Stable Cell Lines


Stable cell lines are crucial laboratory tools that can be used to express large amounts of a protein of interest. This can be necessary for a variety of reasons; high protein production may be necessary for the screening of experimental drugs, studying of gene functions, or production of therapeutic proteins (including recombinant antibodies).  The cell lines indefinitely reproduce and continue to express the transgene consistently during that time period.

Perhaps the most important characteristic of stable cell lines is that they are, as the name implies, genetically homogenous in culture. This means that even after new generations have been produced, the same genetic characteristics are present in all cells. This is particularly relevant to long term genetic studies and industrial production of highly specific proteins. In research settings, the availability of stable cell lines allows for the evaluation of drug treatments and monitoring of the evolution of cellular behavior. Cancer cell lines, for example, must be stable in order for drug testing to be indicative of drug potential.

Stable cell lines contrast strongly with transiently-transfected cells, which do not have stable genetic and protein expression characteristics. Transient transfection results in temporary changes to cell lines, which may aid in one-time production of proteins or short-term experiments. This makes transiently-transfected cell lines ideal for simple experiments, but problematic for studies that last for several days or more. As such, genetic modifications of cells used for longer studies must be permanent (i.e be present in the genome), and hence require advanced transfection techniques.


Cell cultures are usually kept in specialized containers and broth as shown above. These cell-culturing flasks allow for the reproduction of cells, which is supplemented by added nutrients. As a result of the favorable conditions for cell growth, protein production is at a maximum.

Generation of a stable cell line refers to the process of developing homogenous populations of cells that demonstrate expression of a transfected gene insert.  The transfected gene integrates into the genome of the host cell, and as a result, are able to express the transfected genetic material.  This is opposite of transient transfected cells that express the transfected DNA for a short time (e.g. 8 to 96 hours).

Broadly, the generation of stable cell lines is a two step procedure.  First, exogenous plasmid DNA is transfected into the host cell line.  This is followed by applying antibiotics to select only those cells exhibiting the desired plasmid DNA expression.  Nucleic acids to be inserted can be in the form of plasmid DNA, and encode microRNA (miRNA), short interfering RNA (siRNA), or full length mRNA transcripts.

After the cells are selected, they are cultured and divided among several containers to ensure cell survival. Once the cells have been accustomed to a positive environment for growth, they begin to express desired traits. If protein production is desired, then proteins are produced, which can be purified for other research and therapeutic purposes.

There are many attributes to developing stable cell lines, such as the DNA vector used, the protein to be studied, the cell line selected, reagents used, and the transfection method.  Also, certain cell lines respond better to specific transfection methods, culture media or reagents.  Identification of positive clones exhibiting the desired phenotype is extremely laborious and characterization of clonal cell lines takes many weeks.  Also, toxicity due to the protein being expressed may make it impossible to develop a stable cell line.  There are several companies that specialize in generating stably expressing cell lines and provide commercially available service with a  relatively short turnaround period (4-6 weeks).


Utilizing a stable mammalian cell line for production of target molecules may increase the acceptability of the final gene product as pipeline development nears production.  However, the production of these stable cell lines can be expensive, complex and time-consuming.  Biology contract research organizations provide specialized development of stable cell line services, with some companies even providing a 28-day day generation of stable cell line service (see description here).  Scientists use stable cell lines for diverse applications, ranging from studying cell differentiation, gene expression, cellular events, toxicity, and production of recombinant antibodies and proteins. Stable cell lines are also helpful in the evaluation of drugs in specific environments. For example, a genetic mutation may be introduced in a cell line to restrict cell-initiated apoptosis, which may aid in the evaluation of drugs as they affect the division of cells in tumor settings. This can be particularly useful if in vivo models are not available for testing, or if time constraints require faster evaluation of drug potential.

Stable cell lines present an advantage over transiently-transfected lines in that they allow for the mass-production of similar cells. Whereas a generation of transiently-transfected cells cannot pass on modifications to offspring, stably-transfected cells will pass on all genetic modifications to the next generation. As a result, cell cultures can be mass produced, and just a small amount of cells can be grown to generate much larger amounts of identical cells. Such reproductive capacity conveys a benefit to stably-transfected cell lines that makes them far move valuable than their transiently-transfected counterparts.