1) Cell Culture Tutorial – An Introduction

In biology, cell culture is the process by
which cells are grown in controlled conditions outside of its native environment. This technique
has enabled scientists to use cells, the fundamental unit of life, as an imperative model system
for understanding physiological processes in the human body. One of the major contributions of cell culturing
is the development of polio vaccines; back in the early 1930s, researchers had to use
live animals to grow poliovirus, but with cell culture, researchers were able to have
greater control of virus production which eventually led to the creation of the vaccine.
Fast forward to the 1970s, mammalian cell culture was used to create properly folded
and glycosilated proteins, such as interferons and antibodies, that were impossible to make
in prokaryotic E. coli cells because E. coli cells lack post-translational modifications.
Nowadays cell culture is being used widely in laboratory settings, particularly in the
field of vaccine research, cancer research, and protein therapeutics. Cells that are used in laboratories can be
generalized into two categories: primary cells and established cell lines. Primary cells
are cells that are directly prepared from an organism’s tissues. If grown under the
right conditions, primary cells will grow and proliferate, but they are only able to
do so a finite number of times. This is because each time as the cells divide, part of its
DNA’s telomere is lost and after a certain number of cell divisions, the cells would
reach senescence when the cells can no longer divide. Established cell lines, on the other
hand, are able to escape the normal constraints of the cell cycle and grow indefinitely, making
them extremely useful for long-term research. Whereas primary cells are obtained directly
from donor tissues, cell lines can be derived from clinical tumors, or created from transforming
primary cells with viral oncogenes or chemical treatments. These reagents or treatments target
one of the checkpoints in the five phases in the mammalian cell cycle. Checkpoints are
there to ensure that the cell meets the appropriate requirements in order to move on to the next
stage of making daughter cells. One of the checkpoints is at the end of G1, which allows
cells to commit to cell division. The second checkpoint is at the S-phase, and is there
to check the quality of the replicated DNA and to determine if any DNA repair mechanisms
need to be activated. The third checkpoint is after G2, and its purpose is, again, to
ensure that DNA has been replicated completely and is undamaged. Finally, there are several
checkpoints within mitosis to ensure that the cell is in a proper position to complete
cell division. When a cell starts to divide uncontrollably,
it is because they have evaded these checkpoints and the cell cycle is allowed to proceed and
cells continue to proliferate. Many viral oncogenes, such as E6 and E7 from the human
papillomavirus, can degrade controllers of the cell cycle; namely the tumor suppressor
genes, to turn a normal cell cancerous. Primary cells and established cell lines in
culture usually adopt one of the two growing forms: they either grow as monolayers or are
free-floating in culture media. Cells that form monolayers are also often referred to
as adherent cultures while the free-floating cells are called suspension cultures. Knowing
the growth properties of the cells can help scientists choose the appropriate cultureware
for their experiments. To keep the cells alive, it is important to keep the conditions as
close to the physiological conditions as possible, therefore supplements such as amino acids,
inorganic salts, and vitamins, are added into the culture media. Traditionally other vital
supplements including macromolecules, lipids, and growth factors are supplemented in the
form of fetal bovine serum (FBS). However, there are a lot of uncertainties with regards
to the use of FBS. For example, as scientists do not know the serum’s exact composition,
it is always subjected to lot-to-lot variation and this in turn, can affect the cultures.
Recently, defined media, where the exact composition of the media is known, are on the rise to
address this issue. As physiological fluids have a neutral pH,
the culture media also have a buffering system to ensure that the proper pH is maintained.
One way to achieve this is to use sodium bicarbonate to keep the pH of the media between 7.2 and
7.4 with 5-10% gaseous carbon dioxide. Another common buffering system involves the use of
zwitterions, such as HEPES. In both kinds, it is common to have a pH indicator such as
phenol red added to the media so the scientists can visually monitor the changes in pH. Different cell lines generally require different
types of media, and the most commonly used commercial media today are Dulbecco’s Modified
Eagle Medium (DMEM), Roswell Park Memorial Institute-1640 (RPMI), and Ham’s F12 Nutrient
Mixture (F12). Apart from using the appropriate media for
growth, the cells are also maintained in the correct temperature. For example, most mammalian
cells are incubated at 37 degrees Celsius for optimal growth, while cells derived from
cold-blooded animals should be kept at lower temperatures. Other measures to take when working with cell
cultures is to ensure that the cells are kept under a sterile environment. Microbial contaminates
such as bacteria, fungi, or mycoplasma can ruin the culture and lead to invalid experimental
data. A Laminar flow hood is always used for cell culture work. It maintains a steady,
clean airflow and prevents outside contaminants from entering the culture. In addition to
microbial contaminations, the cells can also be contaminated by other cells. Cell lines
can be overgrown and replaced by other fast-growing cells inadvertently introduced into the original
culture. The consequence of using a misidentified cell line is making false scientific conclusions.
Authenticating cell lines using Short-Tandem Repeat profiling, or STR is recommended for
cultures from time to time. abm provides a wide range of cell culture
related products, ranging from the starting materials, such as primary and immortalized
primary cells, to immortalizing agents, media and plastic ware. We also have specialized
PCR-based detection kits and drug treatments for mycoplasma contamination. Lastly, since
many scientific journals and funding agencies now require evidence of cell line authentication
by STR profiling as a requisite for publishing or obtaining funding, we are proud to now
offer this service to our customers. Cell culture technique has advanced our scientific
knowledge since the 1930s and it continues to provide us valuable information about all
aspects of biology. If you’d like to read more, please visit our knowledge base at the
link below. Also, be sure to check out our next video for cell culture techniques. Please
leave your questions and comments below, and we will answer them as soon as possible. Thank
you for watching.

Author: Kennedi Daugherty

Leave a Reply

Your email address will not be published. Required fields are marked *