Understanding Cell Passaging
Cells grown in a laboratory setting require careful management to ensure their continued health and proliferation. As cells divide, their numbers increase, leading to a crowded environment. This overpopulation depletes nutrients, accumulates waste, and reduces growth rate and viability. Many adherent cell types exhibit contact inhibition, where growth slows or stops when cells touch. This poses a challenge in cell culture, where continuous growth is desired.
Cell passaging, also known as subculturing, involves transferring a portion of an existing cell culture into a new vessel with fresh growth medium. This dilution provides space, replenishes nutrients, and removes waste. Regular passaging ensures cells remain in an active growth phase, maintaining their morphology, function, and genetic stability.
While the goal of passaging remains consistent, techniques vary by cell type. Adherent cells, which attach to a surface, require enzymatic or mechanical detachment before transfer. Suspension cells, which float freely, can be directly diluted and re-seeded without detachment.
Preparation for Passaging
Successful cell passaging requires meticulous preparation of the workspace and reagents. Aseptic technique is important to prevent contamination. This involves working within a laminar flow hood, which provides a sterile air environment, and sterilizing surfaces with 70% ethanol.
All materials contacting cells must be sterile, including culture vessels, pipettes, and solutions. Essential reagents include complete growth medium and phosphate-buffered saline (PBS). For adherent cells, a detachment solution like trypsin-EDTA is necessary.
Additional equipment includes an incubator for optimal temperature and CO2, and a centrifuge to pellet cells. A cell counter is important for determining cell concentration. Personal protective equipment, including sterile gloves, a lab coat, and eye protection, should always be worn.
The Passaging Procedure
The process for adherent cells begins with observing confluency under a microscope. Once cells reach 70% to 90% confluency, they are ready for subculturing. The old growth medium is then aspirated and discarded.
Next, the cell monolayer is washed with sterile PBS to remove residual medium and serum, as serum inhibits detachment solutions. A small volume of detachment solution, such as 0.25% trypsin-EDTA, is added to cover the cell layer. The flask is incubated for 2 to 5 minutes at 37°C, allowing the enzyme to break down proteins.
Once cells detach and round up, fresh growth medium is added. The serum in the medium neutralizes the trypsin, preventing cell damage. Cells are gently pipetted to break clumps and create a single-cell suspension. This suspension is transferred to a sterile centrifuge tube.
The cell suspension is centrifuged at 100-200 x g for 3-5 minutes to pellet the cells. The supernatant is carefully aspirated and discarded. The cell pellet is resuspended in a known volume of fresh growth medium for accurate cell counting.
A small aliquot of the cell suspension is taken for cell counting. Based on the desired seeding density, the appropriate volume is added to new culture vessels with fresh growth medium. These cultures are placed back into the incubator. Suspension cells are simply diluted into fresh medium without detachment or centrifugation.
Post-Passaging Care and Troubleshooting
After passaging, monitoring ensures recovery and growth. Within 24 hours, adherent cells should be observed to confirm reattachment and spreading. Regular checks for morphology, growth rate, and signs of contamination are important.
Common issues include poor cell attachment or low viability, often from over-trypsinization or insufficient neutralization. Adjusting trypsin incubation or ensuring adequate serum can mitigate these. Contamination (bacterial, fungal, mycoplasma) is another challenge. Early detection and immediate isolation or disposal of contaminated cultures are important.
The “split ratio” determines the dilution factor. For example, a 1:4 split means one part cell suspension to four parts total volume. This ratio influences passaging frequency; a higher dilution means cells take longer to reach confluency. Proper split ratios ensure cells remain in their optimal growth phase.
For long-term preservation, cryopreservation is used. This involves freezing cells in a cryoprotective agent like dimethyl sulfoxide (DMSO), which prevents ice crystal formation. Storing cells in liquid nitrogen maintains valuable cell lines for extended periods, providing a reliable backup.