A new quantitative test method for cell proliferation based on detection of the Ki-67 protein.

Abstract

A quantitative method to assess cell proliferation is one essential prerequisite for testing biomaterial cytocompatibility in vitro. Currently used methods, e.g. bromodeoxyuridine incorporation, show serious disadvantages concerning either sensitivity, specificity or handling. A new enzyme linked immunosorbent assay (ELISA) system for the quantification of cell proliferation based on detection of the Ki-67 protein is described. This protein has turned out to be strictly correlated with the active parts of the cell cycle but to be absent in G0. The measurement of Ki-67 expression by different human cell types, e.g. endothelial cells and HeLa cells, was evaluated in order to answer the question of whether the data obtained using the Ki-67 ELISA method correlate with the proliferation measured with flow cytometrical DNA analysis and microscopical evaluation. Methods currently used for the evaluation of cell proliferation were compared to the new Ki-67 ELISA method. In addition, the functionality of adherent endothelial cells, and the viability and morphology of the cells were investigated. Cells were treated with standard culture medium with or without the transcription inhibitor, actinomycin D, or growth factors, e.g. endothelial cell growth factor (ECGF), and were exposed to metal ion standard solutions. These solutions were in a cytotoxic-non-cytotoxic range. Ki-67 ELISA was found to be a reliable quantitative method to assess proliferation of cultured human cells in vitro. It has advantages over methods that are currently being used. It is easy to perform and corresponds to the requirements for a test to be selected for biomaterial testing according to ISO standard 10 993.