Fluorescein diacetate for determination of cell viability in 3D fibroblast-collagen-GAG constructs.

Quantification of cell viability and distribution within engineered tissues currently relies on representative histology, phenotypic assays, and destructive assays of viability. To evaluate uniformity of cell density throughout 3D collagen scaffolds prior to in vivo use, a nondestructive, field assessment of cell viability is advantageous. Here, we describe a field measure of cell viability in lyophilized collagen-glycosaminoglycan (C-GAG) scaffolds in vitro using fluorescein diacetate (FdA). Fibroblast-C-GAG constructs are stained 1 day after cellular inoculation using 0.04 mg/ml FdA followed by exposure to 366 nm UV light. Construct fluorescence quantified using Metamorph image analysis is correlated with inoculation density, MTT values, and histology of corresponding biopsies. Construct fluorescence correlates significantly with inoculation density (p  <  0.001) and MTT values (p  <  0.001) of biopsies collected immediately after FdA staining. No toxicity is detected in the constructs, as measured by MTT assay before and after the FdA assay at different time points; normal in vitro histology is demonstrated for the FdA-exposed constructs. In conclusion, measurement of intracellular fluorescence with FdA allows for the early, comprehensive measurement of cellular distributions and viability in engineered tissue.