Analzying HUVEC Alignment Under Flow/1um Bead Shear Calculations

ByAlec

Matlab Generated Endothelial Cell Alignment Distribution

Top and Bottom Gel

Figure 1. Matlab based alignment semi-quantification of HUVECs seeded on top of a collagen I gel. (A) Matlab output of major axis angle (relative to the x-axis) of HUVECs before flow set on top of cell image. (B) HUVEC angle of alignment relative to x-axis after 24 h flow. (C) Histogram plot of A, angles changed to reflect y-axis alignment. (D) Histogram plot of B.

No Gel

Figure 2. Matlab based alignment semi-quantification of HUVECs seeded directly on the NPN chip. (A) Matlab output of major axis angle (relative to the x-axis) of HUVECs before flow set on top of cell image. (B) HUVEC angle of alignment relative to x-axis after 24 h flow. (C) Histogram plot of A, angles changed to reflect y-axis alignment. (D) Histogram plot of B.

Bottom Gel

Run 1

Figure 3. Matlab based alignment semi-quantification of HUVECs seeded directly on the NPN chip with collagen I gel on the reverse side. (A) Matlab output of major axis angle (relative to the x-axis) of HUVECs before flow set on top of cell image. (B) HUVEC angle of alignment relative to x-axis after 24 h flow. (C) Histogram plot of A, angles changed to reflect y-axis alignment. (D) Histogram plot of B.

Run 2

Figure 4. Matlab based alignment semi-quantification of HUVECs seeded directly on the NPN chip with collagen I gel on the reverse side (RUN 2). (A) Matlab output of major axis angle (relative to the x-axis) of HUVECs before flow set on top of cell image. (B) HUVEC angle of alignment relative to x-axis after 24 h flow. (C) Histogram plot of A, angles changed to reflect y-axis alignment. (D) Histogram plot of B.

Statistical Analysis

The next step in this data analysis is to perform some form of statistical analysis. Comparing real alignment data to a set of randomly generated alignments may be a good place to start. Also, comparing the frequency distributions of the before flow and after flow alignment histograms may provide some level of significance.

 

 

Example Shear Calculations with 1um Polystyrene Beads

 

Matlab Video Processing

Using matlab, the video seen above was converted to gray scale frames, which were then converted to binary images (Figure 5). Matlab then tracks the center of the particle over 5 frames and calculates a distance traveled in pixels, distance traveled in um, and velocity in um/s.

Figure 5. (Top) Images showing particle trajectory over 4/30 s. Each picture represents a frame of a video shot at 30 fps. The particle circled in red was used to obtain velocity. (Bottom) Data from matlab analysis on particle velocity.

Using the formula for shear flow through a slit:

τ=6Qμ/(wh^2 )

I calculated a shear at the surface of the gel of about 0.093 dyn/cm^2. The expected shear at this level is around 6.5 dyn/cm^2.

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