Measurement of human tumour cell growth in soft-agar cultures using computer-assisted volume analysis

Growth in soft-agar bilayer cultures of human tumour cells derived from 4 in vitro continuous cell lines, from 21 xenografts carried in athymic mice, and from 197 samples of fresh human solid tumours of various histologic types was analyzed by computer-assisted image analysis. Replicate cultures for each specimen were assessed on successive days of incubation for the number and volume of growth units within multiple size categories. Our results confirm the recent finding of others that there is an upper limit of approximately 10(9) microns 3 to the cumulative growth unit volume obtainable in a 2 ml bilayer soft agar culture system. Since this upper limit to the carrying capacity of the closed culture system exists, the extent of growth within the cultures is determined in a fundamental way by the cumulative volume of growth units initially inoculated into cultures. A growth index of greater than or equal to 16-fold was only seen when initial cumulative growth unit volume was less than 10(7) microns 3 per culture dish. Computer-assisted volume analysis (CAVA) appears to be a useful quantitative method to study the growth of human tumour cells in soft agar cultures.