Assessment of Microgroove-Aligned Endothelial Cells for in vitro Simulations
1
Fischer ,
1
Gray ,
2
Trinkle ,
1
Eitel ,
1
Anderson
Jennifer L.
Lindsay N.
Christine A.
Richard E.
Kimberly W.
1
Department of Chemical and Materials Engineering, University of Kentucky, Lexington
2
Department of Mechanical Engineering, University of Kentucky, Lexington
ABSTRACT
MATERIALS AND METHODS
RESULTS - MORPHOLOGY
RESULTS - CHEMISTRY
The circulatory system is lined with a thin layer of endothelial cells, which
compose the lining of the vessel walls. Adhesion of cancer cells to this
endothelial cell lining is an important step in the metastatic cascade and
researchers are currently using in vitro techniques to investigate these
interactions. Under static culture conditions, endothelial cells grow in a
characteristic cobblestone pattern rather than growing in straight lines due to
the absence of shear stresses that would normally be found in circulatory
vessels. It has recently been suggested that such changes in cell morphology
can affect surface expression profiles, which may alter how frequently or
strongly cancer cells bind to endothelial cells. While flow adapting endothelial
cells is important prior to studying cancer cell binding in vitro, traditional
methods can be cumbersome due to the fact that the cells have to be exposed
to flow for an extended period of time under controlled environmental
conditions. In this study, we are investigating the efficacy of a microgroovealigned flow adaptation method for acquiring a flow adapted phenotype.
Micropatterning and Seeding Protocols
F-actin Staining
VCAM-1 Staining
Endothelial Cells: Cobblestone vs. Unidirectional Growth
 Microgrooves were created on glass slides using the negative epoxy-based
photoresist, SU-8 2000.5, as shown below.
 Human Umbilical Vein Endothelial Cells (HUVECs) were then seeding onto
unmodified (control) glass slides and micropatterned slides as shown below.
 F-actin staining was used to stain the actin fibers in the cellular cytoskeleton so  Initial fluorescent images of antibody tagging to evaluate surface expression
that the aspect ratios and orientation angles could be tabulated more readily
 Primary Antibody: Anti-VCAM-1 Antibody, clone P3C4
 Images show noticeable elongation of HUVECs on micropatterned versus control
 Secondary Antibody: Goat Anti-Mouse IgG (Fc), Fluorescein Conjugated
slides regardless of seeding density
 Staining completed for N=3 slides for both the unmodified control and
Morphology and Surface Chemistry Evaluation Protocols
 Elongation and orientation were compared at high and low seeding densities for
micropatterns. Unmodified slides stimulated with 480U/ml TNF-α
 The HUVEC morphology was characterized by aspect ratios and orientation
unmodified control and micropatterned slides
(tumor necrosis factor alpha) were also used as a positive control (N=3).
angles.
 High density: 62,000cells/cm2, Low density: 25,000 cells/cm2
Three images were analyzed per slide.
 Aspect ratio = length of cell/width of cell
Preliminary results from images and median pixel intensities
Aspect
Ratio
Comparison
 Orientation angle = deviation from horizontal
(histogram peaks) suggest upregulation of VCAM-1 on the surface
 Grooves defined as 0°
of microgroove-aligned HUVECs. This was not statistically
 Positive deviations represent above horizontal, negative represent below
significant in comparisons of mean pixel intensities.
 The surface chemistry was first evaluated by examining vascular cell adhesion
molecule VCAM-1 expression due to its significance in the metastatic cascade.
This was done using the following protocol:
 Using microgrooves created with SU-8 photoresist on glass, HUVECs were
successfully cultured statically in a more elongated and unidirectional form
reminiscent of in vivo morphology
 Initial fluorescent images suggest upregulation of VCAM-1 on
micropatterned glass slides which is more representative of the in vivo
surface chemistry. The average histograms derived from the images also
suggest upregulation (as evidenced by positive shift in median peak) on the
microgrooves, however this effect was not apparent in the means likely
due to the asymmetry from cutoffs of autofluorescence and nonspecific
Atomic Force Microscopy Characterization
staining on the left and the variability in the right tail regions.
 At both seeding densities, statistically higher aspect ratios were obtained for the
micropatterned versus the control slides (p-value <0.05)
Grooves
Glass
 Error bars represent SE
 Examine surface expression of ICAM-1, E-selectin and P-selectin
 In unmodified controls, n= 60. In micropatterned studies, n = 138 and n = 114
 Quantify results of surface chemistry in spectrophotometer for
Orientation Angle Comparison
comparison
 Compare results to cells flow adapted in a parallel plate flow chamber
CONCLUSIONS
RESULTS – APPROACH VALIDATION
OBJECTIVES
Overall Objective
The overall objective of this work was to create a static culture system that
better mimics the chemistry and morphology of endothelial cells grown in
vivo using microfabrication techniques.
FUTURE WORK
Specific Objectives
Investigate and compare morphology of endothelial cells grown on
micropatterned grooves with those grown on a blank glass slide by
quantifying aspect ratios and orientation angles.
Investigate surface expression of cells grown on micropatterned grooves in
comparison to cells grown on a blank slides using fluorescent tagging of
antibodies for the following proteins involved in tumor cell adhesion to the
endothelium: ICAM-1, VCAM-1, and E-selectin and P-selectin.




 The AFM was used to confirm that our methods for patterning SU-8 2000.5 were  Shown above are orientation angle histograms for the high density
micropatterned slides (left) vs. control (right)
correct and resulted in the desired topography.
 Micropatterned slides show small distribution mostly between ± 20°
 Average groove depth was 500 nm as expected
 Control slides show large distribution between ± 100°
 Consistent trench widths and spacing was achieved
Results demonstrate elongated, unidirectional growth achieved
ACKNOWLEDGEMENTS
UKY NCI CNTC (Grant # R25CA153954)
UKY NSF IGERT Grant in Bioactive Interfaces and Devices (NSF Award # 0653710)
CeNSE Laboratory at the University of Kentucky
Support from the National Science Foundation REU Program #EEC-1156667 and the Bucks
For Brains Program, Office of Undergraduate Research, University of Kentucky
REFERENCES
1. MicroChem. http://www.microchem.com/Prod-SU8_KMPR.htm (April 2012).
2. Cines et al. Endothelial Cells in Physiology and in the Pathophysiology of Vascular Disorders. Blood Journal.
1998; vol 91
3. Park, T; Shuler M. Integration of Cell Culture and Microfabrication Technology. Biotechnol. Prog. 2003, 19,
243-253