Methods for separating malignant cells from clinical specimens

Abstract

Fragments of a biopsy sample on the order of about 50 to 5000 cells are preferred for establishing viable tumor cell cultures for purposes such as establishing cell lines, chemotherapeutic assays and the like. Such fragments retain the three-dimensional cellular structure or organization of the original tumor and, therefore, can be cultured more readily. To obtain such fragments suitable for culturing, the biopsy sample can be enzymatically digested in a proteolytic or nucleolytic enzyme, such as collagenase, or by mechanical dissociation, or both where necessary. The fragments can then be suspended in an aqueous medium so that non-aggregated cells (e.g., red blood cells, lymphocytes, macrophages) and cellular debris will form a supernatant while the remaining fragments containing aggregated tumor cells are deposited in a sediment layer. Preferably, the medium is an isotonic tissue culture medium and decantation is conducted at least twice; first in a serum-containing medium and then, secondly, in a serum-free medium. Fragments containing living tumor cells can be selected by fluorochromasia, that is, by contacting the sedimented layer with a fluorogenic substrate such that viable tumor cells take up and hydrolyse the substrate, and then exhibit fluorescence. Cytotoxicity assay protocols employing tumor cell aggregates prepared by the present techniques are also disclosed.