The foundation of modern biomedical research rests on the ability to grow living cells outside of their original host. This practice, known as cell culture, allows scientists to study biological processes, test new medicines, and produce therapeutic proteins in a controlled environment. These microscopic communities require careful maintenance to keep them healthy and actively growing. Cell passaging is the routine procedure that makes long-term cell culture possible, ensuring the cells remain viable for extended periods of study.
Defining the Practice of Cell Passaging
Cell passaging, also called subculturing or cell splitting, involves transferring a small portion of a crowded cell culture into a new vessel filled with fresh growth medium. This technique is necessary because cells in a confined space consume available nutrients and excrete metabolic waste products, which halts growth. The goal of passaging is to reduce cell density and restore the optimal environment for continued division.
The decision to split a culture is based on its confluency, the percentage of the culture dish surface covered by adherent cells. The ideal time to passage is when cells reach 70% to 90% confluency. Allowing cells to become too dense signals them to slow or stop proliferating entirely. Dividing the population and providing fresh surface area ensures the cells remain in their exponential growth phase, maximizing their metabolic activity for experiments.
The Mechanics of Splitting Cells
Detachment and Neutralization
The first step in passaging adherent cells is detaching them from the culture vessel using a gentle enzymatic solution. The enzyme trypsin is typically used to break down the proteins anchoring the cells to the surface. It is often combined with a chelating agent like EDTA to disrupt cell-to-cell connections. The enzyme solution is applied briefly, and the flask is agitated until the cells lift off, creating a suspension of individual cells. After detachment, the enzyme’s activity must be neutralized, usually by adding a serum-containing medium.
Counting and Dilution
A small sample of the cell suspension is then mixed with a stain, such as Trypan blue, to distinguish between living and dead cells. The total number of viable cells is counted using a specialized slide called a hemocytometer to determine the precise cell concentration. The final stage is the dilution and re-plating process into a new, sterile culture vessel. The volume transferred is determined by the required split ratio, such as 1:5 or 1:10. This ratio ensures the cells have adequate space and fresh resources to begin dividing again. The entire procedure must be performed inside a specialized, sterile environment, like a laminar flow hood, to prevent contamination from airborne microorganisms.
Ensuring Cell Health and Experimental Integrity
Preventing Senescence and Death
Consistent and timely cell passaging maintains the biological integrity and quality of the cell line. If cells remain crowded or starved for too long, they can enter a state of reduced growth known as senescence or begin to die off. Regular splitting prevents the buildup of growth-inhibiting signals and toxic byproducts. This keeps the cells in the healthy, actively dividing state necessary for accurate research.
Minimizing Genetic Drift
A concern in long-term culture is genetic drift, where repeated cycles of division unintentionally select for cells with subtle genetic changes. Over many passages, these selected cells may behave differently from the original population, leading to inconsistent experimental results. By tracking the passage number and limiting the number of times a cell line is split, researchers minimize this drift. This ensures that cells used in current experiments are genetically similar to those used previously.
Maintaining Doubling Time
Proper passaging is also necessary to maintain a consistent population doubling time, the time it takes for a cell population to double in number. If a cell line is split too infrequently, its doubling time can slow down, affecting the reliability of time-sensitive experiments. Standardizing the passaging ratio and schedule ensures that all experiments are conducted using cells in the same physiological state, which is required for reproducible scientific data.
Essential Roles in Biotechnology
Successful cell passaging is central to large-scale applications in the biotechnology and pharmaceutical industries.
- Vaccine Manufacturing: Continuous, healthy cell lines, such as Vero or HEK293 cells, are used as biological factories to grow the viruses needed to create both live-attenuated and inactivated vaccines. The constant availability of high-quality, low-passage cells is necessary to ensure a reliable and scalable supply of viral material.
- Therapeutic Protein Production: Passaging is central to producing biologics, which include monoclonal antibodies and certain hormones. Cell lines like Chinese Hamster Ovary (CHO) cells are repeatedly passaged and scaled up to enormous volumes in bioreactors to produce these complex molecules.
- Drug Development: Continuously passaged cell lines are used for high-throughput screening and toxicology testing. This allows researchers to quickly assess how tens of thousands of potential drug compounds affect human cells before proceeding to more complex models.
- Tissue Engineering: The entire field of regenerative medicine relies on the meticulous expansion and maintenance of various human cell types through careful passaging.