Development of an in vitro 3D microfluidic system for maintaining PHH over long time and its possible use for drug testing

Abstract

Traditional toxicity tests are characterized by the use of in vitro and in vivo laboratory animals with the purpose of avoiding potential damage to humans. The use of animals for drug testing raises ethical questions, and involves high costs as well as a lack of reliability. However, the use of PHH as an alternative to animal testing involves two main problems: the limited access to liver tissue and the short life span of these cells. Therefore, hepatoma cell lines and hepatocyte-like cells from extra hepatic cell sources have been investigated as an alternative in toxicity assays, but their low expression of hepatocytic markers and their low metabolization capacity limit their use in drug testing. Polarization plays a key role in drug metabolism. Therefore, the hepatotoxicity in 2D and 3D (sandwich) cultures has been investigated here by using acetaminophen as a model drug. Our data have demonstrated that cell death is higher in 3D cultures than in 2D cultures as there is a higher metabolism of acetaminophen and therefore higher concentration of toxic metabolites (NAPQI) formed after treatment with acetaminophen. During these experiments, it has been observed that the protein expression of CYP2E1 and SOD1 depends on the concentration of acetaminophen. Therefore, the increase of the concentration of acetaminophen has led to a parallel increase of the expression of CYP2E1, but to a decrease of the expression of SOD1 in 3D, but not in 2D cultures. In addition, higher import/export rates have been observed for the multidrug resistance-associated protein-1 (MRP1), which is a specific transporter protein for acetaminophen metabolite transport in 3D cultures. 3D cultures exhibited better comparability of the results to the in vivo situation. However, the limited access to liver tissue and the impossibility of using sandwich culture in small culture plates restrict the use of sandwich cultures for high-throughput screening. Therefore, PHH have been re-suspended in collagen gels. In this 3D model, cell viability, glucose production, and NH4 detoxification have been preserved at a higher level over 28 days than in 2D cultures. Similar results have been observed during the measurements of the phase I and II metabolic activity. Furthermore, PHH have exhibited higher import/export rates in two transporter proteins, MRP1 and multidrug resistance protein-1 (MDR1/P-gp), located at the basolateral and apical membrane respectively. Finally, PHH have exhibited higher toxicity in 3D than in 2D cultures after being stimulated by acetaminophen and diclofenac. The culture of PHH under a continuous flow has demonstrated an improvement of the detoxifying activity of the cells. However, microfluidic systems are expensive and sophisticated devices whose use is restricted in hepatotoxicity prediction. Therefore, a simple and cheap microfluidic system has been tested in this work. The data from the live/dead staining has proven the survival of PHH in the microfluidic system. Similar results have been found during the metabolism of acetaminophen and diclofenac which correlates with the activity of CYP2E1 and CYP2C9 respectively in static and flow cultures. However, these findings could not be corroborated by the use of fluorescence and absorbance assays measured by a plate reader. In order to improve the characterization of the PHH in the µ-slide system, new approaches have to be developed, along with alternative experiments and an improvement of methods. In this thesis, the behaviour of PHH on natural collagen gels in different culture conditions has been studied. However, the liver ECM is not only composed of collagen, but also contains proteins and proteoglycans. In addition, non-parenchymal cells are involved in the inflammatory response. Therefore, future studies should focus on testing hepatotoxicity in co-cultures of PHH and non-parenchymal cells in microfluidic systems with a matrix comprising several ECM proteins in order to achieve in vitro results which are highly comparable to the in vivo situation.