Elastomeric sensor surfaces for high-throughput single-cell force cytometry

Abstract

As cells with aberrant force-generating phenotypes can directly lead to disease, cellular force-generation mechanisms are high-value targets for new therapies. Here, we show that single-cell force sensors embedded in elastomers enable single-cell force measurements with ~100-fold improvement in throughput than was previously possible. The microtechnology is scalable and seamlessly integrates with the multi-well plate format, enabling highly parallelized time-course studies. In this regard, we show that airway smooth muscle cells isolated from fatally asthmatic patients have innately greater and faster force-generation capacity in response to stimulation than healthy control cells. By simultaneously tracing agonist-induced calcium flux and contractility in the same cell, we show that the calcium level is ultimately a poor quantitative predictor of cellular force generation. Finally, by quantifying phagocytic forces in thousands of individual human macrophages, we show that force initiation is a digital response (rather than a proportional one) to the proper immunogen. By combining mechanobiology at the single-cell level with high-throughput capabilities, this microtechnology can support drug-discovery efforts for clinical conditions associated with aberrant cellular force generation.

Figure 1:. Operational principles of the general-use FLECS force cytometer

(a) TOP: Platform schematic showing cells adhered to functionalized adhesive micropatterns embedded into a thin glass-supported elastomeric film. LEFT: Top view showing multiple pattern shapes and a blow-up of a cell contracting an “X” pattern and inwardly displacing its terminals. BOTTOM: Overlay of fluorescent patterns and phase contrast images of adhered contracting cells. RIGHT: Time-lapsed images of a contracting cell and the underlying micropattern. Scale bar represents 25 μm. (b) Well-plate implementation. (c) Image analysis workflow. INPUT: Image sets of the micropatterns (set 1) and the stained cell nuclei (set 2). PROCESSING: Algorithm (i) identifies and measures all micropatterns in image set 1, (ii) cross-references the positions of each micropattern in image set 2 and (iii) determines whether 0, 1, or >2 nuclei (i.e. cells) are present (see: Fig. S2). OUTPUT: Mean center-to-terminal displacements of the micropatterns containing a single nucleus (i.e. 1 cell) are compared to the median of the corresponding measurement of all un-displaced patterns containing 0 nuclei (i.e. un-occupied patterns) and the differences are plotted as a horizontal histogram.

Figure 2:. Whole-cell contractility resolves contractile changes with differentiation and drug treatment

(a) Primary human adipose- or bone marrow-derived mesenchymal stem cells (MSCs) exhibited much higher contractile responses than either committed lineage 8 hrs post-seeding. Non-contractile subpopulations are seen amongst the MSCs indicating heterogeneity and potentially low purity that results from standard separation methods. N represents the number of cells. A typical contracted pattern representing approximately the median case from each distribution is shown below. (b) Overlays of fluorescent images of contracted patterns (green), phalloidin–stained actin (red) and nuclei (blue) of adipose- derived multi-potent MSCs showing three instances of cells fully spread over the patterns and actin stress fibers that route stresses to the vertices of the “X” patterns. Scale bar represents 25 μm. (c) Representative distributions of single-cell responses to increasing doses of blebbistatin. Plots comprise pooled data from 4 technical replicates of each condition. (d) Dose-response curve over 3 decades in which we identify an IC₅₀ of 2.61 μM. Error bars represent SEM. N represents number of cells in each distribution. The Kruskal-Wallis test for non-parametric data was used to perform statistical analysis on the contractile distributions with significance defined as P < 0.05.

Figure 3:. Parallel study of fatal asthma and non-asthma patient-derived airway smooth muscle cells

(a) Experimental workflow. 12 patient derived HASM cell lines were seeded into 8 wells each in a FLECS well-plate. A baseline image is taken. Cells are then all treated with the contractile agonist bradykinin (BK, 10 μM final concentration) and imaged for 16 minutes at 4 min intervals. Half of all cells are treated with 50 μM formoterol (form) and the other half with DMSO vehicle (veh). Cells are imaged an additional 20 minutes at 4 min intervals and finally at 10 minutes later. (b) Distributions of responsive cells from a representative patient. The distribution shifts upward following BK treatment, and is halted by formoterol. (c) Median values of the evolving contractile distributions for each of 12 patient cell lines, with or without formoterol treatment. Each data point of each trace comprises an average of 4 separate well measurements on the well-plate (d) Pair-wise comparison of age-, race-, and gender- matched patients with and without asthma. The first age listed is for the normal patient, the second for the asthmatic patient. The letters denote race and gender. The full characteristics for all patient donors are listed in Table S1. In general, HASM cells from asthma patients exhibit greater tone and/or contraction following stimulation. (e) Collective comparisons of all asthma vs non-asthma HASM cells. Each data point represents a median value of an individual well measurement. All wells (96) were compared in (i), only wells that received vehicle (48) were compared in (ii)-(iii) and only wells that received formoterol (48) were compared in (iv). Tone was statistically greater for asthmatic HASM. Initial rate of response to BK was greater for asthmatic cells, however, the long-term time response over the course of the experiment was similar for the two groups. Rescue via formoterol was also similar for the two groups. A one-tailed student’s t-test was performed on patient pairs in (d) and pooled groups in (e). In (d) * indicates P < 0.05, *** indicates P < 0.001. Error bars represent S.E.M.

