Culturing cells can be tricky business. Considering that they are derived from living organisms, they have type-specific needs that allow them to flourish in favorable yet artificial conditions. Ensuring a nutrient-rich and temperature-controlled environment in their media of choice is a good start. But how do you maintain healthy cells so you can execute a successful experiment?
Given that cell culture is one of the major tools used in basic research and in drug discovery and development, processes and standardized protocols have been generated for various cell types. Cell viability procedures and characterization assays can help maintain the right cells for your experiment. However, cells are living things and so naturally have some variability in how they grow and what makes them happy.
Below are some helpful tips from Alison Killilea, director of the University of California, Berkeley Cell Culture Facility, and Kevin Grady, product line business manager at ATCC, to keep cells healthy and happy based on common challenges that arise in cell culture.
A contaminated culture can cause cells to weaken and die off, or can change expression patterns and transfection efficiencies that affect experimental outcomes. Mycoplasma is a challenge for cell culture labs in that the infection is not easily visible. A PCR-based mycoplasma detection assay can be run, or a nuclear stain must be used to visualize punctate staining on cell membranes where mycoplasma are located. All cultures should be tested for mycoplasma before use, especially if received from another lab.
In order to combat contamination, pen/strep is a common additive to media. However, be cautioned against antibiotic use due to pressure put on the cells, changing their selection and behavior. Pen/strep can also mask a contamination issue, allowing it to spread. Instead, ensure sterile techniques are used to prevent any contamination in the first place.
Aseptic techniques are possibly the single most important aspect of any cell culture protocol. Use 70% ethanol to spray the hood before and after use, spray gloves before entering the hood, and spray media bottles before opening. Aseptic techniques such as these keep cells happy and contamination free without the use of stressful pen/strep.
In order to maintain a happy culture, always use, or at least start with, recommended media. Certain cell types proliferate better in the specific media that is right for them. However, certain experimental conditions can sicken cells regardless of media use. Sparsely plated cells are unhealthy due to the decreased amount of growth factors and other hormones being released by a fewer number of cells. To overcome this issue if sparsely plated cells are needed, use 20% conditioned media combined with 80% new media when passaging cells to provide an initial insurgence of hormones for cells to feel comfortable in their new environment.
Fetal bovine serum (FBS) is another important growth medium for cell culture that contains low antibody levels and more growth factors, allowing flexibility across different cell culture applications. Pretesting and screening FBS helps to ensure healthy cell transfers by keeping the nutrient base stable across passaging.
Freeze and thaw techniques can start new cells out on the right foot, or cause cell death. Freezing cells should occur at a rate of one degree per minute using relevant slow cooling devices. This creates an even freeze across cells and limits ice crystal formation. Reliable freezers will keep cells at a constant state without causing fatal freeze/thaw cycles as well.
Conversely, thawing should occur quickly in a 37 degree water bath followed by resuspension in complete growth medium. Standardized protocols are created to keep cells in their best state. Make sure to follow them precisely without straying from timed procedures causing too much time in a water bath, or other deleterious delays.
Routine environmental monitoring habits are highly recommended to test cleanliness in the lab. Leave an open agar plate in the cell culture hood with good air flow, and then incubate the plate. It will be evident if the hood was cleaned properly. The same test can be done with incubators, storing an open media bottle for a period of time. Ensuring the use of premium biosafety cabinets with good laminar flow is an important feature of any cell culture lab to maintain a healthy cell culture with no external environmental influence.
Performing incubator checks for CO2 and temperature variation can be a significant aspect of keeping cells happy. If their external environment is not stable, then they won’t be either. Thus, choosing a quality incubator that can maintain constant CO2 levels and temperature ranges can save time and potentially cell lines.
Maintaining cell culture takes time, patience, and adaptive techniques to create the right environment for cells to grow well. Once mastered, healthy cell cultures can be the key to a successful experiment.