Proliferation of human ocular fibroblasts. An assessment of in vitro colorimetric assays.

Growing interest surrounds the pharmacologic modulation of ocular wound healing and fibroblastic proliferation. Therefore many investigators have developed assays to screen potential antiproliferative drugs on cultured fibroblasts. Such assays are extremely labor intensive, especially when many compounds are tested. Using proliferating human ocular fibroblasts, the authors explored the feasibility of three in vitro colorimetric assays as rapid, simple alternatives to more tedious proliferation assays, such as Coulter counting. These were: (1) the mitochondrial metabolism of a tetrazolium dye, (2) the cytoplasmic activity of hexosaminidase, and (3) the lysosomal uptake of neutral red, a weak basic dye. Serial dilutions of fibroblasts were assayed with each colorimetric technique; Coulter counting was used as a standard. All three colorimetric methods showed strong linear relationships between optical density and Coulter-cell counts. The MTT and neutral-red techniques were relatively insensitive, unable to quantify reliably fewer than 20,000 or 50,000 cells, respectively. On the other hand, the hexosaminidase assay was far more sensitive, reliably detecting a few hundred cells. Despite their differences and limitations, these three colorimetric techniques are useful as inexpensive screens whenever multiple drugs are tested for antifibroblastic effects.