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Cell Culture Dissociation Techniques

Cell culture dissociation is a critical step in cell culture maintenance. It involves detaching cells from their growth surface to enable subculturing or harvesting. This section outlines two main methods: using enzyme-free dissociation buffers and using enzymatic reagents.

1.       Using Enzyme-Free Cell Dissociation Buffers

This method is gentle and maintains cellular integrity without the use of enzymes:

  1. Preparation
    • Ensure all reagents are warmed to 37°C before use to avoid shock to the cells.
  2. Removal of Growth Medium:
    • Discard the old growth medium from the culture vessel.
  3. Rinsing
    • Rinse cell monolayers with 5 ml of calcium and magnesium-free PBS per T75 flask or 100 mm dish.
    • Gently rock the vessel for 30 to 60 seconds at room temperature.
    • Aspirate and discard the rinse solution.
    • Repeat this rinsing step once more.
  4. Dissociation
    • Add approximately 5 ml of enzyme-free Cell Dissociation Buffer to the vessel.
    • Gently rock at room temperature for 1 to 2 minutes and check for dissociation under a microscope.
    • Tap the flask or dish against the hand to dislodge the cells if necessary.
    • If cells are adherent, allow them to sit at room temperature for an additional 2 to 5 minutes, and tap again if needed, using more dissociation buffer.
    • Once cells are detached, add at least 5 ml of complete growth medium to neutralize the dissociation buffer and resuspend the cells.
  5. Viability Check
    • Monitor cell viability during subculturing, ensuring it remains above 90%.

2.       Using Other Reagents for Dissociation

This method allows for the use of various dissociation reagents:

  1. Removal of Used Medium
    • Discard the old media from the culture vessel.
  2. Washing Cells
    • Wash the cells with a balanced salt solution free from calcium and magnesium, or use EDTA.
    • Add the wash solution gently to the side opposite the cells and rock the vessel for 1 to 2 minutes before discarding the wash.
  3. Dissociation
    • Apply 2 to 3 ml of the chosen dissociation solution per 25 cm² of growth surface, ensuring coverage of the cell sheet.
    • Incubate the vessel at 37°C and rock gently. Dissociation typically occurs within 5 to 15 minutes, depending on the cell line.
    • For stubborn cells, tapping the vessel can expedite the process.
    • Observe the cells carefully to prevent overexposure and potential damage.
  4. Harvesting Cells
    • Once cells are fully detached, allow them to collect at the bottom of the vessel by standing it upright.
    • Add complete media, then disperse and collect the cells by pipetting over the cell layer.
    • Count and proceed with subculturing.

In both methods, it's important to determine the optimal conditions for each cell line through empirical observation. The process should preserve cell viability, which should be routinely checked to exceed 90% during subculturing. These procedures provide a foundation for researchers to adapt based on the specific requirements and characteristics of their cell lines.

3.      Overview of Techniques for Subculturing Adherent Cells

Effective subculturing of adherent cells requires their detachment from the culture vessel. Various methods are employed, each suited for different cell types and conditions. When selecting a dissociation method, it is crucial to consider the specific needs of the cell line and the goals of the experiment. Regular monitoring of cell viability, with an aim for greater than 90% at the time of subculturing, is essential for the continued health and productivity of the culture. Here is an overview of these procedures:


Dissociation Agent

Typical Applications

Mechanical Shake-off

Gentle or vigorous agitation by shaking or pipetting

Used for cells that are loosely adherent or in the mitotic phase.

Mechanical Scraping

Physical tool such as a cell scraper

Suitable for protease-sensitive cells, although it can be harsh and potentially damaging.

Enzymatic Treatment

Trypsin solution

Effective for cells that adhere strongly to the culture vessel.

Enzymatic Treatment

Trypsin + Collagenase

Ideal for dense cell cultures or those with multilayered growth, particularly fibroblasts.

Enzymatic Treatment

Dispase enzyme

Allows for the removal of epidermal cells in intact sheets, maintaining cell-to-cell connections.

Enzymatic Treatment

TrypLE™ enzyme

A strong dissociation option for robustly adherent cells and suitable for protocols requiring animal origin-free reagents.

Enzymatic Treatment


A gentle alternative to trypsin, effective for a wide variety of cell types, including stem cells and primary cells.

Enzymatic Treatment

Trypsin + EDTA

A combination used to enhance cell detachment by chelating divalent cations, facilitating enzyme activity.


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