Subculturing adherent cells using trypsin-EDTA

• Subculturing/passaging can be defined as the preparation of fresh culture by transferring cells from an existing culture.
• Subculturing is done by transferring a small number of cells (usually 1/3 to 1/10 cells of the existing semi confluent culture) from an existing culture dish to a new culture dish containing fresh growth medium.
• The Trypsin-EDTA method, also referred to as trypsinization, is the most commonly used method for passaging adherent cells.
• Trypsin-EDTA method of subculturing of cell culture involves the following steps.
  • Washing of cells with Ca2+- free  and Mg2+ – free PBS
  • Trypsin – EDTA treatment
  • Inactivation of trypsin
  • Preparation of fresh culture dish from the cell suspension

Washing of cells with Ca2+– free  and Mg2+ – free PBS

• This step involves removing the old culture medium from the culture dish, followed by washing with PBS which is free of Ca2+- free and Mg2+ ions.
• This step is intended to remove divalent cations and serum-containing medium from the cell culture. Serum in culture medium has trypsin inactivating activity (trypsin inhibitors) and divalent cations strengthen the cell-cell and cell-substratum interaction by stabilizing them.

Trypsin – EDTA treatment

• This step involves brief incubation (a few minutes, varies from 1 – 5 min for most cell lines) of adherent cell culture with Trypsin EDTA solution at 37°C.
• This step is intended to disrupt both cell-cell and cell-substratum interactions. These interactions are mediated by various proteins (cadherins, integrins, extracellular matrix proteins like fibronectin, vitronectin) and their interactions are strengthen by divalent cations (e.g., Fibronectin-integrin interactions are promoted by Ca2+).
• Trypsin, a serine protease, cleaves the polypeptide at C-terminal of lysine or arginine amino acid, except when either is followed by proline. Trypsin shows optimal activity at 37°C and pH 8.0.
• EDTA, a chelating agent, sequesters divalent cations (e.g., Ca2+, Mg2+).
• Trypsin disrupts cell-cell and cell substratum interactions by digesting proteins and EDTA weakens these interactions by chelating divalent cations.

Inactivation of trypsin

• Since trypsin digests proteins, excessive trypsin treatment can cause high cell death by disrupting the plasma membrane. Therefore, the inactivation of trypsin is an essential step in this method.
• Usually, trypsin is inactivated by adding a serum-containing growth medium. In specific conditions where serum can not be added, other trypsin inhibitors, e.g., Soybean trypsin inhibitor, are used.

Preparation of fresh culture dish from the cell suspension

• This step aimed to distribute cell suspension into fresh culture dishes. Usually, when the purpose is to maintain a culture in a healthy state, a rough estimation of cells called split ratio is used to distribute cells to fresh culture dishes. 
• Split ratio suggests how many culture dishes can be prepared from the existing culture dish. For example, you can prepare 4 – 6 culture dishes if a recommended split ratio is 1:4 to 1:6  for a specific cell line. Alternatively, calls can be counted and a specified number of cells can be transferred to another fresh flask.