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A Comprehensive Explanation of PBMC Clustering

After thawing and culturing cryopreserved PBMCs, cell aggregates sometimes form that cannot be dispersed by gentle pipetting.

At the smallest scale, cell aggregates can be observed under a microscope; at the largest scale, more prominent flocculent or mass-like clumps are visible to the naked eye.

 

Some cell types naturally exhibit a tendency to grow in clusters; as the seeding density increases, it is normal to observe small cell aggregates under the microscope. However, if visible clumps form that cannot be dispersed by pipetting, this warrants attention.

 

The main reasons for clumping of PBMCs after recovery are as follows:

 
 
 
 
 

1. During the cryopreservation and thawing of cells, external stress accelerates cell death; the “sticky” DNA molecules released from dead cells then bind neighboring cells together, resulting in the formation of cell aggregates.

2. Cryopreserved PBMCs typically contain variable amounts of platelets; when the platelet count is excessively high, platelet aggregation can lead to severe cell clumping.

3. If the PBMC seeding density is too high, resulting in an excessive cell density in the culture dish, this may also increase the tendency of cells to aggregate.

In light of the aforementioned causes, we recommend the following methods to alleviate clumping of PBMCs after thawing and culture.

 
 
 
 
 

Through experimental procedures, we observed that when PBMCs were seeded at different densities in 1640 complete medium, some of the cells at higher densities began to aggregate after approximately 1 hour, with aggregation becoming more pronounced by 2 hours. When the seeding density was below 3 million cells/mL, gentle pipetting could fully disperse any flocculent precipitates; however, at densities below 1 million cells/mL, cell clumping was virtually invisible to the naked eye. Therefore, we recommend maintaining a cell density of 1–2 million cells/mL during the culture of PBMCs following recovery.

In addition, the addition of deoxyribonuclease I (DNase I) to the sample can reduce DNA debris and cell clumps. Currently, numerous effective protocols are available for using DNase I to minimize cell clumping. We recommend adding DNase I solution to the cell suspension to a final concentration of 0.1 mg/mL (or 200 Kunitz units/mL), incubating at room temperature for 15 minutes, and then proceeding with downstream applications; however, if your sample is intended for genomic DNA extraction, this step should be handled with particular care.

EDTA is a non-enzymatic dissociant commonly prepared as a working solution in Ca2+- and Mg2+-free PBS. The generally accepted mechanism of action of EDTA is that certain tissues require Ca2+ and Mg2+ to maintain their structural integrity during survival. EDTA chelates these ions from the tissue microenvironment, forming stable complexes, thereby promoting cell–cell dissociation. Consequently, a 1 mM EDTA solution is usually sufficient to effectively prevent cell aggregation.

In addition, filtering the resuscitated cells through a 37-μm cell strainer before seeding can also help alleviate cell clumping.

A very small number of volunteers may experience extensive cell clumping, resulting in pellets that cannot be resuspended. If DNase I fails to achieve complete digestion, this may be due to the action of cell–cell adhesion molecules on the cell surface; in such cases, try trypsin digestion.

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