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Effects of Clumps and Clusters on Survival Measurements with Clonogenic Assays¹

Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06510

The growth of colonies in semisolid medium is receiving widespread attention as a possible test for predicting the clinical response of individual human tumors to specific drugs. One problem encountered in these studies is the difficulty of preparing single-cell suspensions from solid tumors. Often, microscopic examination of the cultures just after plating reveals many clusters or clumps of cells; these are often large enough and numerous enough that some plates are counted or fixed immediately so that this "background" can be subtracted from the "colony" count used to assess cell viability. However, this correction addresses only one of the problems created by the presence of clumps and clusters. It does not eliminate errors and artifacts introduced by multiplicity (multiple clonogenic cells contributing to a colony), abortive clones, and cell volume changes, or by inhomogeneities in the microenvironment, cell metabolism, and drug distribution within clumps. Because of such factors, survival curves determined using suspensions contaminated with clumps and clusters may provide inaccurate assessments of the true drug sensitivity of the individual tumor cells.

¹ This work was supported by Grants CA-06519 and CA-35215 from the National Cancer Institute and by Grant PDT-145 from the American Cancer Society. A preliminary report of this work was presented to The Cell Kinetics Society, March 21 to 24, 1984.

Received 7/30/84. Revised 12/26/84. Accepted 12/31/84.

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