Real time monitoring of oxygen uptake of hepatocytes in a microreactor using optical microsensors

Abstract

Most in vitro test systems for the assessment of toxicity are based on endpoint measurements and cannot contribute much to the establishment of mechanistic models, which are crucially important for further progress in this field. Hence, in recent years, much effort has been put into the development of methods that generate kinetic data. Real time measurements of the metabolic activity of cells based on the use of oxygen sensitive microsensor beads have been shown to provide access to the mode of action of compounds in hepatocytes. However, for fully exploiting this approach a detailed knowledge of the microenvironment of the cells is required. In this work, we investigate the cellular behaviour of three types of hepatocytes, HepG2 cells, HepG2-3A4 cells and primary mouse hepatocytes, towards their exposure to acetaminophen when the availability of oxygen for the cell is systematically varied. We show that the relative emergence of two modes of action, one NAPQI dependent and the other one transient and NAPQI independent, scale with expression level of CYP3A4. The transient cellular response associated to mitochondrial respiration is used to characterise the influence of the initial oxygen concentration in the wells before exposure to acetaminophen on the cell behaviour. A simple model is presented to describe the behaviour of the cells in this scenario. It demonstrates the level of control over the role of oxygen supply in these experiments. This is crucial for establishing this approach into a reliable and powerful method for the assessment of toxicity. Presently, there is a great demand for in vitro test systems for the elucidation of the mode of action of drug candidates and in particular for the assessment of toxic effects of compounds 1-3. The reasoning for this demand is diverse but several points are crucial in this context: Tests based on animals do not fully reflect all the aspect of human nature, animal tests are expensive and the throughput achievable is rather small 4,5. Finally, ethical concerns have a growing impact on the overall discussion of this subject. This is widely acknowledged and as a result, enormous efforts have been made in recent years to develop methods that do not only give meaningful results but are robust in order to enable a thorough validation. The bulk of recent work has been dedicated to two key components, the cell models 6-9 and the formats of the reactors 10-12 , the cells are implemented in. The former is motivated through the necessity to use cells that mimic essential functional details of the related tissue or organs. The format of the cell reactor must enable three major functions: Firstly, it has to provide an environment that is able to maintain status and the vitality of the cells. Secondly, the fluidic system should ensure the supply of medium, the availability of important soluble molecular factors and the controlled immersion of cells with the compounds of interest. Thirdly, access of powerful characterization methods to the cells is crucial for a detailed analysi...

Three-Dimensional Liver Culture Systems to Maintain Primary Hepatic Properties for Toxicological Analysis In Vitro

“…Culture medium perfusion not only mimics blood flow, but also shear stress and a gradient of oxygen and nutrient supply as it can be observed along the lobular axis in the human liver [ 115 ]. Some of those liver-on-a-chip systems even provide the possibility of automated microscopy for live cell imaging [ 116 , 117 , 118 , 119 ]. Such technical advances pave the way for high-throughput screens with those devices, despite they are still difficult to handle and expensive.…”

“…In such devices, HepG2 cells were embedded in a collagen I matrix together with oxygen sensor beads. Cells could be cultured for up to 30 days and proved to be suitable for toxicological analysis as shown by testing hepatotoxic compounds, including acetaminophen, amiodarone and troglitazone [ 116 , 118 , 119 ].…”

Microfluidic organ-on-chip system for multi-analyte monitoring of metabolites in 3D cell cultures

“…In recent years, it has been shown, that the monitoring of metabolic parameters and culture conditions by means of embedded sensors can provide many benefits in terms of information content and quality of drug screening experiments, as well as fundamental metabolic characteristics of tissues. 16 Sensors for many different parameters such as oxygen, [17][18][19][20][21][22][23][24][25][26][27] superoxide, 28 nitric oxide (NO), 29 pH, 20,25,27,[30][31][32] transepithelial electrical resistance (TEER), 33-36 lactate, 19,23,31,37 glucose 19,31,37 and other biochemically relevant substances were developed and used for examination of static and dynamic 2D and 3D in vitro models. Optical sensors measure variations in optical properties, e.g.…”

Progress and Challenges in the Use of a Liver-on-a-Chip for Hepatotropic Infectious Diseases

“…Moreover, the phosphorescence dye loaded in polystyrene microbeads (10–50 µm in diameter) provided kinetic information on oxygen levels in 3D-cultured hepatocellular carcinoma cells and primary mouse hepatocytes upon exposure to acetaminophen. Two possible modes of action of acetaminophen were proposed: one dependent on and another independent of a toxic acetaminophen metabolite [ 99 ].…”