Clonogenic assay
Encyclopedia
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 as well as for titration of Cell-killing Particles (CKPs) in virus stocks.
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.
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.
For Cell-killing Particle assays, the surviving fraction of cells is used to approximate the Poisson Distribution of virus particles amongst cells and therefore determine the number of CKPs encountered by each cell.
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. The cells are put in petri dish
es 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 gray, 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 algorithm
s to analyse images. These are either captured by a image scanner
or an automated microscope that can completely automate the counting process. 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.
, ionizing radiation
, or a combination of the two. 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.
Microbiology
Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...
technique for studying the effectiveness of specific agents on the survival and proliferation of cells. It is frequently used in cancer research
Oncology
Oncology is a branch of medicine that deals with cancer...
laboratories to determine the effect of drugs or radiation on proliferating tumor
Tumor
A tumor or tumour is commonly used as a synonym for a neoplasm that appears enlarged in size. Tumor is not synonymous with cancer...
cells as well as for titration of Cell-killing Particles (CKPs) in virus stocks.
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 cultureTissue cultureTissue culture is the growth of tissues or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar...
vessel and allowed to grow. - The colonies produced are fixed, stained, and counted.
At the conclusion of the experiment, the percentage
Percentage
In mathematics, a percentage is a way of expressing a number as a fraction of 100 . It is often denoted using the percent sign, “%”, or the abbreviation “pct”. For example, 45% is equal to 45/100, or 0.45.Percentages are used to express how large/small one quantity is, relative to another quantity...
of cells that survived the treatment is measured. A graphical representation of survival versus drug concentration or dose of ionizing radiation
Ionizing radiation
Ionizing radiation is radiation composed of particles that individually have sufficient energy to remove an electron from an atom or molecule. This ionization produces free radicals, which are atoms or molecules containing unpaired electrons...
is called a cell survival curve.
For Cell-killing Particle assays, the surviving fraction of cells is used to approximate the Poisson Distribution of virus particles amongst cells and therefore determine the number of CKPs encountered by each cell.
Any type of cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
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
Biopsy
A biopsy is a medical test involving sampling of cells or tissues for examination. It is the medical removal of tissue from a living subject to determine the presence or extent of a disease. The tissue is generally examined under a microscope by a pathologist, and can also be analyzed chemically...
of a tumor in a patient. The cells are put in petri dish
Petri dish
A Petri dish is a shallow glass or plastic cylindrical lidded dish that biologists use to culture cells or small moss plants. It was named after German bacteriologist Julius Richard Petri, who invented it when working as an assistant to Robert Koch...
es 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
Gray (unit)
The gray is the SI unit of absorbed radiation dose of ionizing radiation , and is defined as the absorption of one joule of ionizing radiation by one kilogram of matter ....
of radiation, 500 cells might be plated, but at 4 or 5 gray, 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
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...
and is quite tedious. Recently, machines have been developed that use algorithm
Algorithm
In mathematics and computer science, an algorithm is an effective method expressed as a finite list of well-defined instructions for calculating a function. Algorithms are used for calculation, data processing, and automated reasoning...
s to analyse images. These are either captured by a image scanner
Image scanner
In computing, an image scanner—often abbreviated to just scanner—is a device that optically scans images, printed text, handwriting, or an object, and converts it to a digital image. Common examples found in offices are variations of the desktop scanner where the document is placed on a glass...
or an automated microscope that can completely automate the counting process. 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 drugMedication
A pharmaceutical drug, also referred to as medicine, medication or medicament, can be loosely defined as any chemical substance intended for use in the medical diagnosis, cure, treatment, or prevention of disease.- Classification :...
, ionizing radiation
Ionizing radiation
Ionizing radiation is radiation composed of particles that individually have sufficient energy to remove an electron from an atom or molecule. This ionization produces free radicals, which are atoms or molecules containing unpaired electrons...
, or a combination of the two. Some current research studies the potentiation of drug effects by concurrent irradiation—a synergistic
Synergy
Synergy may be defined as two or more things functioning together to produce a result not independently obtainable.The term synergy comes from the Greek word from , , meaning "working together".-Definitions and usages:...
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
Gênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
or receptors on the cell, the responses of different cell types, or synergistic effects of multiple drugs.