Measurement of single-cell adhesion strength using a microfluidic assay

Abstract

Despite the importance of cell adhesion in numerous physiological, pathological, and biomaterial-related responses, our understanding of adhesion strength at the cell-substrate interface and its relationship to cell function remains incomplete. One reason for this deficit is a lack of accessible experimental approaches that quantify adhesion strength at the single-cell level and facilitate large numbers of tests. The current work describes the design, fabrication, and use of a microfluidic-based method for single-cell adhesion strength measurements. By applying a monotonically increasing flow rate in a microfluidic channel in combination with video microscopy, the adhesion strength of individual NIH3T3 fibroblasts cultured for 24 h on various surfaces was measured. The small height of the channel allows high shear stresses to be generated under laminar conditions, allowing strength measurements on well-spread, strongly adhered cells that cannot be characterized in most conventional assays. This assay was used to quantify the relationship between morphological characteristics and adhesion strength for individual well-spread cells. Cell adhesion strength was found to be positively correlated with both cell area and circularity. Computational fluid dynamics (CFD) analysis was performed to examine the role of cell geometry in determining the actual stress applied to the cell. Use of this method to examine adhesion at the single-cell level allows the detachment of strongly-adhered cells under a highly-controllable, uniform loading to be directly observed and will enable the characterization of biological events and relationships that cannot currently be achieved using existing methods.

Appendix

The results of the finite element analysis demonstrate that the channel deformation is greatest at the entrance and decreases along the channel length. Across, the channel width, the deformation is roughly parabolic, with the maximum at the center. The channel deforms by over 100% at the channel entrance for a flow rate of 400 ml/hr. Along the length, the deformation decreases in an approximately linear fashion, resulting in a slight taper of the channel ceiling. The height of the deformed channel was averaged across the width at every node along the channel length. This height was then related to the pressure drop and channel position for varying flow rates by fitting a surface function to the data. A simple polynomial fit was used:

$$ {h_{corr}} = {a_1} + {a_2}\left( {\Delta P} \right) + {a_3}(x) + {a_4}\left( {\Delta P} \right)(x), $$

(7)

where ΔP is the pressure drop in Pa, x is the distance along the channel in m, and a _i are fitting coefficients. The maximum relative error of the fit was 0.3%. During the experiments, the channel ceiling deforms, but the flow rate supplied by the syringe pump is constant (for a particular step in the ramp). The pressure drop corresponding to the new, deformed geometry will therefore be less than that of the original channel. The pressure drop across a channel with a tapered ceiling (assuming w>>h) is

$$ \Delta P = \left( {\frac{{6\mu \,Q\,L}}{w}} \right)\left( {\frac{{{h_{\max }} + {h_{\min }}}}{{\,h_{\max }^2\,h_{\min }^2}}} \right), $$

(8)

where h _max is the height at the beginning of the taper, and h _min is the height at the end. From the FE results, it was predicted that the height of the deformed channel remained small in comparison to the channel length and width. Therefore, in the derivation of Eq. 8, it was assumed that the velocity components normal to the streamwise component were insignificant in comparison. As a consequence, the pressure gradient along the deformed channel did not deviate significantly from the linear pressure drop of a rectangular channel. To couple the mechanical deformation to the flow mechanics, Eq. 7 was substituted into Eq. 8, with x set as the channel distance corresponding to either h _max or h _min. The equation was solved for ΔP for a series of flow rates, Q, and a new relationship between the pressure and flow rate was computed (R ² = 0.999):

$$ \Delta {P_{corr}} = - 0.11\,{Q^2} + 252.96\,Q + 6941.65, $$

(9)

where ΔP _corr is in units of Pa and Q is in units of ml/hr. Eq. 9 provides a first-order approximation of the pressure drop along the deformed channel. Substitution of Eq. 9 into Eq. 7 yields an expression for the deformed height of the channel as a function of channel position and flow rate as given in Eq. 6 in the Materials and Methods section.