Subculturing adherent cells using trypsin-EDTA

  • Subculturing/passaging can be defined as preparation of fresh culture by transferring cells from an existing culture.
  • Subculturing is done by transferring a small amount 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.
  • Trypsin-EDTA method, also referred to as  trypsinization, is a most commonly used method for passaging adherent cells.
  • Trypsin-EDTA method of subculturing of a cell culture involves 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 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 (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 is 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 agents, sequesters divalent cations (e.g., Ca2+, Mg2+).
  • Trypsin disrupts cell-cell and cell substratum interactions by digesting proteins and EDTA weakened these interaction 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, inactivation of trypsin is an essential step in this method.
  • Usually trypsin is inactivated by adding 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 suggest that 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 containing  medium.