Evaluation of fluorescein diacetate for flow cytometric determination of cell viability in orthopaedic research.

Accurate estimation of cellular viability is important both in research and in aspects of orthopaedic clinical practice. We have been interested in the potential for flow cytometric application of fluorescein diacetate (FDA) in evaluating chondrocyte survival following cryopreservation of osteochondral allografts as well as in the assessment of sarcoma necrosis following preoperative chemotherapy. In order to evaluate the suitability of this method for cell viability assays, this study compared FDA with more traditional methodology (trypan blue, clonigenic assay, metabolic activity analysis, measurement of DNA synthesis, and histological assessment of necrosis). Both chondrocytes and sarcoma cells were exposed to various experimental injuries prior to viability analysis. Although it is evident from these experiments that FDA accurately reflects cell survival after physical injury, it underestimates the effect of chemotherapy on cell reproductive potential in vitro. However, FDA is highly correlated with histological assessment of tumor viability after chemotherapy in vivo. It is apparent that the methodology chosen for determination of viability should be appropriate for the type of experimental injury and should analyze the cell function (i.e., metabolic activity or reproductive capacity) that is appropriate for the experimental model.