Establishing three-dimensional cell culture models to measure biotransformation and toxicity
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A great proportion of new chemical entities will be terminated from the clinical drug development pipeline as a result of deficiencies in drug absorption, distribution, biotransformation and elimination as well as potential to cause toxicity. Exposure of the liver (and other organs) to hepatotoxins, may potentially interact with cellular constituents, causing toxicity and various lesions. The pre-clinical assessment of hepatotoxic potential of new drug entities and herbal compounds are investigated on a tissue, cellular and molecular level by employing various in vitro and in vivo techniques. The in vitro models currently available mainly involve traditional two-dimensional (2D) cell culture techniques; however, these models lack various tissue specific properties found in the in vivo environment. As a result, pre-clinical assessment of drug hepatotoxicity and biotransformation still rely predominantly on in vivo animal models. To reduce the use of animal models, more reliable and readily available in vitro models are needed, capable of bridging the gap between the existing models and the in vivo situation. Three-dimensional (3D) spheroid cell cultures offer higher physiological relevance than traditional 2D cell cultures, overcoming many of the shortcomings associated with traditional 2D cell cultures. Specifically, the dynamic micro-tissue 3D spheroid cell culture system produced in micro-gravity bioreactors has attracted attention, although several other types of multi-cellular spheroid systems are also currently under investigation. This study investigated the potential of the 3D HepG2/C3A spheroid model to evaluate the acute and sub-chronic hepatotoxic potential of a crude aqueous Xysmalobium undulatum (Uzara) extract. Acute hepatotoxic effects were investigated in 2D and 3D HepG2/C3A cell cultures at concentrations of 200, 350, 500, and 750 mg/kg. Parameters evaluated included cell proliferation, glucose uptake, intracellular adenosine triphosphate (ATP) levels and adenylate kinase (AK) release. Furthermore, sub-chronic hepatotoxicity of crude Uzara aqueous extract was investigated during a sub-chronic 21-day study in the 3D HepG2/C3A spheroid model as well as in Sprague Dawley rats. The results from the in vitro study clearly indicated hepatotoxic effects and possible liver damage following treatment with valproic acid (the positive control group) as indicated by the growth inhibition observed, the loss of cell viability and the increased cytotoxicity as indicated by the reduced intracellular ATP levels and increased AK levels. The results also indicated that crude Uzara water extract had dose-dependent hepatotoxic potential, although the effects appeared to be exaggerated in the 2D cell cultures compared to the 3D spheroid cultures. The results was also supported by the increased in vivo levels of AST, ALT and LDH and the slight increase in triglycerides, following treatment of the Sprague Dawley rats with valproic acid. This is indicative of hepatic cellular damage, possibly resulting in hepatotoxicity. Similarly, following treatment with the crude Uzara aqueous extract, results obtained from the in vivo Sprague Dawley model indicated moderate hepatotoxic potential. The results confirmed the potential of the 3D HepG2/C3A spheroid model to effectively and reliably predict the long-term outcomes of possible hepatotoxicity. A novel 3D spheroid model for biotransformation applications was also developed, employing the LS180 cell line and micro-gravity bioreactors. The human colon carcinoma cell line, LS180, is often used as a biotransformation model to study inhibition and induction of CYP450 enzymes in vitro. The new three-dimensional cell culture model combined the dynamic rotating micro-gravity bioreactor technique with the micro-encapsulation technique, using sodium alginate. These encapsulated LS180 spheroids have the potential to be employed as a novel long-term culturing model for future in vitro biotransformation studies.
- Health Sciences