Mechanistic Understanding of Single-Cell Behavior is Essential for Transformative Advances in Biomedicine

Abstract

Most current efforts to advance medical technology proceed along one of two tracks. The first is dedicated to the improvement of clinical tasks through the incremental refinement of medical instruments. The second comprises engineering endeavors to support basic science studies that often only remotely relate to human medicine. Here we survey emerging research approaches that aim to populate the sprawling frontier between these tracks. We focus on interdisciplinary single-live-cell techniques that have overcome limitations of traditional biological methods to successfully address vital questions about medically relevant cellular behavior. Most of the presented case studies are based on the controlled manipulation of nonadherent human immune cells using one or more micropipettes. The included studies have (i) examined one-on-one encounters of immune cells with real or model pathogens, (ii) assessed the physiological role of the expandable surface area of immune cells, and (iii) started to dissect the spatiotemporal organization of signaling processes within these cells. The unique aptitude of such single-live-cell studies to fill conspicuous gaps in our quantitative understanding of medically relevant cause-effect relationships provides a sound basis for new insights that will inform and drive future biomedical innovation.

Keywords: adhesion; calcium; chemotaxis; host-pathogen; immune cell; micropipette; neutrophil; phagocytosis; signaling; single-cell.

Figure 1

Single-live-cell, single-target pure-chemotaxis assay. a. Sketch of a dual-micropipette experiment to examine interactions between a single immune cell and a single pathogenic particle. b. Photograph of a dual-micropipette setup as used on an inverted microscope. c. Sketch of the microscope chamber including water reservoirs used to control and measure the pipette-aspiration pressure. d. Illustration of pure-chemotaxis experiments to test the response of human neutrophils to two forms of C. posadasii. The underlaid color gradient depicts the local concentration of chemoattractant produced by the host’s complement system at the surface of the differently sized target particles. e. Summary of the results of pure-chemotaxis assays to test the chemotactic response of human neutrophils to 11 different pathogenic and model targets. f-h. Examples of pure-chemotaxis experiments testing human neutrophils against clusters of S. Typhimurium (f), C. albicans cells (g), and endospores and spherules of C. posadasii (h). The positive neutrophil response is triple-checked by positioning the target at three different sides of the neutrophil. All scale bars denote 10 µm.

Figure 2

Expansion of the surface area of immune cells during phagocytosis. a-c. Scanning electron micrographs of J774 macrophages attached to a substrate (a) and during phagocytosis of 10-µm (b) and 30-µm (c) IgG-coated beads. These snapshots of fixed cells demonstrate how cell-surface wrinkles are progressively “ironed out” as the cells increase their apparent surface area. Note that the images in c are shown at a reduced scale compared to a and b. d,e. Fluorescence and transmitted-light images of human neutrophils (actin labeled red) that were fixed during phagocytosis of several 5-µm IgG-coated beads (labeled green). Each panel includes two different confocal slices, allowing us to determine the correct count of internalized beads. f,g. Graphs of the time course of the apparent surface area of human neutrophils during phagocytosis of C. posadasii endospores (f) and frustrated phagocytosis of C. posadasii spherules (g) (reproduced from [15]). Each plot includes the results from 3 different experiments. The inset in g shows the surface area over an extended time period. Included are videomicrographs recorded during representative dual-micropipette, single-live-cell experiments. All scale bars denote 10 µm.