Localized Cell-Surface Sampling of a Secreted Factor Using Cell-Targeting Beads

Abstract

Intercellular communication through the secretion of soluble factors plays a vital role in a wide range of biological processes (e.g., homeostasis, immune response), yet identification and quantification of many of these factors can be challenging due to their degradation or sequestration in cell culture media prior to analysis. Here, we present a customizable bead-based system capable of simultaneously binding to live cells (through antibody-mediated cell tethering) and capturing cell-secreted molecules. Our functionalized beads capture secreted molecules (e.g., hepatocyte growth factor secreted by fibroblasts) that are diminished when sampled via traditional supernatant analysis techniques (p < 0.05), effectively rescuing a reduced signal in the presence of neutralizing components in the cell culture media. Our system enables capture and analysis of molecules integral to chemical communication that would otherwise be markedly decreased prior to analysis.

Figure 1.

Bead-based approaches to quantifying cell-secreted factors for communication, with the highest concentration immediately after secretion being near the cell surface. (Bi) Traditional methods transfer the cell culture supernatant to another well plate and use beads for bulk solution analyte sampling while (Bii) our new approach utilizes cell-taggeting beads in situ for analyte sampling.

Figure 2:

Calibration curves for mono-(MF) and dual-functionalized (DF) beads demonstrate ability of both beads to capture HGF. Per MagPlex manufacturer’s recommendation, a five-parameter logistic fit was used to fit calibration curves and demonstrate the HGF-bead binding ability for each independent experiment (A: Experiment 1; B: Experiment 2; C: Experiment 3; D: Experiment 4) in duplicate (duplicates are shown as two plotted circles or triangles). Curves follow the expected trend based on manufacturer’s technical notes. Decreased signal is observed for DF beads as the surface has roughly half the binding sites for HGF compared to MF beads. Insets are of first four points of the calibration curves. All curves are plotted on a logarithmic scale for visualization.

Figure 3.

Dual-functionalized (DF) beads (functionalized with anti-CD90 and anti-HGF) target CD90⁺ cells (NHDFn). (A and B) Representative images of CD90⁺ cells stained with CellTracker Green (green) and CD90-cells stained with CellTracker Blue (blue); cells were seeded at a density of 2.6×10⁴ cells/mL, cultured for 3 days, and incubated with DF beads (pink) for 2 h. Images of wells were taken after washing to determine successful bead-binding to the cell surface receptor CD90. (A) CD90⁺ cells show an increased bead retention compared to (B) CD90⁻ cells. (C) An average of 71% of DF beads were retained when incubated with CD90⁺ cells, significantly higher bead retention than for CD90⁻ cells or no cells (empty well). Some nonspecific binding was observed for beads placed in an empty well (<5% of beads retained). Bar graphs represent mean ± SD of n=3 independent experiments. Brown-Forsynthe and Welch ANOVA tests were used followed by Dunnett’s T3 test. * p < 0.05. 10X Scale bar = 100μm. 20X Scale bar = 50μm.

Figure 4.

Localized cell-surface sampling recovers HGF signal in the presence of a neutralizing factor. Schematics showing workflow of (Ai) traditional supernatant sampling and (Aii) localized cell-surface sampling of a cell-secreted factor. Mono-functionalized (MF) beads (functionalized with anti-HGF) were used for supernatant sampling while dual-functionalized (DF) beads (functionalized with anti-HGF and anti-CD90) were used for localized cell-surface sampling. (Ai) MF beads are added to supernatant samples collected from cell cultures after 2 h incubation. (Aii) DF beads are added to directly to cultures for a 2 h sampling period. (Bi) Supernatant analysis shows a decrease in recovered HGF when cells are incubated with free anti-HGF whereas (Bii) localized cell-surface sampling with DF beads shows no change in recovered HGF since beads capture HGF before neutralization by free anti-HGF. Error bars are SEM. Experiments were performed in the absence (−) or presence (+) of free anti-HGF and are represented as circles or squares, respectively; each point represents an averaged technical replicate (supernatant sampling) or culture replicate (localized cell-surface sampling). († HGF recovered using DF beads is captured at the surface of the cell, rather than the bulk, leading to increased overall HGF capture. The absolute values in (Bi) should not be directly compared to the absolute values in (Bii), but rather the trend within each graph between control and treated conditions.) Unpaired Student’s t-test. * p = 0.016; ns p = 0.428.