UC Irvine

A procedure was developed to directly measure the self-renewal capacity of clonogenic cells from biopsies of metastatic human malignant melanoma. A culture of colony-forming cells was performed with bilayer agar in microtiter wells. The number of live tumor cells from biopsies of melanoma tissue was determined and was used to calculate plating efficiencies. Sequential photography showed that cells did not migrate in agar, thereby documenting that all the cells within colonies were direct descendants of clonogenic cells. A calibrated pneumatically controlled micropipet attached to a micromanipulator was used to quantitatively remove melanoma colonies without removing adjacent cells or agar. Plucked primary colonies were mechanically disaggregated into single cells; viability was greater than 95% as determined by trypan blue dye exclusion. Dose-related formation of secondary colonies was observed after replating of cells from pooled primary colonies. Cells from individual colonies were replated, and secondary colonies formed. These techniques allowed a simple and direct assessment of the self-renewal capacity of colony-forming melanoma cells.