The capillary cloning system has been shown to have advantages over conventional cloning of human
tumor cells in Petri dishes. In the present study a further optimization towards homogeneous colony distribution and high cloning efficiency is described. For reasons of reproducibility the study focused on cell lines, i.e. three human linew (MDA-231, HT-29, L363) and one rodent line (CHO-AB). Major variables investigated were the gel length, the capillary tube diameter, the tube sealing and
buffer system, and the cell number. Criteria for optimal
tumor colony growth included homogeneous colony distribution along the gel, mean colony size and cloning efficiency. It was found that colony distribution as well as overall colony growth depended largely on the gel length, i.e. on the volume of
tumor cell containing
agar applied per capillary tube. The results showed that optimal
tumor cell colony growth was achieved in 100 ul capillary tubes of 1.2 mm internal diameter filled with 30ul, yielding a gel length of 27 mm. Colony formation did not significantly differ between sealed and unsealed tubes, provided that
HEPES buffer was added. It was concluded that, for practical reasons, sealing of tube ends and therefore utilization of
HEPES buffer is not necessary. In a head to head comparison, cloning efficiency was equal or higher in capillary tubes than in Petri dishes. The capillary cloning system is an alternative for
drug development as well as for predictive
drug testing. Its major advantage is the utilization of fewer
tumor cells.