Clonogenic assay
A clonogenic assay is a microbiology technique for studying the effectiveness of specific agents on the survival and proliferation of cells. It is frequently used in cancer research laboratories to determine the effect of drugs or radiation on proliferating tumor cells.[1]
Although this technique can provide accurate results, the assay is time-consuming to set up and analyse and can only provide data on tumor cells that can grow in culture. The word "clonogenic" refers to the fact that these cells are clones of one another.
Procedure
The experiment involves three major steps:
- The treatment is applied to a sample of cells.
- The cells are "plated" in a tissue culture vessel and allowed to grow.
- The colonies produced are fixed, stained, and counted.
At the conclusion of the experiment, the percentage of cells that survived the treatment is measured. A graphical representation of survival versus drug concentration or dose of ionizing radiation is called a cell survival curve.[2]
Any type of cell could be used in an experiment, but since the goal of these experiments in oncological research is the discovery of more effective cancer treatments, human tumor cells are a typical choice. The cells either come from prepared "cell lines," which have been well-studied and whose general characteristics are known, or from a biopsy of a tumor in a patient.[3] The cells are put in petri dishes or in plates which contain several circular "wells." Particular numbers of cells are plated depending on the experiment; for an experiment involving irradiation it is usual to plate larger numbers of cells with increasing dose of radiation. For example, at a dose of 0 or 1 gray of radiation, 500 cells might be plated, but at 4 or 5 grays 2500 might be plated, since very large numbers of cells are killed at this level of radiation and the effects of the specific treatment would be unobservable.
Counting the cell colonies is usually done under a microscope and is quite tedious. Recently, machines have been developed that use algorithms to analyse images.[4] These are either captured by a image scanner or an automated microscope that can completely automate the counting process.[5] One such automated machine works by accepting certain types of cell plates through a slot (not unlike a CD player), taking a photograph, and uploading it to a computer for immediate analysis. Reliable counts are available in seconds. As of 2004, these machines are expensive, with basic models retailing for over USD$30,000.
Variables
The treatment is usually a drug, ionizing radiation, or a combination of the two.[6] Some current research studies the potentiation of drug effects by concurrent irradiation -- a synergistic effect -- and in this situation two groups are studied: a control group, which is not treated with the drug; and a treatment group, which is treated with the drug. Both groups are irradiated. If the slopes of their survival curves differ significantly, then a potentiating effect may be evident and could be studied further.
A thorough discussion of the promising research being conducted with the aid of this technique is beyond the scope of this text, but some studies involve the effect of the expression of particular genes or receptors on the cell, the responses of different cell types, or synergistic effects of multiple drugs.
See also
References
- ↑ Hoffman RM (1991). "In vitro sensitivity assays in cancer: a review, analysis, and prognosis". J. Clin. Lab. Anal. 5 (2): 133–43. PMID 2023059.
- ↑ Franken NP, Rodermond HM, Stap J, Haveman J, van Bree C. (2006). "Clonogenic assay of cells in vitro". Nature Protocols. 1: 2315–2319. doi:10.1038/nprot.2006.339.
- ↑ Hamburger AW (1987). "The human tumor clonogenic assay as a model system in cell biology". Int. J. Cell Cloning. 5 (2): 89–107. PMID 3553356.
- ↑ Niyazi M, Niyazi I, Belka C (2007). "Counting colonies of clonogenic assays by using densitometric software". Radiation oncology (London, England). 2: 4. PMID 17212832.
- ↑ Dahle J, Kakar M, Steen HB, Kaalhus O (2004). "Automated counting of mammalian cell colonies by means of a flat bed scanner and image processing". Cytometry A. 60 (2): 182–8. PMID 15290719.
- ↑ Carney DN, Winkler CF (1985). "In vitro assays of chemotherapeutic sensitivity". Important Adv. Oncol.: 78–103. PMID 3916747.