Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype

Abstract

Valvular interstitial cells (VICs) actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis. To better understand and quantify how microenvironmental cues influence VIC phenotype and myofibroblast activation, we compared expression profiles of VICs cultured on poly(ethylene glycol) (PEG) gels to those cultured on tissue culture polystyrene (TCPS), as well as fresh isolates. In general, VICs cultured in hydrogel matrices had lower levels of activation (<10%), similar to levels seen in healthy valve tissue, while VICs cultured on TCPS were ~75% activated myofibroblasts. VICs cultured on TCPS also exhibited a higher magnitude of perturbations in gene expression than soft hydrogel cultures when compared to the native phenotype. Using peptide-modified PEG gels, VICs were seeded on (2D), as well as encapsulated in (3D), matrices of the same composition and modulus. Despite similar levels of activation, VICs cultured in 2D had distinct variations in transcriptional profiles compared to those in 3D hydrogels. Genes related to cell structure and motility were particularly affected by the dimensionality of the culture platform, with higher expression levels in 2D than in 3D. These results indicate that dimensionality may play a significant role in dictating cell phenotype (e.g., through differences in polarity, diffusion of soluble signals), and emphasize the importance of using multiple metrics when characterizing cell phenotype.

Robust Generation of Quiescent Porcine Valvular Interstitial Cell Cultures

“…Other approaches for reducing aSMA expression have involved 3D VIC culture in soft hydrogels. 10,[23][24][25] Interestingly, VIC encapsulation in synthetic hydrogels decorated with peptides derived from collagen I led to the lowest percentage of aSMA-positive VICs compared with fibronectinand elastinderived peptides. 23 In many of these cases, the ability to induce the quiescent phenotype has been hypothesized to depend on the soft mechanical properties of these materials.…”

3D matrix‐embedding inhibits cycloheximide‐mediated sensitization to TNF‐alpha‐induced apoptosis of human endothelial cells

“…Others and we demonstrated that three‐dimensional (3D) matrix‐embedding influences the phenotype and genotype of a variety of human cells mimicking the in vivo cell type to a greater extent than simply 2D tissue culture plating (Baharvand, Hashemi, Kazemi Ashtiani, & Farrokhi, ; Benya & Shaffer, ; Luca et al, ; Mabry, Payne, & Anseth, ; Methe et al, ; Nelson & Bissell, ). 3D cell culture systems have gained increasing interest in drug discovery and tissue engineering due to their evident advantages in providing more physiologically relevant information and more predictive data for in vivo tests (Edmondson, Broglie, Adcock, & Yang, ).…”

Complementary, Semi-automated Methods for Creating Multi-dimensional, PEG-based Biomaterials

“…6,33,[37][38][39][40] The amount of fibrillar F-actin in cells cultured on the 2D hydrogels was much lower in both cell lines than on TCPS (Figure 3c). [41][42] Comparing the 2D hydrogels by qualitative visual inspection, we observed that more cells could adhere to the "collagen-rich" substrate over the collagen I only substrate for both MDA-MB-231 and Hs578T cell lines. This result is likely due to the variety of integrin-binding sites provided by the protein mixture.…”