Search for improved culture conditions for clonogenic growth of human colorectal cancer cells in vitro

In order to analyze and define potentially better growth conditions for colonic stem cell proliferation, we chose four established human colorectal cancer cell lines that differed in biologic cell properties. We studied variables of standard cloning conditions including culture medium, serum supplement, solidifying agent, addition of specific growth factors and use of capillaries as an alternative culture vessel. While modulation of serum concentration as well as use of various standard formulations of culture base media did not result in a reproducible increase of plating efficiencies (PEs), a significant increase in colony formation (when compared to the conventional assay procedure) was achieved; 1) by use of 0.3% agarose or boiled agar as semisolid matrix and 2) by culturing of cells in enriched ‘GMF medium’. Specific growth factors, such as EGF or glucagon resulted in “occasionally better” in vitro growth. This suggests a retention of the ability of cells in culture to respond to physiologic regulators of growth. To verify and extend these initial results obtained with continuous cell lines, growth enhancing modifications of the original cloning technique were subsequently applied to in vitro growth of 15 human colorectal cancer specimens obtained directly from patients. Specimens that grew 30 or more colonies under standard plating conditions displayed a more than two‐fold increase in PEs which was reproducible for the two specific variables mentioned above, but the overall success rate of the assay could not be improved. In addition to the possibility that several deficient basic requirements for achieving optimal environmental conditions for colonic stem cell growth have not been defined, we believe a major reason for failing to improve the number of drugassayable specimens is related to an inherent interneoplastic diversity in terms of growth requirements of human colorectal malignancies.