Liquid-overlay culture of cellular spheroids.

The capacity of different types of human cells to form and grow as spheroids was tested by using suspension culture over a bottom surface, which prevented cell attachment (liquid-overlay culture). The best and most convenient bottom surface so far tested was the bottom of a normal culture dish covered with a thin film of agarose. The agarose film was allowed to dry prior to the addition of medium. Several different human cell lines were tested, and it was found that many types of tumor cells formed and grew as spheroids, Large variations in growth rate, cell morphology, thickness of the viable cell layer and PO2 profiles were found among the tested spheroids. This variability parallels the variability seen in solid tumors. The morphology and the growth rate of solid tumors vary a great deal, depending on the type and position of the tumors (Ackerman and Rosai 1975; Steel 1977). Some tumors have a slow growth rate, with a few proliferating cells, while others grow faster and have higher proportions of proliferative cells (Charbit et al. 1971; Malaise et al. 1973; Friedman 1974; Lightdale and Lipkin 1975; Steel 1977). Local variations in both morphology and proliferation are usually found even within single tumors (Denekamp and Kallman 1973; Ackerman and Rosai 1974). Considering the variability in morphology, growth rate, and radiosensitivity between different types of spheroids, it is not difficult to imagine that wide variations may exist in these variables between different types of tumors or even between different regions within single tumors. The use of a group of different spheroids, showing a spectrum of growth rates, radiosensitivities, etc., will, it is hoped, be helpful in elucidating factors of importance in cancer therapy.