Passaging/Subculturing Methods for Cells

• Several methods have been developed for passaging cells. Each method has its own advantages and drawbacks. Always check the cell line instruction manual and relevant literature for the optimal procedure.
• Most passaging methods aim to obtain single-cell suspension by breaking cell-substratum and cell-cell contacts. The single-cell suspension is further diluted by the addition of fresh growth medium and allowed to grow in its optimal growth environment.
• Depending on the experimental requirement, type and growth mode of cell culture, and nature of the cell line, one should choose a particular method.
• Depending on growth mode, cell culture can be adherent, semi-adherent, and non-adherent. Since nonadherent cells grow in suspension, such culture can be passaged essentially by diluting the existing cell culture. In contrast, semi-adherent and firmly adherent cells need to be detached from the substratum for passaging.
• Several methods have been developed for passaging cell culture. These methods can be broadly classified into three types based on the nature of treatment used to dissociate cells.
  • Enzymatic methods (e.g., Trypsin, Collagenase, Dispase)
  • Mechanical methods (Scraping by rubber policeman, Dislodging cells by vigorous shaking)
  • Treatment with chelators (e.g., EDTA or EGTA treatment)
• The most commonly used method, the Trypsin-EDTA method for all regular adherent cell cultures, combines both enzymatic and chelator treatment.

Enzymatic treatments (e.g., Trypsin, Collagenase, Dispase)

• Enzymatic methods are routinely used in most cell culture laboratories for passaging adherent cells.
• A brief treatment of cells with enzyme solution and subsequent gentle pipetting results in single-cell suspension.
• Enzymatic methods are very efficient at disrupting both cell-substratum as well as cell-cell contacts, leading to a single-cell suspension. Cell number can be determined easily in single-cell suspension, therefore, enzymatic methods are valuable for those experiments where accurate cell number is one of the requirements of the experimental procedure. However, enzymatic treatments result in the digestion and cleavage of cell surface proteins (e.g., receptors), therefore, not suitable for experiments which aim to analyze cell surface proteins.
• The sensitivity of cell lines for a particular enzymatic method may differ. Therefore, one needs to standardize treatment conditions. Overtreatment with enzyme solution can lead to cell death, whereas, under treatment can cause inefficient detachment of cells and lead to cell clumps.
• Often inhibitors (e.g., serum, soybean trypsin inhibitor, etc.) are added to stop the enzyme action.

Mechanical means (Scraping by rubber policeman, Dislodging semi-adherent cells by vigorous shaking)

• Mechanical means involved disrupting cell-substratum interaction by mechanical force like scraping using rubber policeman or vigorously tapping the culture dish or extensively pipetting culture medium over the cell layer.
• This method often leads to cell death and is not recommended for tightly adherent cells.
• Cell clumps are often observed therefore determining cell number using haemocytometer counting is difficult. However, this method is suitable for semi-adherent cells.

Treatment with chelators (e.g., EDTA or EGTA treatment)

• Chelators sequester the divalent cations, e.g., Ca2+ or Mg2+, which are required for adhesion of the cell to substratum as well as cell-cell contacts.
• This method takes a long-time incubation with a chelator solution. Most cell lines partially lose their contact with the substratum and subsequently extensive pipetting or tapping of culture dishes detaches cells from the substratum.