Cell culture is the process of growing and maintaining cells outside of their natural organism in a controlled laboratory setting. This technique moves cells from a complex living system to a simplified, highly managed environment, consisting of a nutrient-rich medium, controlled temperature, and specific gas atmosphere. Cell culture is a foundational tool for biomedical research, used for disease modeling, drug toxicity testing, and developing cell-based therapies. Obtaining a population of viable, healthy cells is the fundamental first step required to begin any experiment.
Categorizing Cell Sources: Primary Versus Continuous Lines
Cells taken directly from a living organism, such as a biopsy or organ, are known as primary cells. These cells are the most physiologically relevant because they closely mirror the characteristics of the tissue they came from. Primary cells have a finite lifespan, meaning they can only divide a limited number of times before they cease proliferation (the Hayflick limit).
In contrast, continuous or immortalized cell lines are cultures that proliferate indefinitely. This immortality is the result of a genetic mutation or modification that bypasses the Hayflick limit. These established cell lines are easy to handle, highly reproducible, and are routinely stored for long-term use in specialized facilities called cell banks.
Harvesting Cells from Living Tissue
Acquiring primary cells requires a multi-step process to break down solid tissue into a suspension of individual cells. The procedure begins with aseptic technique and rapid transport of the tissue sample to minimize microbial contamination and cell death. Once in the lab, the tissue is subjected to mechanical dissociation, involving physically cutting or mincing the sample into small fragments to increase the surface area available for the next step.
This mechanical disruption is followed by enzymatic digestion, which dissolves the extracellular matrix that holds the cells together. Researchers use enzymes such as trypsin, a protease that breaks down proteins responsible for cell-to-cell adhesion. Another common enzyme is collagenase, which is used to cleave collagen, a major component of connective tissue. The tissue fragments are incubated with the enzyme solution, often at 37°C, for a specific period to release the cells without causing excessive damage.
To complete the harvest, the resulting suspension is gently pipetted (trituration) to ensure the tissue is completely dispersed into single cells. The mixture must then be purified. Centrifugation is employed to separate the heavier cells, which form a compact pellet, from the lighter supernatant containing the waste material. The supernatant is aspirated, and the cell pellet is resuspended in fresh culture medium, ready to be plated in a flask for the initial culture phase.
Obtaining and Preparing Established Cell Lines
Established cell lines are obtained from long-term storage in liquid nitrogen (cryopreservation), which keeps the cells metabolically inert at temperatures below -135°C. The first step is to retrieve the cryovial and thaw it rapidly, usually by plunging it into a 37°C water bath. This quick warming maximizes cell viability by preventing the formation of damaging ice crystals.
The cryopreservation medium contains a cryoprotective agent, commonly Dimethyl Sulfoxide (DMSO), which prevents cellular dehydration and ice formation during freezing. Since DMSO is toxic, the thawed cell suspension is immediately transferred to a tube containing pre-warmed fresh culture medium to dilute the concentration.
The diluted cell suspension is then subjected to low-speed centrifugation to pellet the cells. The supernatant containing the diluted DMSO is aspirated and discarded, and the cells are resuspended in a final volume of fresh, complete growth medium. The cells are plated into a culture flask at a high density to promote recovery and allow them to begin proliferating. Before use, the line’s identity is verified, often using Short Tandem Repeat (STR) profiling, and checked for microbial contaminants like Mycoplasma.