A hypo-osmotic medium to disaggregate tumor cell clumps into viable and clonogenic single cells for the human tumor stem cell clonogenic assay.

A hypo-osmolar medium and tissue processing technique is described which is useful for disaggregation of residual human tumor cell clumps persisting after mechanical or enzymatic treatment of solid tumors and malignant effusions. The addition of the hypo-osmolar procedure to the standard methods for disaggregation increased the viable single cell yield in solid tumors by 47% and in malignant effusions by 67%. In 5 of the 26 solid tumor specimens tested in the human tumor stem cell assay, clonogenic single cells were obtained with the hypo-osmolar procedure, whereas no growth was observed using standard methods. Overall, the success rate for clonogenicity increased from 46% to 65% for the 26 solid tumors, with the major improvement occurring in ovarian cancer. Clonogenicity was obtained in 80% of malignant effusions both by standard methods and the hypo-osmolar techniques. The increased total yield of clonogenic cells obtained with this procedure enhances the opportunity for experimental versatility and in vitro drug testing.