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Cell aggregation is most commonly caused by the presence of free DNA and cell debris released into the culture medium after cell lysis. The sticky nature of extracellular DNA promotes the aggregation of cells and cellular debris, resulting in the formation of larger cell clusters.
The following factors may contribute to cell lysis and the release of DNA into the culture medium:
1. Over-digestion: Excessive use of proteolytic enzymes (such as trypsin commonly used for cell dissociation) may damage cells and promote cell aggregation.
2. Environmental stress: Mechanical stress, repeated freeze–thaw cycles, and other environmental stresses can accelerate cell death and increase cell debris accumulation.
3. Tissue dissociation: Preparation of single-cell suspensions from primary tissues through chemical, mechanical, or enzymatic dissociation may cause partial cell disruption. During tissue dissociation, collagenase digestion is often required to degrade the extracellular matrix and release individual cells.
4. Overgrowth: When cells reach over-confluence, excessive accumulation of cell debris and extracellular DNA released from lysed cells may promote cell aggregation.
Preventive Measures
1. Use MaxACA according to the recommended protocol: MaxACA is designed as an anti-aggregation reagent for CHO, HEK293, insect cells, and vaccine cell lines in both batch and fed-batch culture processes.
2.Divalent cation chelation: Chelating agents such as citrate and EDTA can be used to remove calcium ions from the culture medium and reduce cell aggregation.
3. Cell density control: Before passaging, refer to cell line-specific literature or established cell characteristics to determine the recommended seeding density.
4. Handling practices: During cell dissociation, handle cells gently and use appropriate centrifugation conditions. Small aggregates can often be dispersed through gentle pipetting or mechanical trituration.
5. Mycoplasma contamination: Discard contaminated cell cultures, disinfect the cell culture room and incubators, and follow good laboratory practices (GLP) to minimize contamination risks.
