Best Practices – Cell Culture Techniques
The ability to take cells from their natural environment and culture them artificially in the lab has been an invaluable tool for researchers to study cell physiology, metabolic pathways as well as look at cellular responses to drugs and toxic compounds. Additionally, cells cultured in vitro have been successfully utilized for the large-scale production of biological compounds (e.g., vaccines, therapeutic proteins).
That said, culturing cells in the laboratory is not a trivial task and success is dependent on a number of factors ranging from having the proper equipment, sterile technique, culture media and conditions, to utilizing the appropriate protocols for handling the cells during passaging and freezing/thawing.
The Gibco Cell Culture Basics Handbook is an excellent resource for anyone embarking on cell culture and summarizes the important aspects and considerations for successful cell culture techniques. Some of the important cell culture best practices from the handbook are summarized here.
The establishment of a dedicated cell culture workspace with the correct equipment and safety protocols is essential to the success of your cell culture, which include (at minimum):
- A cell culture hood (most commonly a biological safety cabinet II)
- Incubators to maintain the correct temperature and humidity for your cell type
- Refrigerators and freezers to store culture media components
- Cryogenic storage to maintain cell stocks
- Automatic cell counter or microscope and hemocytometer
Using proper aseptic technique ensures that contaminating microbes such as bacteria, fungi, and viruses are not introduced into your cell culture. The key tenants of aseptic technique are having a sterile work area, good personal hygiene, sterile reagents (i.e. culture media, dissociation enzymes) and handling practices to reduce the probability of contamination. It is important to emphasize that the routine use of antibiotics to mitigate contamination is undesirable since it can hide low level contamination from mycoplasma and other cryptic sources as well as lead to the development of antibiotic-resistant strains.
The cell culture environment encompasses the physiochemical (i.e. pH, temperature and CO2) and physiological conditions (i.e. growth factors, vitamins and other nutrients) necessary for cell propagation in vitro. Aside from the temperature, all of these components are provided by the cell culture media.
Culture Media. The most important aspect of the cell culture environment is selecting the appropriate culture media for your cell type of interest since each cell type has unique metabolic requirements. It not only provides the necessary nutrients, growth factors, and hormones for cell growth, but also aides in regulating the pH and the osmotic pressure of the culture.
Serum. As an additive to basal media, serum is widely used to provide growth and adhesion factors, hormones, lipids, and minerals for cells. Many of the lipids and micronutrients are bound to the albumin in serum to facilitate their entry into and use by the cell. Fetal bovine serum (FBS) is by far the most common animal sera used for cell culture because it has reduced levels of gamma globulins and complement proteins that can result in the unwanted binding and lysis of cells in culture. However, not all sera are equal and able to support the proliferation of cells in culture. FBS suffers from high cost, variability, and unwanted biological effects on certain cell types are common hurdles. This is why it is critically important to obtain sera from a reputable supplier.
Cultureware. The use of plastic cultureware has become commonplace for cell culture where polystyrene (PS) is the most frequently used plastic in labs today. The type of cultureware will depend on whether you are growing adherent or suspension cells as well as the scale of your cell culture, which will dictate the type and size of the culture vessel. PS is hydrophobic in nature making it ideal for suspension cells but many cells are anchorage-dependent (adherent) cells that require a tissue culture (TC)–treated surface to promote cell adhesion and spreading. In this case, a negative charge is imparted to the PS surface by plasma gas which increases its hydrophilicity for cellular attachment. Furthermore, this coating can be optimized for your cell type of interest by coating the TC- surface with extracellular matrix molecules like peptides (e.g., poly-D-lysine or PDL), proteins (e.g., collagen), or polysaccharides can better mimic the in vivo environment.
Culture Conditions. The pH, CO2 and temperature are other physiochemical parameters that require consideration as these requirements are also cell type dependent. The pH and CO2 levels are closely linked and are modulated by media components. As cell metabolize, they produce CO2, which causes the pH of the culture media to decrease (become acidic). Buffers such as HEPES or bicarbonate (HCO3-) in the culture media help counteract these changes in pH. As well, changes in atmospheric CO2 can alter the pH of the medium thereby necessitating the use of exogenously delivered CO2, typically controlled by the cell culture incubator alongside the temperature. Typically, cells are cultured in 4-10% atmospheric CO2 but the specific conditions are dependent on your cell type.
Having optimized your cell culture environment for cell proliferation, the next consideration is subculturing or passaging the cells. Cells in culture require passaging when
- a) Cells in adherent cultures occupy all the available substrate and have no room left for expansion
- b) Suspension cells have expended the nutrients in the culture medium and can no longer support further proliferation/metabolism
The timing of when to passage the cells takes an experienced hand but clues from visualizing the cultures both macro- and microscopically can provide valuable information. Adherent cells will stop dividing once they reach confluence due to contact inhibition, therefore passaging prior to this point in necessary to maintain optimal logarithmic growth. Furthermore, decreases in pH, as shown by pH indicators in many cell culture media, tend to signal an increase in cellular metabolism and therefore density. Establishing a passaging schedule is vital to understand the growth characteristics and monitor the health of your cell culture.
Cryopreservation/Thawing of Cells
Cell in continuous culture have a finite lifespan unless they have been immortalized and are thus prone to genetic instability and senescence as their passage number increases. It is, therefore, worthwhile to prepare and preserve working stocks of the cells in cryogenic storage that allow you to initiate fresh, low passage cells as required. The best method for cryopreserving cultured cells is storing them in liquid nitrogen in complete medium in the presence of a cryoprotective agent such as dimethyl sulfoxide (DMSO) that reduce the freezing point of the medium and also allow a slower cooling rate, mitigating the formation of ice crystals that can damage cells reducing cell viability and subsequent recovery.
Conversely, equally important is the proper thawing of cryopreserved cells ensures high viability and recovery. As with cryopreservation, the thawing procedure is stressful to frozen cells, and using good technique and working quickly ensures that a high proportion of the cells survive the procedure.
The more you work with your cells, the better you will understand their requirements. Proper technique, adherence to protocols, suitable equipment and high-quality reagents are invaluable to the success of your cell culture. The Gibco Cell Culture Basics Handbook in itself is a great detailed resource and the supplementary “Cell culture basics instructional videos” can be used for training and lab practice refresher courses as well.