Figure 1.
Schematics of the microfluidic device and the experimental setup for the cell separation process.
(A) Schematic of channel dimensions. The channel height is . The ridge is inclined at angle
and the ridge width
. The spacing
of ridge period is
. The gap
between the ridges and the substrate is varied depending on cell stiffness. (B) SEM images of the permanent mold (top) and microfluidic device made of PDMS showing ridges and outlet divider (bottom). (C) Schematic of a microfluidic channel. The channel is
in length and
in width. (D) Schematic of the experimental setup for cell separation process. A mixture of two types of cells with difference in stiffness is input into the device and the separated cells are collected at two outlets. (E) Images of separated cells with nuclear cell stains illustrate untreated K562 cells (green) and
cytochalasin D softened K562 cells (blue) undergoing separation.
Figure 2.
Cell trajectories are a function of cell stiffness.
(A) Overlay of still frames from a video of an untreated and 2 CD softened K562 cells flowing in a channel. Each micrograph is an overlay of 10 still frames at equal 10 ms time intervals from a video taken at 1200 fps. Green and red solid lines represent numerical simulations of the flow trajectory stiff and soft capsules. (B) Cell transverse displacement per ridge (n = 110 cells for each cell population) for untreated K562 cells and 2
CD softened K562 cells are
and
respectively. (C) Young's modulus (
for each cell population) for untreated K562 cells and 2
CD treated K562 cells are
and
respectively. The error bars represent the standard deviation. Nonparametric Wilcoxon signed-rank tests were used to test statistical significance between the two cell populations, with ** indicating a p<0.0001.
Figure 3.
Numerical simulations that demonstrate the separation principle.
(A) Cells experience both a hydrodynamic force, , and an elastic force,
, as the cells are deformed by the ridges. The elastic force varies with cell stiffness. The net transverse displacement is a result of interplay between the hydrodynamic force and stiffness-dependent elastic force. (B) The free energy associated with cell compression,
, increases to a maximum as the cell passes through the ridge and varies as a function of cell Young's modulus. The difference in the gradient of free energy of soft and stiff cells gives rise to different transverse forces that deflect cell trajectories in the microchannel perpendicular to the ridge and dependent on cell mechanical stiffness. (C) Simulation of velocity field and the resulting streamlines. The diagonal ridges create secondary flows (blue arrows represent velocity vector of the flow) that circulate underneath the ridges which propels soft cells in the negative transverse direction. The trajectory of soft cells follows closely to the streamline due to the minimal elastic force.
Figure 4.
HeyA8 cells and Jurkat cells have similar cell diameters but different stiffnesses and can be separated.
(A) Flow cytometry analyses of the initial mixture of cells and the cells collected at the stiff and soft outlets show the enrichment for HeyA8 cells (E = kPa) was 5.7-fold and for Jurkat cells (E =
kPa) was 3.1-fold (N = 6). HeyA8 cells were fluorescently labeled green for these studies and Jurkat cells were labeled red. (B) AFM measurement of Young's modulus of Jurkat cells and HeyA8 cells initially, before mixing and flowing, show that HeyA8 cells and Jurkat cells differ greatly in Young's modulus (
cells for each cell type). (C) HeyA8 cells and Jurkat cells are similar in cell diameter when suspended (
,
respectively). (D) Separated cells at outlets were measured by AFM (
for each outlet). Nonparametric Wilcoxon signed-rank tests were used to test statistical significance, with * indicating a p<0.001 and ns indicating no significance.
Figure 5.
Hey cells and K562 cells separation.
(A) Flow cytometry analyses of the initial mixture of cells and the cells collected at the stiff and soft outlets show an enrichment for Hey cells (E = kPa) of 5.3-fold and for K562 cells (E =
) of 1.8-fold (N = 2). (B) Cell stiffness was measured with AFM (
cells for each cell type) and quantified in terms of Young's modulus. A nonparametric Wilcoxon signed-rank test was used to test statistical significance, with ** indicating a p<0.0001.
Figure 6.
K562 cells and stiffened K562 cells separation.
(A) Flow cytometry analyses of the initial mixture of cells and the cells collected at the stiff and soft outlets show an enrichment of both cell types at the stiff and soft outlet respectively. 4% formaldehyde treated K562 cells (E = kPa) were enriched 6.7-fold at the stiff outlet and untreated K562 cells (E =
) were enriched 2.3-fold at the soft outlet (N = 2). (B) Cell stiffness was measured with AFM and quantified in terms of Young's modulus. A nonparametric Wilcoxon signed-rank test was used to test statistical significance, with ** indicating a p<0.0001.
Table 1.
Cell size is weakly correlated to cell stiffness and cell transverse displacement.
Table 2.
Effect of channel flow rate on Jurkat and HeyA8 cell separation.