Poster BB3 - Role of Extracellular Matrix Components in Regulating Mechanical Phenotype of Cells in a 3D Microtissue

Introduction: The perivascular matrix is comprised of various matrix proteins and cellular components that are responsible for the regulation and maintenance of vasculature in tissues. However, despite the advances in tissue engineering, our understanding of how mechanical forces generated by cells in the perivascular matrix modulate blood vessel growth is limited. Our laboratory has previously characterized the mechanical phenotype of various stromal cells by tracking matrix distortions generated by the inherent contractility of cells and that such mechanical forces can activate the VEGFR-2 receptor to drive angiogenesis. Cadherins are mechanosensors that maintain cell-cell interactions and blood vessel stability; the balance of neural cadherin (N-Cad) and vascular endothelial cadherin (VE-Cad) is an important regulator of endothelial cell activity for maintaining quiescent vasculature or undergoing angiogenesis. However, it is unclear how stromal mechanical phenotypes are related to cadherin regulation. Research has shown that decreasing the amount of calcium available to cells can deplete N-Cad. Our objective is to investigate the mechanical phenotype of cells in 3D microtissue models in different matrix compositions and with varying amounts of calcium to alter N-Cad regulation.

Materials and

Methods: We investigated the mechanical phenotype of human umbilical vein endothelial cells immortalized with an hTERT insertion (HT2), normal human ventricular cardiac (NHCF), normal human dermal (NHDF), and normal human lung (NHLF) fibroblasts. First, cells and fluorophore beads were embedded within a 3D microtissue model made of 10mg/mL fibrin or 9mg/mL fibrin + 1mg/mL collagen-I using a polydimethylsiloxane ring to house the microtissues. Gels were allowed to polymerize for 30min prior to addition of media. After 24hr, the control samples received media with physiological levels of Ca2+ (1.8 mM) and the experimental samples received media with low Ca2+ (0.45 mM). Systems were cultured overnight prior to live-cell imaging inverted epifluorescence microscope over a 30-minute interval. Image data was collected, and bead displacements were analyzed to measure the matrix distortions caused by cell contractility events. The microtissues were then fixed and stained for N-Cad (Abcam, ab19348) at a 1:500 concentration. DAPI was used to stain the nuclei. The samples were re-imaged after staining. All images collected were 100µm z-stacks with a step-size of 2.

Results, Conclusions, and Discussions: The study aimed to explore the impact of different matrix compositions on cell-matrix distortions, which we refer to as mechanical phenotype. Previous studies indicated stiffer matrices correlated with smaller average matrix displacements while maintaining the phenotypic trend of cell types across various matrix compositions. Our findings revealed that microtissues containing HT2s, NHCFs, and NHDFs exhibited significantly reduced average matrix distortions when cultured in low calcium compared to the control with physiological calcium levels (Figure 1a). Conversely, NHLFs showed a small but significant increase in matrix distortion magnitudes under the same conditions in fibrin-only gels but not fibrin+collagen gels (Figure 1a, b). In addition to suppressing N-Cad function, lowering calcium levels for these microtissue cultures alters actin/myosin contractility events, thus decreasing matrix distortions in most of our samples. These studies are critical pilot steps to defining how the interactions between matrix compositions and soluble molecules in the media regulate the mechanical phenotype of our cells. Among the cell types, the highest matrix distortions were observed in NHCFs, while the lowest in NHLFs. Overall, adding a small amount of collagen decreases the average size of matrix distortions generated by each cell type compared to fibrin-only gels (Figure 1b). However, the relative differences between stromal lines were maintained, with NHCFs having the highest average matrix distortions. These results also highlight the heterogeneous nature of mechanical phenotype within a single cell type. Staining for N-Cad shows somewhat decreased levels for HT2, NHCF, NHDF, and NHLF samples in low calcium conditions (Figure 2). However, these decreases do not appear to be dramatic, which could mean low calcium may suppress N-Cad functionality but not overall expression in our model. This suggests that alterations in N-Cad in 3D in vitro systems may influence fibroblast interactions with neighboring cells and matrix partially through mechanical phenotypic changes. Future studies will quantify N-Cad in microtissue models to compare the mechanical phenotype with the expression of N-Cad in relation to matrix composition and calcium levels. Overall, our microtissues represent a combinatorial platform to study mechanical forces in tissue engineering when developing large, vascularized scaffolds to study diseased states and healthy tissues.

Acknowledgements (Optional): The authors would like to acknowledge funding for this project: R00-CA230202 (M.K.S.L), IMPACT Award, O’Neal Comprehensive Cancer Center (M.K.S.L), and Blazer Graduate Research Fellowship (V.B.).