Improved optical detection of colony enlargement and drug cytotoxicity in primary soft agar cultures of human solid tumour cells

Abstract

Summary The presence of cellular aggregates in cell suspensions derived from human solid tumours often complicates subsequent evaluation of colony formation in primary soft agar cultures (Agrez et al., 1982b). In the present study, performance of a conventional colony formation assay was observed to lack sufficient sensitivity to identify growth and active chemotherapeutic agents in the majority of specimen cultures. Modification of conventional methodologies to include filtration of cell suspensions, use of "proliferation control" and "cytotoxicity control" cultures as well as vital staining were found to be essential for the valid assessment of primary soft agar cultures in our laboratory. In addition, application of drugs to culture surface in place of culture incorporation appeared to facilitate culture performance and drug sensitivity testing.

Measurement of human tumour cell growth in soft-agar cultures using computer-assisted volume analysis

“…Tumour acquisition, digestion andfiltration Methods of tumour acquisition, digestion, and filtration have been described previously (Alley & Lieber, 1984a Soft agarose cell culture and culture surface drug application While the basal layer of cultures was prepared with 0.5% agar, the cellular (upper) layer was formulated with 0.3% Seaplaque agarose. A single, "bulk" cell suspension was prepared for each specimen culture; inoculation of 1 ml aliquots (500,000 nucleated cells) in replicate dishes was performed with a constantvolume step-syringe.…”

Soft agarose culture human tumour colony forming assay for drug sensitivity testing: [3H]-thymidine incorporation vs colony counting

“…Since the preparation of cell suspensions from human tumour xenografts and, especially, from primary human cancers, is generally complicated by the presence of small cell aggregates (Agrez et al, 1982a;Umbach & Spitzer, 1983;Alley & Lieber, 1984); then use of this or related assays for measuring tritiated thymidine incorporation may prove to be more sensitive and reliable than optical counting for detecting anticancer drug effects in soft agar cultures. …”

“…This extensive experience has generated concern about technical problems which occur in the performance of soft agar colony forming assays using samples of cells isolated from primary human tumours; publications on this topic have appeared in this journal previously (Agrez et al, 1982a; Alley & Lieber 1984& Lieber , 1985. Consequently, it was of interest to investigate whether the use of a different way of assessing cell proliferation and drug effects in such soft agar tumour colony forming assays might be as effective or more effective than simply optically counting the number of colonies formed.…”

A feasibility study of the MTT assay for chemosensitivity testing in ovarian malignancy

“…The assay, which measures the conversion of a tetrazolium salt (MTT) to formazan, has already been successfully applied to drug screening in cell lines (Alley et al, 1988;Carmichael et al, 1987) and fresh leukaemic cells (Sargent & Taylor, 1989;Twentyman et al, 1989;Pieters et al, 1988). Most chemosensitivity testing previously carried out in ovarian malignancy used the clonogenic assay (Alberts et al, 1980;Bertoncello et al, 1982;Alley & Lieber, 1984). This method is technically difficult.…”

“…The intervening development in semi-automated, microtitre techniques has made his method rapid and simple. The assay, which measures the conversion of a tetrazolium salt (MTT) to formazan, has already been successfully applied to drug screening in cell lines (Alley et al, 1988; Carmichael et al, 1987) and fresh leukaemic cells (Sargent & Taylor, 1989;Twentyman et al, 1989;Pieters et al, 1988 (1982). Once a cell suspension was obtained these samples were also separated by density gradient centrifugation and washed in HBSS before resuspending in culture medium as described above.…”