Cell culturing is a foundational process in modern biomedical science, defined as the technique of growing cells outside of their natural, living environment in a controlled setting, known as in vitro. This ability to maintain and propagate living cells—whether human, animal, or plant—is a powerful tool for studying fundamental biological processes, disease mechanisms, and cellular responses to new medicines. By isolating cells from the complexity of an organism, scientists can manipulate variables individually, making cell culture an adaptable technology that underpins much of contemporary medical research.
The Essential Environment for Growth
Maintaining living cells outside the body requires artificially reproducing the precise physical and chemical conditions they experience naturally. This relies on a specialized incubator, which regulates the environment to mimic conditions inside a mammalian body. Temperature is held at 37°C, and the air is humidified to prevent the culture media from evaporating.
A controlled level of carbon dioxide, typically 5%, is maintained. This CO2 concentration works with a bicarbonate buffering system in the culture liquid to stabilize the medium’s pH between 7.2 and 7.4. If the pH shifts, cells can suffer stress or cease to grow.
The cells are sustained by cell culture media, a liquid formulation containing:
- Inorganic salts for osmotic balance.
- Amino acids and vitamins for protein synthesis and metabolic reactions.
- Energy sources like glucose.
- Growth factors and hormones, often from Fetal Bovine Serum (FBS), which stimulate cell division.
Cells grow in two primary formats. Traditional two-dimensional (2D) culture involves growing cells in a single layer (monolayer) on a flat plastic surface. Three-dimensional (3D) culture systems, such as scaffolds or gels, allow cells to interact in a configuration that more closely resembles natural tissue architecture.
Types of Cells Used
Researchers primarily use two distinct categories of cells: primary cells and established cell lines. Primary cells are isolated directly from a living tissue, such as a biopsy. Because they retain the biological characteristics of the original tissue, they are highly relevant for studying normal physiological function.
A limitation of primary cells is their finite lifespan, adhering to the Hayflick limit. After a certain number of divisions (typically 40 to 60), these cells enter senescence, stop dividing, and eventually die. This makes them challenging to maintain long-term and often necessitates fresh isolation for new experiments.
Established cell lines, in contrast, are cells that have been transformed to divide indefinitely (“immortalized”). These cells can be grown reliably for years, making them convenient and highly reproducible for large-scale studies. The HeLa cell line, derived from a cervical tumor in 1951, is a famous example used worldwide today.
The drawback of immortalized cell lines is that they often accumulate genetic changes, meaning they may not perfectly represent the behavior of normal cells. Researchers must choose between the physiological accuracy of short-lived primary cells and the robust, unlimited supply of established cell lines.
Standard Laboratory Techniques
Maintaining cell cultures requires diligent, standardized laboratory procedures to ensure cell health and experimental integrity. Preventing contamination by airborne microbes, fungi, or bacteria is achieved through aseptic technique. This involves performing all manipulations within a laminar flow hood, which maintains a constant flow of filtered air over the workspace.
Before and after procedures, the hood surfaces and all materials are wiped down with 70% ethanol to eliminate contaminants. Researchers wear gloves and limit movement to avoid disrupting the sterile airflow, protecting the cultures from environmental exposure.
Cultures require regular monitoring and feeding. Scientists routinely check cells under a microscope to evaluate their morphology and density, ensuring they are healthy and contamination-free. The culture medium is changed every few days to replenish depleted nutrients and remove accumulated metabolic waste products.
When cells cover the available surface area—a state called confluency—they must be split or subcultured (passaging). For adherent cells, an enzyme like trypsin is used to detach them from the plastic surface. The resulting cell suspension is then diluted into new flasks with fresh medium, allowing continued growth.
For long-term preservation, cells undergo cryopreservation to halt metabolic activity indefinitely. Cells are mixed with a cryoprotectant, such as dimethyl sulfoxide (DMSO), which prevents damaging ice crystals during freezing. Vials are cooled at a controlled rate before transfer to a liquid nitrogen storage tank, where the ultra-low temperature keeps the cells viable for decades.
Real-World Applications
Cell culture has significantly contributed to the development of pharmaceuticals and medical treatments. In drug discovery, cell cultures are the foundation of high-throughput screening (HTS). This method allows scientists to test thousands of potential drug compounds simultaneously, identifying molecules effective against target cells (like cancer cells) and those that cause toxicity in normal cells before expensive animal trials begin.
Cell culture technology is also fundamental to producing biological medicines and vaccines. Large-scale culture occurs in massive, sterile containers called bioreactors.
Bioreactor Production
Genetically modified cells are grown in bioreactors to produce therapeutic proteins, such as monoclonal antibodies used to treat autoimmune diseases and cancer. Many viral vaccines, including those for polio and viral vector vaccines, are also manufactured by growing the virus in specific cell lines within these bioreactors.
Regenerative Medicine
Cell culture drives the emerging field of regenerative medicine and tissue engineering. Scientists can culture a patient’s own skin cells to grow large, transplantable sheets of epidermis for skin grafts to treat severe burns. More complex applications use advanced techniques like 3D bioprinting to arrange multiple cell types in three-dimensional structures, paving the way for creating complex tissue models and replacement organs.