Cell culture, or tissue culture, is the process of growing cells outside their natural organism (in vitro) by providing a highly controlled, artificial environment that mimics conditions found inside the body. Scientists utilize this technique extensively for fundamental research, drug testing, and manufacturing complex biological products like vaccines and therapeutic proteins. Success requires supplying the correct nutrients and maintaining an environment free from contamination.
Essential Infrastructure and Sterility
The physical environment for cell culture must be meticulously controlled to support cell life and prevent contamination. Aseptic technique is the paramount concern, involving procedures designed to create a barrier between the cultured cells and environmental microorganisms. This technique relies on specialized equipment and careful handling practices.
Working surfaces are managed within a Laminar Flow Hood, or biological safety cabinet, which continuously filters air to provide a sterile workspace. The hood maintains a constant, unidirectional flow of clean air over the working area, acting as a barrier against airborne microbes. To maintain sterility, the interior of the hood and all materials must be wiped down with a disinfectant, typically 70% ethanol, before and after use.
Once prepared, cell cultures are placed inside a CO2 Incubator, which provides the precise environmental conditions required for growth. This chamber maintains a constant temperature, usually 37°C for mammalian cells, matching internal body temperature. The incubator also regulates humidity and maintains a specific concentration of carbon dioxide (typically 5%) necessary to stabilize the pH of the growth medium via a bicarbonate buffer system.
Sourcing the Biological Material
Before growth begins, the biological starting material must be selected, falling into two main categories: primary cells and established cell lines. Primary cells are isolated directly from living tissue, such as a biopsy or surgical sample, and retain the characteristics and functions of their original tissue. While they provide highly relevant data because they mimic the body’s environment, they have a finite lifespan and divide only a limited number of times in culture.
In contrast, established cell lines are cells modified to proliferate indefinitely, making them effectively immortal. They are easier to work with, can be maintained for long periods, and offer consistency between experiments, which is beneficial for large-scale screening and manufacturing. However, cell lines may accumulate genetic changes over time and can differ in function from the original tissue, making them less representative of the body’s natural state.
Cells are often stored long-term through cryopreservation, a process where they are carefully frozen in a cryoprotective agent like dimethyl sulfoxide (DMSO) and stored in liquid nitrogen vapor to halt metabolic processes. When a new culture is needed, cryovials are rapidly thawed in a warm water bath and immediately transferred to fresh medium.
Crafting the Perfect Growth Medium
The growth medium is the liquid life-support system providing all nutritional and chemical requirements for cells to survive and multiply in vitro. The foundation is a basal nutrient mixture containing essential components like inorganic salts, amino acids, and vitamins. Inorganic salts, such as sodium and potassium, maintain the correct osmotic balance and support cellular functions.
A primary energy source, most often glucose, is supplied to fuel the cell’s metabolism. Amino acids are also supplied as the building blocks for protein synthesis, including essential amino acids the cells cannot produce themselves. To mimic the natural environment, the medium is typically supplemented with Fetal Bovine Serum (FBS).
FBS supplies a complex mixture of hormones, growth factors, and binding proteins that stimulate cell growth and adhesion. The medium also contains a pH indicator, usually phenol red, which allows for visual monitoring of the culture’s health. If the cells are growing too rapidly, they produce acidic metabolic byproducts, causing the medium to turn yellow, which signals a need to change the medium and replenish the environment.
Long-Term Culture Maintenance
Maintaining a healthy, long-term cell culture requires routine monitoring and a procedure called subculturing, or passaging. This is performed when cells become too dense, either covering the available surface area (confluency) or exhausting the medium’s nutrients. Overgrowth stresses the cells and reduces viability, requiring them to be physically separated and diluted into new vessels.
For adherent cells (those attached to the surface), a detaching agent like the enzyme trypsin is used to gently release them from the flask. The detached cells are then counted to determine viability and density. A small, precise number are transferred to a new culture vessel containing fresh growth medium, ensuring the cells remain in the logarithmic growth phase where they are most active.
Regular visual inspection under a microscope is performed to check cell morphology and monitor for contamination. Microbial contaminants (bacteria, yeast, or mold) appear as foreign particles or cloudy patches in the medium, often accompanied by a rapid color change due to excessive acid production. If contamination is suspected, the affected culture must be immediately discarded to prevent spreading to other healthy cultures.