Trypsinization is a laboratory procedure designed to release adherent cells from culture vessel surfaces. This process prepares cells for various downstream applications, including subculturing, cell counting, or setting up new experiments. It provides a method to obtain a single-cell suspension from a confluent cell monolayer.
The Purpose of Trypsin in Cell Culture
Many cell types are adherent, meaning they require a surface to attach to for growth and proliferation. These cells anchor themselves to the culture flask and to each other through specialized proteins known as cell adhesion molecules (CAMs) and by interacting with components of the extracellular matrix. These proteins maintain the cell’s attachment and structural integrity within the culture.
To detach these cells, a specific enzyme called trypsin is introduced into the culture environment. Trypsin is a type of protease, an enzyme that functions by breaking down proteins. Its purpose in cell culture is to specifically cleave the peptide bonds within these cell adhesion proteins, effectively dismantling the connections that bind cells to the flask surface and to neighboring cells. This enzymatic action leads to the gentle release of cells, allowing them to float freely in the culture medium.
The Step-by-Step Trypsinization Protocol
Trypsinization begins by removing the old growth medium from the cell culture vessel using aspiration. This step is important because residual serum in the medium contains protease inhibitors that would otherwise neutralize the trypsin, preventing it from detaching the cells.
Following media removal, the cell layer is rinsed with Phosphate-Buffered Saline (PBS). This wash step ensures the removal of any remaining serum proteins, creating an optimal environment for the trypsin to act efficiently on the cell adhesion molecules. After rinsing, the PBS is aspirated from the vessel.
A thin layer of trypsin-EDTA solution is then added to cover the cell monolayer. The volume used is typically just enough to coat the bottom surface. The culture vessel is then placed into an incubator, usually at 37°C, for a short period, commonly ranging from 2 to 5 minutes.
During this incubation, the trypsin begins to digest the adhesion proteins. Cell detachment is confirmed by observing the culture under a microscope, where detached cells will appear rounded and begin to float. A gentle tap on the side of the flask can also help dislodge any loosely attached cells.
Once sufficient detachment is observed, fresh, serum-containing culture medium is added to the vessel. This step is important as the serum components effectively neutralize the trypsin’s enzymatic activity, preventing prolonged exposure that could damage the cells’ surface proteins. The cells are then gently pipetted up and down to create a homogeneous single-cell suspension.
Key Variables and Troubleshooting
Over-trypsinization occurs when cells are exposed to the enzyme for too long or at too high a concentration. This can lead to excessive digestion of cell surface proteins, potentially causing cell damage or reduced viability.
Conversely, under-trypsinization results in incomplete cell detachment, leaving many cells still adhered to the flask or forming large clumps. This incomplete dissociation makes it difficult to obtain an accurate cell count and can negatively impact downstream experiments. The presence of residual serum from an insufficient wash can also contribute to under-trypsinization by inhibiting the enzyme.
Ethylenediaminetetraacetic acid (EDTA) is included with trypsin in dissociation solutions. EDTA functions by chelating, or binding, calcium ions, which are often required for the integrity of cell-to-cell junctions and some cell adhesion molecules. By removing these calcium ions, EDTA helps disrupt cell-to-cell connections and aids the overall detachment process, making the trypsin more effective. The optimal incubation time and concentration of trypsin can vary considerably depending on the specific cell type being cultured, requiring some optimization for new cell lines.
Alternatives to Trypsin
Gentler enzymatic alternatives to trypsin include Accutase and TrypLE, which are often preferred due to their milder action and sometimes broader range of effective temperatures. These enzymes cause less cellular stress and maintain higher cell viability.
A cell scraper can physically dislodge robust and firmly adherent cells from the culture surface. This mechanical method is quick but may cause more physical stress to the cells. Specialized non-enzymatic dissociation buffers are also available, which work by altering the ionic environment or chelating ions to disrupt cell adhesion without using proteolytic enzymes.