QUANTITATION OF FIBROBLAST POPULATION-GROWTH RATE IN-SITU USING COMPUTERIZED IMAGE-ANALYSIS

The development of computer-assisted image analysis has provided the t echnology to rapidly determine the population size of cultured cell mo nolayers in situ. We have adapted this technology to determine the pop ulation growth rate of cultured fibroblasts for use in a high-replicat e format. Human lung fibroblasts were seeded into 1/2 A 96-well plates that had one-half the culture area of standard 96-well plates. The ce lls were cultured in medium supplemented with different concentrations of FBS and on days 0, 1, 2, 3, 5, and 7, and their nuclei were staine d with propidium iodide. A microscopic field representing one-quarter of a well of fluorescent nuclear images was captured onto a Macintosh computer, and the number of nuclei were counted using an image analysi s software program. There were no significant differences between the number of nuclei counted manually and the number counted using compute r-assisted software, until day 7 where the cells were multilayered (P < 0.05). This image analysis method was compared to other assays typic ally used to estimate cell proliferation or population size, namely he mocytometer counting, a rapid colorimetric staining assay using naphth ol blue-black, and [H-3]-thymidine incorporation. The growth rates der ived using image analysis were in close agreement with results derived from hemocytometer counts and [H-3]-thymidine incorporation. However, the growth rates of cells grown in high concentrations of FBS as dete rmined using naphthol blue-black were substantially lower than results from image analysis. We conclude that this adaptation of computer-ass isted image analysis provides a method to derive accurate growth curve s by directly counting the number of cells in a large number of replic ates. (C) 1995 Wiley-Liss, Inc.