Mitotic Activity in Human Lymphocyte Cultures as a Measure of Cytotoxicity

Abstract

We have examined the feasibility of using the mitotic activity of human lymphocytes in vitro as an index for determining cytotoxicity. Untreated replicate cultures agreed closely in mitotic index if the initial cell inoculum was in the range of 0.6 to 1.0×10⁶ cells/ml of culture medium. Smaller size inocula led to erratic results in mitotic index and low mitotic indices. Microcultures, made with two drops of whole blood, yielded mitotic indices lower than was convenient to use in testing for cytotoxicity, although replicate cultures showed good agreement in mitotic activity. Mitotic indices varied between donors but also varied in the same donor when blood was drawn on different days. Several chemical agents were used to test the sensitivity of the system to inhibition over a 24 hour period. Mitomycin C completely inhibited mitosis in the concentrations used (1.0 and 0.1μgm/ml), bacitracin did the same in high concentration (500 units/ml) but lower concentrations were less inhibitory. Fluorouracil and fluorodeoxyuridine inhibited growth 60 or 70% during 24 or 6 hour periods of treatment but did not inhibit at all during the last three hours of growth. The late-S or G₂ phase of the lymphocyte cell cycle are insensitive to mitotic inhibition with either FU or FUdR. The mitotic activity of lymphocyte cultures has been found to be a suitable criterion, if judiciously employed, to study cytotoxicity and to measure events in the cell cycle that serve as prerequisite for mitosis.