Optimizing Photo-Encapsulation Viability of Heart Valve Cell Types in 3D Printable Composite Hydrogels
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Photocrosslinking hydrogel technologies are attractive for the biofabrication of cardiovascular soft tissues, but 3D printing success is dependent on multiple variables. In this study we systematically test variables associated with photocrosslinking hydrogels (photoinitiator type, photoinitiator concentration, and light intensity) for their effects on encapsulated cells in an extrusion 3D printable mixture of methacrylated gelatin/poly-ethylene glycol diacrylate/alginate (MEGEL/PEGDA3350/alginate). The fabrication conditions that produced desired hydrogel mechanical properties were compared against those that optimize aortic valve or mesenchymal stem cell viability. In the 3D hydrogel culture environment and fabrication setting studied, Irgacure can increase hydrogel stiffness with a lower proportional decrease in encapsulated cell viability compared to VA086. Human adipose derived mesenchymal stem cells (HADMSC) survived increasing photoinitiator concentrations in photo-encapsulation conditions better than aortic valve interstitial cells (HAVIC) and aortic valve sinus smooth muscle cells (HASSMC). Within the range of photo-encapsulation fabrication conditions tested with MEGEL/PEGDA/alginate (0.25–1.0% w/v VA086, 0.025–0.1% w/v Irgacure 2959, and 365 nm light intensity 2–136 mW/cm2), the highest viabilities achieved were 95, 93, and 93% live for HASSMC, HAVIC, and HADMSC respectively. These results identify parameter combinations that optimize cell viability during 3D printing for multiple cell types. These results also indicate that general oxidative stress is higher in photocrosslinking conditions that induce lower cell viability. However, suppressing this increase in intracellular oxidative stress did not improve cell viability, which suggests that other stress mechanisms also contribute.
KeywordsExtrusion bioprinting Oxidative stress Mesenchymal stem cells Photo-polymerization Bio-ink Biofabrication
CellTracker™ Red CMTPX
5-(and-6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester CM-H2DCFDA
Compressive modulus calculated from 5 to 15% strain
Human adipose derived mesenchymal stem cells
Human aortic valve sinus smooth muscle cells
Human aortic valve interstitial cells
Hank’s balanced salt solution
High powered light emitting diode
- Irgacure 2959
Light emitting diode
Phosphate buffered saline
Poly-ethylene glycol diacrylate
Tissue engineered heart valves
We thank Jhalak Agarwal and Jennifer Richards who helped develop hydrogel and cell handling protocols. We thank Shivaun Archer, Claudia Fischbach, Jennifer Puetzer, Jeffery Ballyns, Lawrence Bonassar, Paula Miller, Michael Shuler, and Sam Portnoff (Widetronix Inc.) for their assistance and sharing of equipment. We thank Luke and Naomi Shirk for providing porcine tissue for the mechanical testing. This research was supported by the Morgan Family, Felton Family Endowment for Human Heart Valve Research at Seattle Children’s Hospital, Hartwell Foundation, National Science Foundation (CBET-0955172), NSF Graduate Research Fellowship, and American Heart Association (AH0830384N and 13POST17220071).
Conflict of interest
No competing financial interests exist.
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