Cell culture is the process of growing cells outside of their natural environment under controlled laboratory conditions. This technique allows scientists to cultivate cells from animals or plants in an artificial setting for research. It is a foundational tool in biology, used to study the normal functions of cells, understand the mechanisms behind diseases, and test the effects of drugs. The process is also instrumental in the large-scale production of biological materials like vaccines and therapeutic proteins.
Essential Equipment and Aseptic Technique
A dedicated and controlled environment is prepared before any cells are handled. The primary workspace is a biological safety cabinet, which provides a sterile area by constantly bathing the interior with filtered air. Within this cabinet, scientists perform all manipulations of the cells and related materials.
To mimic the conditions inside a living organism, cells are housed in a CO2 incubator. This device maintains a constant temperature and a controlled atmosphere with elevated carbon dioxide levels to keep the culture medium at the correct pH. For routine checks, an inverted microscope is used, which allows for viewing cells through the bottom of the transparent culture flask without disturbing the sterile environment.
The entire process is governed by aseptic technique to prevent contamination from microorganisms like bacteria, fungi, and viruses. This includes sterilizing all equipment and reagents before use and handling materials carefully to avoid contact with non-sterile surfaces. Scientists wear dedicated lab coats and gloves, and all liquids and tools that come into contact with the cells must be sterile.
Initiating a Cell Culture
The first step is preparing the specialized liquid known as culture medium. This begins with a basal medium containing a mixture of salts, sugars, and amino acids. This base is then supplemented with other components to create a “complete” medium, most commonly fetal bovine serum (FBS), which provides a rich source of growth factors. Antibiotics are often added as a protective measure against potential bacterial contamination.
With the medium prepared, a small vial of cells is retrieved from long-term storage in liquid nitrogen. The cryovial is thawed quickly in a 37°C water bath to minimize damage to the cells from ice crystal formation. This step is delicate, as the cryoprotectant agent used for freezing can be toxic to the cells.
Once thawed, the cell suspension is immediately transferred into a culture flask containing pre-warmed complete medium. This seeding process dilutes the toxic cryoprotectant and provides the nutrients needed for recovery and attachment. The flask is then placed into the incubator to begin the growth phase, officially initiating the culture.
Routine Culture Maintenance and Expansion
As cells grow, they consume nutrients from the medium and release metabolic byproducts. To maintain a healthy environment, the depleted medium must be periodically replaced with a fresh supply. During a medium change, the old liquid is carefully aspirated and replaced with pre-warmed, fresh medium, taking care not to disturb the layer of growing cells.
The growth of the culture is monitored by its confluency, which is the percentage of the available surface area covered by cells. For adherent cells, passaging should occur when they reach about 80-90% confluency. If cells become too crowded, they will stop dividing due to contact inhibition or exhaust the nutrients in the medium too quickly.
This process of subculturing, or passaging, is how cell populations are expanded. For adherent cells, this involves adding a reagent like trypsin to detach them from the flask surface. This cell suspension is then collected, and a small fraction of it is seeded into a new, larger flask with fresh medium. Suspension cells, which grow floating in the medium, are passaged by simply diluting the cell suspension into a new vessel.
Monitoring Culture Health and Contamination
Regular monitoring with an inverted microscope is a routine part of the workflow to assess the health of the culture. Scientists visually inspect the cells for proper morphology (shape and appearance). Healthy adherent cells should appear spread out and firmly attached to the vessel surface, while unhealthy cells may look rounded and detached.
The most significant threat to a cell culture is contamination by microorganisms. Bacterial contamination often causes the culture medium to become cloudy overnight and induces a rapid drop in pH, indicated by a color change in the medium. Fungal contamination appears as fuzzy, filamentous growths, while yeast shows up as small, budding particles.
A more difficult threat is contamination by mycoplasma, a type of bacteria so small it cannot be seen with a standard light microscope. Lacking a cell wall, mycoplasma is resistant to many common antibiotics and can alter cell function without causing visible signs of contamination. For this reason, cultures are often periodically tested for mycoplasma using specific molecular methods like PCR to ensure the integrity of experimental results.
Harvesting and Preserving Cells
When the cell population has expanded sufficiently, the culture cycle concludes in one of two ways. The first is harvesting the cells for experimental use. The cells are collected from the flask, often using the same detachment method as in passaging, and can then be used for a wide range of downstream applications, such as extracting DNA or analyzing protein expression.
The second endpoint is cryopreservation, which is the process of freezing cells for long-term storage. After being collected, the cells are resuspended in a special cryoprotectant medium. This medium contains a substance like dimethyl sulfoxide (DMSO) or glycerol, which prevents damaging ice crystals from forming inside the cells during the freezing process.
The vials containing the cell suspension are placed in a controlled-rate freezing container and slowly cooled to -80°C before being transferred to liquid nitrogen. This process creates a cell bank, a frozen stock of cells that can be thawed and cultured again. This practice ensures a consistent and reliable source of cells, preserving the cell line’s characteristics for future experiments.