The cultivation of mammalian cells in lab, or tissue culture as it is commonly called, is a
critical tool for many scientists. Mammalian cells provide scientists with the means to
study biological processes on the cellular level, instead of having to work with a whole organism.
In addition, mammalian cells can be used as a means to produce vital tools in the lab, such as
antibodies or viruses. While undeniably valuable, learning basic tissue culture technique can be a
daunting task for those new to the lab and veteran scientists alike. In this instructional video, we
will provide some best practices and advice for those new to tissue culture. Before beginning any
tissue culture experiment, ensure that the lab is equipped with all of the necessary materials.
Some key instruments include: a carbon dioxide incubator, an inverted microscope, and a class
2 biosafety cabinet. Preventing contamination is critical for any tissue culture experiment.
Contamination prevention begins with establishing and maintaining a sterile work environment. Open the
biosafety cabinet sash to the proper operating height, and turn on the blower. The proper operating
height is usually indicated on the instrument, and tends to be about 8 to 10 inches.
An incorrect sash height will disrupt the airflow in the cabinet, and compromise your cells. Examine
the unit and make sure that the vents are not blocked. Allow the unit to warm up for at least
5 minutes before use. This allows the unit to purge unwanted particulates in the cabinet.
Once the unit has warmed up, wipe all pipettes and surfaces with 70% alcohol and allow it to evaporate.
Thoroughly decontaminate all reagent
bottles and bags of plasticware with alcohol before transferring to the biosafety cabinet.
Before beginning an experiment, position all of the materials that you will need, such as plasticware,
waste bins, and media.
...as this can introduce contaminants.
In addition, we recommend using filtered serological pipettes and tips,
as this will reduce the risk of contaminating common equipment, such as pipettes and pipettemen.
Body floor are a main source of tissue culture contamination. To prevent unwanted contamination,
wear a clean lab coat and gloves when working in a hood. Before entering the biosafety cabinet,
check your lab coat to ensure that your wrists and arms are completely covered. If you find that skin
becomes exposed while working, wear disposable sleeves.
by turning to the side or coughing and sneezing
into your shoulder or upper arm. Before handling cells, you should examine your cultures both macro
and microscopically for signs of contamination, such as bad odors in the incubator, or a change
in media color. Often, contaminated media will turn yellow, due to the acidic byproducts of microbial
growth. In addition, media may appear turbid or cloudy. Before working with your culture,
check for signs of microscopic contamination with the high power objective of your inverted
microscope. Often, low levels of contamination can be observed in the microscope before they
can be observed macroscopically.
In addition to microbial contamination...
Also, be sure to change
your serological pipette or pipette tips anytime they contact your culture, to ensure that you are
not contaminating your media or other reagents with cells. We recommend using different reagent
bottles for different cell lines to limit the chance of cross contamination. When you're ready
to switch to a new cell line dispose of all trash, rinse the aspirator line with bleach,
and wipe down all equipment and surfaces with alcohol. Lastly, before handling the new line,
change your gloves.
We recommend screening all cell lines two to three
weeks after thawing, and rescreening every two to three months thereafter. As cells are cultured,
they will begin to accumulate genotypic variations over time. Consequently, after several months or
years of continuous culturing, your cells have probably diverged quite a bit from the parental
vial. This can influence the phenotype and the reproducibility of your data. To overcome this
challenge, users should create a cell bank of early passage cells. Once cells are received and thawed,
expand them for the first few passages until you have several flasks of early passage cells that
can be prepared for freezing. Store the vials in a cryogenic freezer for future use. As you
use your cell line, keep track of the passage number. Once the cells have been used for 30 or 40
passages, discard the flask and thaw a new vial of early passage cells. Once growth procedures
are established, the user should take care to treat their cells gently.
When you're ready to aspirate, place the pipette in a corner of the flask to prevent disrupting the cells. When tripsinyzing,
or detaching our cells from the flask, do not add the enzyme directly to the model air
and try not to excessively bump the flask
Instead, hold the flask at an angle and pipette
onto the top surface of the flask. Gently rotate your flask back and forth to evenly disperse the
liquid. When you begin to seed a new culture vessel, be sure to evenly distribute the cells
throughout the growth surface. Insufficient mixing will lead to patches of cells that are
over or under dense, and negatively affect the overall health of the culture. We recommend using
a serological pipet to thoroughly mix the cell suspension to ensure even distribution.
When seeding multiple dishes or multiple wells, consider preparing a master mix containing all
of the cells and media for all of the dishes, rather than seeding each vessel individually.
This tends to result in better uniformity across plates and therefore more reliable data. We hope
you've enjoyed this video on getting started with tissue culture. For additional videos protocols
and useful blog posts, please visit Addgene's website.
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