Morphological observation of trypan blue rupturing dead or dying cells leading to over-estimation of viability

Background & Aim Determining cell viability is an essential component in immuno-oncology and cellular therapy. The traditional method for cell viability measurement is the trypan blue exclusion assay, which utilizes a hemacytometer to manual enumerate trypan blue stained cells. However, trypan blue exclusion assays can be time-consuming, tedious, and have user-dependent variation. We have previously showed that trypan blue can alter the morphology of dead immune cells to a diffuse shape, which can lead to over-estimation of viability. Here, we investigate the origin of dim and diffuse objects after trypan blue staining. Methods, Results & Conclusion Utilizing image and video acquisition, we show real-time transformation of cells into diffuse objects when stained with trypan blue. The formation of these diffuse objects is monitored and counted over time as cells die outside of the incubator. Results showed that as cells begin to die in a cell culture, dying cells displayed large blue haze when stained with trypan blue that may be missed during manual cell count. The same phenomenon was not observed when staining cells with propidium iodide. We also demonstrated co-localization of trypan blue and propidium iodide, confirming these diffuse objects as cells that contain nuclei. We hypothesize and demonstrate that rapid water influx may have caused the cells to rupture and disappear. In conclusion, we were able to observe morphological changes in immune cells using image-based cytometry. Since some dead cells disappear after trypan blue staining, the total can be under-counted, leading to over-estimation of cell viability. This inaccuracy could affect outcomes of cellular therapies, which require accurate measurement of immune cells that will be infused back into patients.