Advantages and disadvantages of using mammalian cell cultures vs bacterial

Transfection using cationic lipid polymers

Immunoprecipitation using flagtag
Detection mechanism- enzyme horseradish peroxidase conjugated secondary antibody and
Chemiluminescence.

Background
Artificially growing cells of an organism outside the host by providing the required media is
known as tissue culture. Tissue culture is a proven technique which is in application from many
years and this technique has many implications to test the nature of the cells. Scientists used this
technique to test the nature and behavior of cells outside the host environment, (1) because the
systemic environment of a body is not stable and is subjected to change by many factors which
may result in the deviation of the results for a hypothesis to be tested. On contrary to that, tissue
culture helps in maintaining the growth environment stable for cells, which helps in obtaining
better results without any deviation and a better understanding of the hypothesis. Using
mammalian cell culture over the bacterial culture has its own advantages and disadvantages.
Advantages
Parameters such as temperature, osmotic pressure, pH, O2, CO2 and surface tension of cells can
be controlled of the environment in which they grow (1) and the intracellular and extracellular
environment of the cells can be altered according to the requirements of the experiment. Whereas
the advantages of the bacterial cell culture are that they are replicate relatively faster than
mammalian cell culture they can continuously grow until and unless there is a nutrient depletion
and there wouldnt be a huge risk of cross contamination when compared to mammalian cell
culture.
Disadvantages
The media used for mammalian cell culture has all the required things for a cell to grow such as
hormones, growth factors and other supplements but as the culture proliferates batch by batch its
nutrient requirement may change, we have to be aware of this fact to avoid any interference in
the cellular activities (1). Mammalian cell culture should be done in complete aseptic
environment as there is quite a chance for cross contamination whenever we handle culture. The
reason behind this is, bacterial cultures grow faster when compared to the animal cell culture.
The output of the cell culture is very less, usually in milligrams and requires tremendous skills
and time to invest. Another major disadvantage of this technique is that whenever the cell culture
becomes confluent, we do a process called passage, a passage is nothing but the enzymatic
digestion of the culture from which some part of the culture is transferred to the fresh media (1).
After several passages, there is a fair chance that the cells of interest might get a change of
phenotype when relative to the primary cell lines. This alteration could be avoided only if a

researcher keeps record of the cultures regularly when a passage is done and carefully selects the
culture to be grown.
Transfection is a process of inserting genetic material such as DNA or double stranded RNA into
eukaryotic cell. The method of introduction of genetic material varies depending upon the
requirement of the process. The normal methods used for transfection are Lipid-mediated,
calcium phosphate mediated, DEAE- dextran mediated, Electroporation, biolistic (1). The two
types of lipid mediated transfections are cationic and anionic. The method which we use in our
laboratory is Cationic Lipid-mediated transfection. The cationic lipids are created by using the
cationic lipid reagents and these lipids have the capacity to directly interact with the plasmid
membrane or could be taken up by non-receptor mediated endocytosis (1). The process of
transfection is usually starts with the construction of the plasmid DNA which can express the
desired gene, PTEN in our case and the constructed is transfected into the mammalian cell
culture, HEK 293(Human Embryonic Kidney 293) in our case. The process which we do in our
lab is Transient Cationic Lipid Mediated Transfection (1). The major limitation of this process
is that the process is the cost effectiveness of the process and the advantage of this process is the
higher efficiency, can culture different cell lines and low toxicity. If the cell culture expresses
PTEN then the next step is to precipitate the protein using immunoprecipitation from cell lysate.
Immunoprecipitation is a technique which is used to determine the molecular weight, study the
protein/protein interactions, specific enzymatic activity, and to detect the presence &
quantification of proteins (1). This is followed by SDS-PAGE and immunoblotting (1).