Force microscopy of nonadherent cells: a comparison of leukemia cell deformability

Abstract

Atomic force microscopy (AFM) has become an important tool for quantifying mechanical properties of biological materials ranging from single molecules to cells and tissues. Current AFM techniques for measuring elastic and viscoelastic properties of whole cells are based on indentation of cells firmly adhered to a substrate, but these techniques are not appropriate for probing nonadherent cells, such as passive human leukocytes, due to a lateral instability of the cells under load. Here we present a method for characterizing nonadherent cells with AFM by mechanically immobilizing them in microfabricated wells. We apply this technique to compare the deformability of human myeloid and lymphoid leukemia cells and neutrophils at low deformation rates, and we find that the cells are well described by an elastic model based on Hertzian mechanics. Myeloid (HL60) cells were measured to be a factor of 18 times stiffer than lymphoid (Jurkat) cells and six times stiffer than human neutrophils on average (E(infinity) = 855 +/- 670 Pa for HL60 cells, E(infinity) = 48 +/- 35 Pa for Jurkat cells, E(infinity) = 156 +/- 87 for neutrophils, mean +/- SD). This work demonstrates a simple method for extending AFM mechanical property measurements to nonadherent cells and characterizes properties of human leukemia cells that may contribute to leukostasis, a complication associated with acute leukemia.

FIGURE 1

Microfabricated wells for force microscopy of nonadherent cells. (A) Schematic diagram of the microwells showing SU-8 photoresist structures on a glass wafer in which nonadherent cells sit. Cells resting inside the microwells are mechanically immobilized for force microscopy with an AFM cantilever. (B) Scanning electron micrograph of microwells fabricated in 8 × 8 arrays. Scalebar is 50 μm. (C) Scanning electron micrograph of a single microwell showing the vertical sidewalls of the SU-8. Scalebar is 2 μm.

FIGURE 2

Geometry of a pyramid-tipped cantilever indenting a cell. (A) Schematic diagram of a cell sitting in a microwell. P_c, P_o, and P_t are the pressures of the cell, surrounding fluid, and cantilever tip, respectively. T is cortical tension. The projected contact area between the cell and the cantilever tip is the square defined by S. R_c is the cell radius. R₀ is the radius of the projected cross-sectional area of an arbitrary cell section taken away from the microwell walls and the cantilever tip. θ is the half-angle of the arc of this arbitrary cell shell section. (B) Expanded view of the tip-cell interaction showing that δ, the penetration depth of the pyramidal tip into the cell, is the sum of δ_t, the indentation of the tip beyond the plane described by S, and δ_c, the indentation described by the distance between the sphere shell and the projected contact area; α is the cantilever tip half-angle.

FIGURE 3

A typical deflection-position curve of a cantilever indenting a HL60 cell in a microwell. Indentation is in the direction of the arrow, and negative piezo position indicates extension after contact with the cell. The contact point is denoted by the circle. The piezo extension rate in this experiment was 1506 nm/s. Deflection of the cantilever is small compared to the indentation of the cell due to the greater stiffness of the cantilever when compared to the cell. Inset: illustration of the relationship between piezo movement z, indentation δ, and deflection d. The deflected cantilever is solid, and the undeflected cantilever is dashed.

FIGURE 4

Effect of piezo extension rate on apparent stiffness of HL60 cells. (A) At increasing piezo extension rates, the viscosity of the HL60 causes increased cantilever deflection for the same piezo position. All legend values are in nanometers/second. At rates up to 415 nm/s, the deflection curves overlay each other. This indicates that the indentation rate was slow enough for viscosity not to be a factor. (B) HL60 apparent stiffness determined by Hertzian mechanics remains constant at low rates. Data in panel B are from the same cell as panel A. (C) The apparent stiffnesses of eight HL60 cells at low piezo extension rates were normalized and averaged. Normalization was performed by averaging the stiffness of each cell across the experimental extension rates and then dividing the stiffness at each rate by this average. At rates of 415 nm/s and below, apparent stiffness remained constant. Error bars represent the standard deviation.

FIGURE 5

Comparison of mechanical models with cell deformation data. (A) An HL60 cell was indented at 24 nm/s. The Hertzian mechanics model (dashed line) fits the data (gray line) better than the liquid droplet model (dotted line). Contact point is denoted by the circle. (B) A sphere shaped indenter with a diameter of 10 μm was attached to the end of the cantilever and pushed into a different cell at 415 nm/s. The Hertzian mechanics (dashed line) and liquid droplet (dotted line) models were modified for a spherical punch and were fit to the data (gray line). Again, the Hertz model was a better fit to the data.

FIGURE 6

Comparison of myeloid and lymphoid cell line and neutrophil stiffness at low piezo extension rates. With a piezo rate of 415 nm/s, HL60 cells have an average apparent stiffness of 855 Pa with a standard deviation of 670 Pa (n = 60), whereas Jurkat cells are significantly softer (p < 0.001) with an average apparent stiffness of 48 Pa and a standard deviation of 35 Pa (n = 37). Neutrophils have an average apparent stiffness of 156 ± 87 Pa (n = 26, mean ± SD), significantly softer than HL60 cells and significantly stiffer than Jurkat cells (p < 0.001 for both).

FIGURE 7

As seen with Wright-stained cells, the nucleus/cytoplasm ratio in HL60 cells (A) and Jurkat cells (C) is larger than it is for normal neutrophils (E). The cortical actin cytoskeleton density of HL60 (B), Jurkat (D), and neutrophil (F) cells does not appear significantly different. Scale bar is 5 μm.