3D-Bioprinting of Valvular Interstitial Cells of Ovine Aortic Valves: Impact of Printing Parameters on Cell Viability

Objectives: Calcific aortic valve disease (CAVD) is a frequent cardiac pathology in the aging society. Current treatment strategies involve the implantation of valvular bio-prostheses, which are limited in several dimensions, particularly regarding long-time durability. Mechanisms of CAVD and graft degeneration are not fully understood and three-dimensional models to study valvular interstitial cells (VIC) physiology and remodelling are needed.

Methods: Fresh VIC were isolated from ovine aortic valves and cultured in Dulbecco's Modified Eagle's Medium (DMEM). VIC of passages six to ten were dissolved in a hydrogel consisting of 1.0% alginate and 7.5% gelatine with a concentration of 2 × 106/mL. VIC-laden hydrogels were printed in six-well cell culture plates (15 wells per procedure) with a 3D-bioprinter (3D-Bioplotter Developer Series, EnvisionTec, Gladbeck, Germany) at different temperatures (20–37°C), with different pressures (0.1–0.8 mbar) and speeds (20–80 mm/s) with a 250-µm nozzle in orthogonal three-layer pattern. After cross-linking with 100 mM CaCl2 solution for 3 minutes and washing with Dulbecco's phosphate-buffered saline (DPBS), 3D cell constructs were cultured in DMEM for up to 30 days. Cell viability was tested by life/dead staining and microscopy.

Result: 3D-bioprinting of cell-laden hydrogels was most accurate at a printing temperature of 30°C and with a platform temperature of 21°C. Changes of the printing pressure were directly related to the post-printing cell viability. With a pressure of 0.1 to 0.2 mbar, cell stress was minimized and cells started to divide approximately 6 days after the printing procedure. Speed adjustment between 45 and 60 mm/s was capable of both preserving printing accuracy and redeeming cell viability. Microscopy and life/dead staining proved living constructs after 30 days of culture in more than 50% of printed wells.

Conclusion: 3D-bioprinting of aortic VIC is capable of developing novel models to examine CAVD initiation and progression. By using low pressure printing methods, we were able to successfully culture bioprinted cell constructs for up to 30 days. Future options include in vitro testing of potential pharmacological interventions as well as tissue engineering applications for personalized valve prosthesis.

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Artikel online veröffentlicht:
19. Februar 2021