The determination of growth rates of individual colonies in agarose using high‐resolution automated image analysisx

Abstract

This paper describes the evaluation of a colony formation assay using automated image analysis, which permits the tracking of growth at the individual colony level, such that a growth rate can be estimated for each colony followed. In principle, this will permit quantitative characterization of cellular heterogeneity in growth rate and cellular heterogeneity in response to proliferation-modifying agents. In addition, we have demonstrated the possibility of using correlative microscopy to relate growth rate to other parameters, using metabolic viability as an example. This should be useful for determining cellular characteristics associated with proliferative behavior and response to proliferationmodifying agents.Key terms: Proliferation rate, correlative microscopy, tetrazolium dye, growth factors, antiproliferative drugs, drug response, computer simulation, heterogeneityThe presence of variant cells within a population derived from a common ancestor is known as cellular heterogeneity (8,27). This situation represents a significant problem in cancer therapeutics, for example, in that diversely responsive subpopulations within a target tissue may allow for selection of resistant cells during therapy (5). To study cellular heterogeneity directly, it is necessary to have methods that are capable of defining cellular characteristics and behavior a t the level ofthe individual cell. Advances in flow cytometry and image analysis are making such studies increasingly possible.Proliferation is an important cell behavior in cancer research, inasmuch as cancer is primarily a disease of growth control, and anticancer therapies are usually aimed at killing, or otherwise arresting the growth of, malignant cells. Unusual cell proliferation also plays a role in arteriosclerosis (7), and thus is a cell behavior with relevance to more than one significant health problem. Although cytometric systems, such as flow approaches, permit multiple parameter measurements on individual cells virtually instantaneously, such measurements do not permit quantification of cell proliferation.A method commonly used for evaluation of proliferative potential of individual cells is the colony formation assay, wherein immobilized cells are allowed to proliferate, and growth is quantified by enumeration of progeny. Computer-assisted evaluation of colony formation assays has been employed for colony counting (10,141, for total colony volume analysis (29,311, and for histogram analysis of colony sizes (12). These approaches permit assessment of growth kinetics of populations and estimation of growth rate of individual colonies by inference. A manual approach involving colony isolation and cell enumeration has been used to determine numbers of cells in colonies in soft agar (29); a similar approach has been used to describe heterogeneity in growth rate among individual colonies growing on plastic (15). Time-lapse cinematographic approaches (1 1,211 and time-lapse video approaches (6,7) have been used to determine the growth rate of individual cells throu...

Growth slow‐down and growth arrest of human colon carcinoma cells HCT‐8 in vitro after exposure to 5‐fluoro‐2′‐deoxyuridine

“…Cells were plated for analysis of proliferation rate of individual colonies as previously described (Slocum et al 1990). Briefly, cells plated in 0.3% agarose were observed at daily intervals, observations were digitally recorded, and area measurements made using a Quantimet Q970 image analyser.…”

“…Experiments in which drug was administered for 3 h in the agarose layer were performed as previously described (Slocum et al 1990). Briefly, cells were plated in an agarose island approximately 160 p1 in volume, with a liquid overlayer of 3 ml of complete medium containing the appropriate drug concentration.…”

“…Since drug exposure before plating allows for the possibility of losing or damaging some cells during drug removal and preparation for plating, we designed a technique for exposing cells in situ to drugs after plating, with removal of drug after timed exposure by changing the liquid medium (Slocum et al 1990). In preliminary experiments using radiolabelled drug, it was established that the FdUrd is reduced 8970-fold by the washing process (Malmberg, Slocum & Rustum 1991).…”

Automatic analysis of growth onset, growth rate and colony size of individual bone marrow progenitors

“…Time-lapse video images allowed dynamic descriptions of the growth in the assay (2, 3). Slocum and co-workers developed a method to monitor and evaluate the growth history of individual cells plated in semi-solid medium (6)(7)(8). The technique is based on measurement of the colony diameter using pseudo dark field microscopy and thus limited to more or less coherent, non-overlapping aggregates.…”

Time-lapse video reveals immediate heterogeneity and heritable damage among human ileocecal carcinoma HCT-8 cells treated with raltitrexed (ZD1694)

“…Time‐lapse recording methods and sophisticated mathematical analyses have been applied to colony formation assays, to characterize inheritance of proliferative capacity and heterogeneity in growth comparing normal and tumor cells (8–14). We undertook a study of the response of individual cells of a human ileocecal carcinoma cell line (HCT‐8), using a time‐lapse video recorder with semiautomated computer analysis, to follow up on earlier work conducted at the individual colony level (15–18).…”