Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues

Abstract

We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method.

Figure 1. Schematic representations of the MCM and BAM.

As depicted on the left with the MCM, single cell suspensions are first layered over Ficoll to obtain mononuclear cells, and then the latter are depleted of T- and B-cells with CD3 and CD19 microbeads, respectively, and magnetic depletion columns. As depicted on the right with the BAM, single cell suspensions are first incubated with allogeneic leukocyte-depleted RBC and the bivalent antibody cocktail followed by Ficoll density centrifugation separation. Enriched target mononuclear cells are then obtained from the Ficoll/supernatant interface. We thank Ashley Smith for her illustrative assistance with this figure.

Figure 2. Comparison of the MCM and BAM.

(a) Flow cytometry of pre-enriched (left column) versus MCM-enriched (middle column) versus BAM-enriched (right column) ILC populations from a representative donor. (b) Pre and post enrichment composition comparison (left) and assessment of total yields of ILC3 and NK cells per gram of tonsil (right). non- significant (ns) (n = 5). (c) Cytokine production by enriched ILC3 and NK cells following MCM and BAM enrichment. White boxes indicate unstimulated, black boxes indicate stimulated samples (n = 6). (d) CD8+ T cell and CD14+ monocyte enrichments from a pediatric tonsil using the corresponding bivalent antibody cocktails. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (b,c) indicates data from 2 independent experiments with at least two donors. Linear mixed effects models were used for statistical comparison and adjusted for multiple comparisons using Holm’s procedure. Error bars indicate s.e.m.