concentrations after repeated exposures. In contrast, cryopreserved PHH in 2Dsw configuration exhibited substantially lower sensitivities to most of them after long-term exposure when using the chosen treatment regimen. Furthermore, the ratio between concentrations at which toxicity of some of the hepatotoxins were observed is significantly higher in 3D culture compared with 2Dsw, and indicative of elevated specificity of PHH in spheroid configuration.

Hepatotoxicity is one of the most crucial steps in drug development, and it’s one of the most common reasons for drug failures at their late stages. During hepatotoxicity analysis, in vitro animal testing is also done, yet sometimes human cell testing contradicts the results done on animals due to the different structure and interactions in the human body in regard of the drug absorption, distribution, metabolism, and excretion (ADME). This makes in vitro testing essential for showing the safety and efficiency in cells. Which makes the proper culturing of hepatocytes necessary to show safety and efficacy in humans.

To conclude, several studies have proved that 3D cultures provide a more accurate representation of the human liver, therefore this method could be used for further testing processes to ensure more accurate hepatotoxicological responses. However, 2D cultures still remain great tools for the advancement of hepatotoxicology.

References used:

Andria, B., Bracco, A., Cirino, G., & Chamuleau, R. A. F. M. (2010). Liver Cell Culture Devices. Cell Medicine, 1(1), 55–70. https://doi.org/10.3727/215517910x519274

Bell, C. C., Dankers, A. C. A., Lauschke, V. M., Sison-Young, R., Jenkins, R., Rowe, C., Goldring, C. E., Park, K., Regan, S. L., Walker, T., Schofield, C., Baze, A., Foster, A. J., Williams, D. P., van de Ven, A. W. M., Jacobs, F., van Houdt, J., Lähteenmäki, T., Snoeys, J., … Ingelman-Sundberg, M. (2018). Comparison of hepatic 2D sandwich cultures and 3d spheroids for long-term toxicity applications: A multicenter study. Toxicological Sciences, 162(2), 655–666. https://doi.org/10.1093/toxsci/kfx289 Godoy, P., Hewitt, N. J., Albrecht, U., Andersen, M. E., Ansari, N., Bhattacharya, S., Bode, J. G., Bolleyn, J., Borner, C., Böttger, J., Braeuning, A., Budinsky, R. A., Burkhardt, B., Cameron, N. R., Camussi, G., Cho, C.-S., Choi, Y.-J., Craig Rowlands, J., Dahmen, U., … Hengstler, J. G. (2013). Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Archives of Toxicology, 87(8), 1315–1530. https://doi.org/10.1007/s00204-013-1078-5

Vorrink, S. U., Ullah, S., Schmidt, S., Nandania, J., Velagapudi, V., Beck, O., Ingelman-Sundberg, M., & Lauschke, V. M. (2017). Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics. FASEB Journal, 31(6), 2696–2708. https://doi.org/10.1096/fj.201601375R

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