In situ assessment of cell viability.

Cryobiological studies of tissues often require the simultaneous assessment of tissue structure and in situ cellular function. Localization of damage during cryopreservation occurs as a consequence of tissue structure and morphology and as a result of biophysical constraints imposed by diffusion and heat transfer. This study used five experimental model tissue systems: cells in suspension, cells attached to a substrate, a monolayer of cells attached to a substrate, porcine corneas, and intact porcine articular cartilage to examine the efficacy of assessing cell recovery using a novel fluorescent stain (SYTO-13). A graded freezing protocol was used to induce varying degrees of tissue damage. Recovery was assessed in the different tissue model systems using SYTO with ethidium bromide, fluorescein diacetate (FDA) with ethidium bromide, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In each of the tissue model systems, the SYTO/EB assessment technique was shown to be equally effective as the existing techniques for the determination of cell recovery. In addition, the properties of fluorescence intensity and rate of release for SYTO were significantly better than those obtained using FDA. Assessment of in situ cell viability was clearly demonstrated using porcine corneas and articular cartilage. The SYTO/EB assay is superior to the existing techniques used for the localization of cell damage in tissues after cryopreservation.