Common Cell Culture Problems: Cell Clumping

Cell clumping: an overview

Cells in suspension may attach to one another and form clumps for a variety of reasons. The most common cause of cell clumping is the presence of free DNA and cell debris in the culture medium, which occurs following cell lysis. The sticky nature of DNA causes cells and other debris to aggregate into large clumps. Below are some causes of cell lysis and DNA release into culture media.

  • Overdigestion: excessive treatment with proteolytic enzymes— like trypsin commonly used for cell detachment—can cause cells to clump
  • Environmental stress: mechanical force, repeated freeze/thaw cycles and other environmental stresses can accelerate cell death, an early symptom of which may be cell-cell adhesion.
  • Tissue disaggregation: preparing a single-cell suspension from primary tissue via chemical, mechanical or enzymatic protocols can lead to some cell rupture. Collagenase is typically needed to digest the extracellular matrix when creating a single-cell suspension from tissue.
  • Overgrowth: When cells reach confluency, there is excessive buildup of cell debris and free DNA from cell lysis.

Cells in suspension may attach to one another and form clumps for a variety of reasons
Troubleshooting

Cell clumping reduces access to critical nutrients and, as a result, hinders overall cell growth. Additionally, cell clumps compromise downstream assays that require clean preparation of single cells (e.g., flow cytometry). Depending on the underlying cause of clumping, we suggest solutions for its prevention in the table below.
 

 

 

Cause of clumping Solution
Cell lysis and DNA release DNase I can be used during tissue disaggregation and regular cell culturing to prevent clumping in a variety of cell types. Note: DNase I is maximally active in the presence of bivalent ions (Mg2+, Ca2+)
Bivalent cations Chelators such as citrate and EDTA can be used to remove calcium from cell culture media. Serum-free, Ca2+/Mg2+-free balanced salt solution may also be used.
Cell density Refer to cell line literature or published characteristics for recommended cell densities prior to passage. 
Handling Handle cells with care and use the appropriate centrifuge settings when pelleting. Minor clumping can be resolved by trituration, or gentle up and down pipetting of cells.
Mycoplasma contamination Discard contaminated cells and disinfect culture hood and cell culture incubator. Follow good laboratory practices to avoid contamination.