Monitoring local cell viability in engineered tissues: a fast, quantitative, and nondestructive approach.

Assessment of cell viability is a key issue in monitoring in vitro engineered tissue constructs. In this study we describe a fully automated, quantitative, and nondestructive approach, which is particularly suitable for tissue engineering. The approach offers several advantages above existing methods. Living and dead cell numbers can be separately determined for both isolated cells and cells that form networks during tissue formation. Moreover, viability can be locally monitored in time throughout the three-dimensional tissue. The viability assay is based on a dual fluorescent staining technique using CellTracker Green (CTG) for detection of living cells and propidium iodide (PI) for dead cells. CTG and PI images are created with a confocal laser scanning microscope. To determine the number of living cells, CTG fluorescence intensity is determined from the CTG image. Thereby, novel image-processing techniques have been developed, normalizing for various undesired influences that alter measurements of absolute CTG fluorescence intensities. Dead cell numbers are determined from the PI image, using an improved computerized counting method. The approach was first evaluated on C2C12 monolayers, of which images were taken directly after probe addition and 24 h later. Results show that at both times, computed living and dead cell numbers highly correlate with manually counted cell numbers (r > 0.996). Next, the approach was applied for monitoring viability in three-dimensional engineered skeletal muscle tissue constructs, which were subjected to unfavorable environmental conditions. This example illustrated that local viability can be quantitatively, nondestructively, and locally monitored in three-dimensional tissue constructs, making it a promising tool in the field of tissue engineering.