Cardiotoxicity testing using pluripotent stem cell-derived human cardiomyocytes and state-of-the-art bioanalytics: a review.

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state-of-the-art bioanalytical methods and sensors is reviewed. The focus is on hESC-derived cells and their refinement into competent testing cells, but the access and utility of other relevant cell types are also discussed. Recent developments in sensor techniques and bioanalytical approaches for measuring critical cardiotoxicity parameters are highlighted, together with aspects of data evaluation and validation. Finally, recommendations for further research are given.

Multiplex bacterial growth monitoring in 24-well microplates using a dual optical sensor for dissolved oxygen and pH.

Non-invasive, simultaneous optical monitoring of oxygen and pH during bacterial cultivation in 24-well microplates is presented using an integrated dual sensor for dissolved oxygen and pH values. The dual sensor is based on oxygen-sensitive organosilica microparticles and pH-sensitive microbeads from a polymethacrylate derivative embedded into a polyurethane hydrogel. The readout is based on a phase-domain fluorescence lifetime-based method referred to as modified frequency domain dual lifetime referencing using a commercially available detector system for 24-well microplates. The sensor was used for monitoring the growth of Pseudomonas putida bacterial cultures. The method is suitable for parallelized, miniaturized bioprocessing, and cell-based high-throughput screening applications.

Toward Microbioreactor Arrays: A Slow-Responding Oxygen Sensor for Monitoring of Microbial Cultures in Standard 96-Well Plates.

In this study, a slow-responding chemo-optical sensor for dissolved oxygen (DO) integrated into a 96-well plate was developed. The slow response time ensures that the measured oxygen value does not change much during plate transport to the microplate reader. The sensor therefore permits at-line DO measurement of microbial cultures. Moreover, it eliminates the necessity of individual optical measurement systems for each culture plate, as many plates can be measured successively. Combined with the 96-well format, this increases the experimental throughput enormously. The novel sensor plate (Slow OxoPlate) consists of fluorophores suspended in a polymer matrix that were placed into u-bottom 96-well plates. Response time was measured using sodium sulfite, and a t90 value of 9.7 min was recorded. For application, DO values were then measured in Escherichia coli and Saccharomyces cerevisiae cultures grown under fed-batch-like conditions. Depending on the DO sensor's response time, different information on the oxygenation state of the culture plate was obtained: a fast sensor variant detects disturbance through sampling, whereas the slow sensor indicates oxygen limitation during incubation. A combination of the commercially available OxoPlate and the Slow OxoPlate enables operators of screening facilities to validate their cultivation procedures with regard to oxygen availability.

Gas sensing in microplates with optodes: influence of oxygen exchange between sample, air, and plate material.

Microplates with integrated optical oxygen sensors are a new tool to study metabolic rates and enzyme activities. Precise measurements are possible only if oxygen exchange between the sample and the environment is known. In this study we quantify gas exchange in plastic microplates. Dissolved oxygen was detected using either an oxygen-sensitive film fixed at the bottom of each well or a needle-type sensor. The diffusion of oxygen into wells sealed with different foils, paraffin oil, and paraffin wax, respectively, was quantified. Although foil covers showed the lowest oxygen permeability, they include an inevitable gas phase between sample and sealing and are difficult to manage. The use of oil was found to be critical due to the extensive shaking caused by movement of the plates during measurements in microplate readers. Thus, paraffin wax was the choice material because it avoids convection of the sample and is easy to handle. Furthermore, without shaking, significant gradients in pO2 levels within a single well of a polystyrene microplate covered with paraffin oil were detected with the needle-type sensor. Higher pO2 levels were obtained near the surface of the sample as well as near the wall of the well. A significant diffusion of oxygen through the plastic plate material was found using plates based on polystyrene. Thus, the location of a sensor element within the well has an effect on the measured pO2 level. Using a sensor film fixed on the bottom of a well or using a dissolved pO2-sensitive indicator results in pO2 offset and in apparently lower respiration rates or enzyme activities. Oxygen diffusion through a polystyrene microplate was simulated for measurements without convection--that is, for samples without oxygen diffusion through the cover and for unshaken measurements using permeable sealings. This mathematical model allows for calculation of the correct kinetic parameters.