Cells form Clumps

When cells form clumps in culture, it can hinder accurate cell counting, lead to uneven cell distribution when passaging, and affect experimental outcomes. Here are some possible reasons for cell clumping and potential solutions:

  1. Cell type and characteristics: Some cell types, such as neuronal cells or certain cancer cell lines, have a natural tendency to form cell aggregates or clusters. For these cell types, clumping might be an inherent characteristic and may not necessarily indicate a problem.
  2. Incomplete dissociation during passaging: Inadequate trypsinization or mechanical dissociation can lead to incomplete cell detachment and clumping. To prevent this, ensure that the trypsin-EDTA solution is evenly distributed across the culture vessel, and incubate the cells for an appropriate amount of time to allow for complete detachment. Gently tapping the flask can also help to dislodge the cells.
  3. Trypsin inactivation: If trypsin is not adequately inactivated by adding an appropriate volume of complete growth medium after cell detachment, it can continue to digest cell surface proteins, promoting cell clumping. Ensure that enough complete medium is added to neutralize trypsin activity effectively.
  4. Cell damage or stress: Suboptimal culture conditions, such as extreme temperatures, incorrect CO₂ levels, or prolonged incubation times, can induce cell stress or damage, causing cells to aggregate. Ensure that the incubator is maintained at 37°C and 5% CO₂, and check the cells regularly to avoid overgrowth.
  5. Contamination: Bacterial or fungal contamination can cause changes in cell behavior, including clumping. If contamination is suspected, examine the culture medium for any turbidity, color changes, or floating particles. Contaminated cultures should be discarded, and proper aseptic techniques should be followed to prevent future contamination.
  6. Serum quality: The quality of the serum used in the culture medium can influence cell behavior. Low-quality or expired serum can cause cell clumping. Ensure that the serum is of high quality, and store it according to the supplier’s recommendations.
  7. Static charge: Plastic culture vessels can develop a static charge, causing cells to aggregate. To minimize the static charge, avoid rubbing the plastic surface and handle the culture vessels gently.

To reduce cell clumping, you can also try the following:

  • Gently pipette the cell suspension up and down several times during cell passaging to break apart cell aggregates.
  • Use cell strainers to remove clumps before seeding the cells in a new culture vessel.
  • Add an anti-clumping agent, such as Pluronic F-68, to the culture medium. However, be cautious when using additives, as they may affect cell behavior and experimental outcomes.

It is crucial to identify the cause of cell clumping and address it to maintain healthy cell cultures and ensure reliable experimental results.