Passaging Cells: A Comprehensive How‑to for Culturing Success

Culturing cells is fundamental in biological research and biotechnology, offering insights into cellular functions and enabling advancements in medical science. Successful cell passaging—transferring cells from one culture vessel to another—is crucial for maintaining healthy cultures and ensuring accurate experimental results.

The following guide delves into essential aspects of cell passaging, offering practical advice to enhance your culturing techniques.

Setup And Equipment

Establishing an optimal environment for cell passaging begins with selecting the appropriate equipment and ensuring a well-organized workspace. A sterile laminar flow hood is essential, providing a contamination-free zone where cells can be handled safely. The hood should be regularly cleaned with 70% ethanol and UV-sterilized to maintain its sterility.

The choice of culture vessels varies depending on the type of cells being cultured. T-flasks, multi-well plates, and culture dishes are commonly used, each offering distinct advantages. T-flasks are ideal for scaling up cultures due to their larger surface area, while multi-well plates allow for high-throughput screening. The material of these vessels, often polystyrene treated for cell attachment, should be compatible with the specific cell line to ensure optimal growth conditions.

The selection of culture media is critical, as it provides the necessary nutrients and growth factors for cell proliferation. Media such as DMEM, RPMI-1640, or MEM are frequently used, each tailored to support different cell types. Supplements like fetal bovine serum (FBS) are often added to enhance growth, though the concentration must be carefully controlled to avoid unwanted differentiation or growth inhibition. It is advisable to source media and supplements from reputable suppliers and verify their quality through batch testing.

A reliable incubator is necessary to maintain the appropriate temperature, humidity, and CO2 levels, typically set at 37°C, 95% humidity, and 5% CO2 for mammalian cells. Regular calibration and maintenance of the incubator are vital to prevent fluctuations that could stress the cells. A microscope equipped with phase contrast is invaluable for monitoring cell morphology and confluency, providing real-time feedback on the health and density of the culture.

Steps For Passaging

Passaging cells, often referred to as subculturing, requires timing, technique, and precision. The process begins with observing the cells under a phase contrast microscope to assess their confluency, a measure of how much of the culture vessel’s surface is covered by cells. Generally, cells are passaged when they reach 70-90% confluency, ensuring they are proliferating but not overcrowded.

Once the decision to passage is made, the first step is to aspirate the old media, which contains waste products and depleted nutrients. This is done carefully to avoid disturbing the cell monolayer. Washing the cells with a balanced salt solution like PBS helps remove residual serum, which can inhibit subsequent detachment. Detachment itself can be achieved through various methods, with trypsinization being the most common for adherent cells. Trypsin, an enzyme that cleaves cell adhesion proteins, is added just enough to cover the cell layer, and the flask is then incubated briefly to facilitate detachment. The timing of this incubation is critical; over-trypsinization can damage cells, while insufficient exposure leaves cells attached.

Once detached, the cells are resuspended in fresh culture medium to neutralize the trypsin. This step is essential as it halts enzymatic activity that could otherwise harm the cells. The cell suspension is then gently pipetted to ensure a single-cell suspension, which is crucial for even distribution in new culture vessels. Cell counting, often performed using a hemocytometer or automated cell counter, follows to determine the concentration of cells. This allows for precise seeding in new vessels, maintaining consistent cell density across passages.

In transferring the cells to new culture vessels, it is important to use the appropriate seeding density, which varies with cell type but typically ranges between 1×10^4 to 1×10^6 cells per cm². The freshly seeded cultures are then placed back into the incubator, where they resume growth under controlled conditions. It is advisable to monitor the cultures regularly, checking for contamination and ensuring the cells are adhering and spreading as expected.

Enzymatic, Non-Enzymatic, And Mechanical Methods

Cell detachment varies depending on the cell type and desired outcome. Enzymatic methods, such as trypsin, are widely used for their efficiency in disrupting cell adhesion proteins. Trypsin, a serine protease, is favored for its specificity, but its use requires careful control to prevent cellular damage. Alternatives like collagenase or dispase offer gentler detachment by targeting specific extracellular matrix components, preserving cell integrity.

Non-enzymatic methods provide alternatives when enzymatic detachment may compromise cell viability. These methods often contain chelating agents that disrupt calcium-dependent cell adhesion. They are useful for sensitive cell lines or when maintaining surface antigens is crucial. Though generally less harsh, non-enzymatic methods may require longer incubation times or additional mechanical assistance.

Mechanical methods, including scraping and pipetting, offer another avenue for cell detachment. Cell scrapers can physically dislodge cells from the substrate, though this approach is usually reserved for cells that are resilient to mechanical stress. Pipetting involves gently washing the cell layer with media to encourage detachment. While mechanical methods can be effective, they often result in higher cell stress and potential damage.

Adherent Versus Suspension Cultures

Cell culture techniques often revolve around two primary types: adherent and suspension cultures. Adherent cultures, also known as anchorage-dependent cells, require a surface to attach and grow, mimicking the in vivo environment of tissues. Common examples include fibroblasts and epithelial cells. The choice of substrate is crucial, with options ranging from standard polystyrene treated for cell attachment to specialized coatings like collagen or fibronectin.

Suspension cultures consist of cells that exist in a free-floating state, such as hematopoietic cells. These cultures offer scalability, as they can grow in bioreactors to produce large quantities of cells or cellular products. Unlike adherent cells, suspension cells do not require trypsinization for passaging, simplifying the process and reducing the risk of enzymatic damage. However, maintaining homogeneous distribution and preventing cell aggregation can be challenging.

Safety Considerations

Ensuring safety in cell culture practices is paramount to protect both the researcher and the integrity of the experiments. Culturing cells involves handling biological materials that may carry risks of contamination or infection. Adhering to biosafety guidelines, such as those outlined by the CDC or WHO, is fundamental. Personal protective equipment (PPE), including gloves, lab coats, and eye protection, should be worn at all times. Proper waste disposal procedures must be followed to mitigate environmental contamination risks.

The sterility of the work environment is another critical aspect. Regular cleaning and decontamination of surfaces with ethanol and other disinfectants help maintain a contamination-free workspace. The use of sterile techniques minimizes the risk of introducing contaminants. Cross-contamination between different cell lines is a significant concern, as it can lead to misleading experimental results. To avoid this, work with only one cell line at a time, and thoroughly clean equipment between uses. Regular testing for mycoplasma and other contaminants is advisable. Maintaining detailed records of cell line provenance, passage number, and any manipulations performed is essential for traceability and reproducibility.