AFM Measurements of Glycocalyx in the Upside Down µSiM

Slide deck from Large Group Meeting 12-21-12 (PPT)

Access requirements for the AFM cantilver to contact the cells requires the cells to be grown on the membrane surface in what is typically the bottom channel of A-line devices (Typical A-line configuration is shown in slide 2).  A PDMS block is attached to the well side of the A-line to provide access ports to the well and to the lower channel (slide 3).  Flow in the channel between upstream and downstream reservoirs is maintained by gravity, with the height difference between upper and lower reservoirs maintained using a peristaltic pump.  Cells are either grown with low/no flow (static) or under a shear stress of 10 dyn/cm^2.  To perform the AFM measurements, the PDMS bock is removed, and the bottom surface of the A-line is peeled off to reveal the cells (slides 4 and 5).  In early experiments a perforated plug was inserted into the well of the A-line in an effort to stabilize the membrane and prevent a “trampoline” effect from the membrane during indentation experiments.  This was found to cause morphological changes to the cell monolayers and was subsequently shown to be unnecessary, and its use was discontinued.

A key innovation for making these measurements possible was the construction of a holding device to position and stabilize the modified A-line device on the AFM platform while maintaining hydration of the cells.  The holding device is shown schematically in slide five, depicted in Slide 6, and shown positioned under the AFM head in Slide 7.

As of this posting, we are able to  grow cell monolayers reliably in this device and to perform AFM indentation experiments on the monolayers (Slide 8).  The cantilever tip is outfitted with a 6.0 um glass bead at the tip to provide a large enough surface area to detect the soft surface of the cell.  An example of data from a successful measurement is shown in Slide 8.  The data are fit to a model of the cell treated as a thin elastic layer (the endothelial glycocalyx layer, EGL) on an elastic half-space (the cell body).  Values for the elastic modulus of the cell, the elastic modulus of the EGL and the thickness of the EGL are determined from the least squares regression between data and theory. (See https://pubmed.ncbi.nlm.nih.gov/32135082/ for details.)

Using these approaches (as of this posting), we have completed tests of 155 cells from 17 different devices from 6 different cell seedings.  The calculated values for the moduli and thickness are sensitive to anomalies in the measurement curves, and care must be taken to avoid including nonsense measurements.  As a criterion for data quality we require that the coefficient of variation (CV = the half width of the 95% confidence interval divided by the fitted value) for the fitted coefficients be less than 0.5.  Summaries of the measured values are shown graphically in Slides 9-11.  There were no statistical differences across different devices, and no statistical differences between values obtained for cells grown statically or under flow.  This latter result is surprising because shear stress is thought to increase glycocalyx production and was expected to result in a thicker glycocalyx.  Values for the fitted coefficients are as follows: (Note (data shown in the slides for 12/15 were not included in the statistics here because of anomalies in the the data (Slide 12).

Static Conditions

Flow Conditions (10 dynes/cm^2)