Figure 4:. Simultaneous measurements of calcium release and contractility in patient-derived HASM single-cells

(a) Experimental workflow. Adhered cells labeled with Fluo-8 are imaged in their tonic state. Agonists are then added and calcium-sensitive dye intensity is recorded for 30s at 100 ms intervals. The same set of micropatterns are then imaged for 25 minutes at 1 minute intervals. Calcium release and contractility traces are extracted from these image series. The black triangle on the individual calcium trace denotes addition of agonist. (b) All traces obtained from cells treated with BK, n = 503 cells. (c) Population-averaged traces for each agonist. Peak values from the two traces do not correlate, indicating high-intensity calcium signal does not necessarily translate to robust contraction. Error bars represent S.E.M.(d) Correlations between peak calcium release and peak contraction for the same single cells. Histograms displayed horizontally and longitudinally correspond to the isolated contractility and calcium measurements, respectively, and the colored bar in each distribution identifies the bin containing the median value. While each agonist induced calcium release and contraction to some extent, we did not observe any strong correlations between these measurements.

Figure 5:. Measuring phagocytic forces generated by individual human macrophages

(a) Representative images of hMDMs on hIgG cross patterns showing a range of phagocytic responses. (b) Representative image of actin-stained hMDMs spread over circular patterns in an array. High rates of single-cell pattern coverage are achieved. (c) Phagocytic contraction of i) ring, ii) cross and iii) filled hIgG circular patterns. The three distributions of single- cell responses were not significantly different. (d) Opsonin-dependence in phagocytic contraction. Vitronectin, fibrinogen, BSA, and hIgG were patterned in 50 μm cross shapes on a stiffer, 67:1 base:crosslinker (B:C) (top) and softer 71:1 (bottom) substrate. HIgG elicited the most contractile response from the largest fraction of macrophages, consistent with the role and urgency of antibody opsonization in immunity. Left Y-axis represents displacement in μm; Right Y-axis represents applied forces in nN. A typical pattern representing each distribution in the 67:1 B:C case is shown below. N represents number of cells in each distribution shown in (c) and (d). (e) Finite element method modeling of forces exerted by a phagocytosing macrophage. Forces were modeled as boundary loads on a linear elastic material, exerted between all pairs of adjacent terminals of the cross pattern. The shape of the non-displaced pattern is outlined in white and the 5μm by 10 μm area over which force is applied is shaded. LEFT: Complete geometry comprising a 150 μm by 150 μm elastic material with 90 μm thickness. MIDDLE: The top view showing the the direction of applied tangential forces, indicated by the arrows. RIGHT: Cross-sectional view of one quarter of the geometry at 50% opacity highlighting the response of the material to the boundary load and indicating the direction of net displacement. Note: Internal columns of material are depicted only to emphasize the displaced geometry due to applied forces and do not represent real boundaries in the material. The Kruskal-Wallis test for non-parametric data was used to perform perform statistical analysis on the contractile distributions for the opsonin-dependence experiment, with significance defined as P < 0.05. For the density dependence experiment, a one-way ANOVA ruled out any significant differences.

Figure 6:. Effects of chloroquine, cytochalasin D and CAL-101 on hMDM contractile force

(a) Contraction distributions of hMDMs engaging IgG-opsonized micropatterns pre-treated with DMSO or with three doses of each drug. (b) Contraction distributions of hMDMs engaging IgG-opsonized micropatterns incubated with DMSO or with three doses of each drug for 15 minutes after reaching steady-state contraction. In (a) and (b) Data are pooled from 4 technical replicates. A bimodal distribution was observed reflecting an “active” phagocytosing population (red curve) and a weakly adhered, inactive population (blue curve). A mixed Gaussian distribution is fitted to each plot to obtain information about the active populations which is used for quantification. N represents the number of cells in each distribution shown in (a) and (b). (c) and (d) median contraction levels of the active populations in (a), (b), respectively. Bars represent mean of 4 replicates + SEM. of no treatment and treatment with DMSO control. Measurements were compared using ANOVAs followed by two-tailed Bonferoni corrected t-tests.