3D-Bioprinting of Ovine Aortic Valve Endothelial and Interstitial Cells for Development of Multicellular Tissue Engineered Scaffolds

Background: Calcific aortic valve disease (CAVD) is a frequent cardiac pathology but still not fully understood. Novel three-dimensional (3D) models containing both valvular interstitial cells (VIC) and valvular endothelial cells (VEC) are urgently needed to investigate the complex cellular mechanisms involved in the pathogenesis of CAVD. 3D-bioprinting can offer new possibilities by designing 3D scaffolds and combining conventional cell culture methods with tissue engineering principles.

Method: Fresh VIC and VEC were isolated from ovine aortic valves and cultured in either Dulbecco's modified eagle's medium (DMEM) for VIC or endothelial cell growth medium (MV) for VEC. Cells were dissolved in a hydrogel consisting of 2.0% alginate and 8.0% gelatine. Cell-laden hydrogels of VIC and VEC coculture were printed in six-well cell culture plates with a 3D-bioprinter (3D-Bioplotter Developer Series, EnvisionTec, Gladbeck, Germany). 3D-printed scaffolds were cultured for up to 21 days. 3D-architecture, composition of cell culture medium components, and hydrogels were altered and cell viability tested by life/dead staining, microscopy and CCK-8 cell viability assay.

Results: Microscopy revealed highest printing accuracy for hydrogels dissolved in DMEM with stable strains for the whole incubation period in all printed scaffolds. In contrast, hydrogels containing MV medium or PBS dispersed throughout the 21-day culture period. Changes in the 3D-architecture of the scaffolds did not affect cell viability of VIC and VEC co-culture. Both side-by-side incubation of VIC and VEC as well as layered design (VEC-laden hydrogel on the outside and VIC-laden hydrogel on the inside) showed similar relative cell viability after 21 days (p = 1.00). Medium composition directly affected cell viability within the scaffolds. Co-culturing with a composition of 70% DMEM and 30% MV medium components reached the highest viability with approximately two times more living cells after 21 days of incubation compared with pure DMEM according to CCK-8 assay (p = 0.01).

Conclusion: 3D-bioprinting of scaffolds consisting of both VEC and VIC is a feasible new approach for aortic valve research to investigating mechanisms of initiation and propagation of CAVD. By combining gelatine and alginate hydrogels and both DMEM and MV medium components, aortic valvular cells proliferated during the 21-day incubation and showed good viability as well as accurate and durable printing architecture.

Publication History

Article published online:
28 January 2023