Scaffold-free high throughput generation of quiescent valvular microtissues

Aims: The major challenge of working with valvular interstitial cells in vitro is the preservation or recovery of their native quiescent state. In this study, a biomimetic approach is used which aims to engineer small volume, high quality valve microtissues, having a potential in regenerative medicine and as a relevant 3D in vitro model to provide insights into valve (patho)biology.

Methods and results: To form micro-aggregates, porcine valvular interstitial cells were seeded in agarose micro-wells and cultured in medium supplemented with 250μM Ascorbic Acid 2-phosphate for 22days. Histology showed viable aggregates with normal nuclei and without any signs of calcification. Aggregates stained strongly for GAG and collagen I and reticular fibers were present. ECM formation was quantified and showed a significant increase of GAG, elastin and Col I during aggregate culture. Cultivation of VIC in aggregates also promoted mRNA expression of Col I/III/V, elastin, hyaluronan, biglycan, decorin, versican MMP-1/2/3/9 and TIMP-2 compared to monolayer cultured VIC. Phenotype analysis of aggregates showed a significant decrease in α-SMA expression, and an increase in FSP-1 expression at any time point. Furthermore, VIC aggregates did not show a significant difference in OCN, Egr-1, Sox-9 or Runx2 expression.

Conclusion: In this study high quality valvular interstitial cell aggregates were generated that are able to produce their own ECM, resembling the native valve composition. The applied and completely cell driven 3D approach overcomes the problems of VIC activation in 2D, by downregulating α-SMA expression and stimulating a homeostatic quiescent VIC state.