Chapter 31
Vitrification of Heart Valve Tissues
Kelvin G. M. Brockbank, Zhenzhen Chen, Elizabeth D. Greene,
and Lia H. Campbell
Abstract
Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has
evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples (1–3 mL
total volume) where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically
provides extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and
3, permits preservation of large samples (80–100 mL total volume) with several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at
À80
C; better matrix preservation than freezing with retention of material properties; very low cell
viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination
associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic
immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is
not used; and reduced manufacturing costs. More recently, we have developed Protocol 4 in which VS55 is
supplemented with sugars resulting in reduced concerns regarding nucleation during cooling and warming.
This method can be used for large samples resulting in retention of cell viability and permits short-term
exposure to À80
C with long-term storage preferred at or below À135
C.
Key words Heart valves, Vitrification, Tissue banking
1 Introduction
Three types of heart valves are employed for replacement of defective valves in patients: mechanical, xenogeneic tissue, and allogeneic
human valves derived from donors’ postmortem. Most patients
receive either xenogeneic tissue or mechanical valves; however,
the use of cryopreserved human valve allografts became established
during the 1970s and 1980s for certain patient subsets. Two
tri-leaflet valves, the aortic and pulmonary, with associated arterial
conduit and cardiac muscle band, are dissected from cadaver donor
hearts for patient use. Following cryopreservation, aortic and pulmonary valve allografts have typically been used to reconstruct the
left or right ventricular outflow tract for repair of diverse congenital
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_31, © Springer Science+Business Media, LLC, part of Springer Nature 2021
593
Vitrification of Heart Valve Tissues
Kelvin G. M. Brockbank, Zhenzhen Chen, Elizabeth D. Greene,
and Lia H. Campbell
Abstract
Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has
evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples (1–3 mL
total volume) where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically
provides extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and
3, permits preservation of large samples (80–100 mL total volume) with several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at
À80
C; better matrix preservation than freezing with retention of material properties; very low cell
viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination
associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic
immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is
not used; and reduced manufacturing costs. More recently, we have developed Protocol 4 in which VS55 is
supplemented with sugars resulting in reduced concerns regarding nucleation during cooling and warming.
This method can be used for large samples resulting in retention of cell viability and permits short-term
exposure to À80
C with long-term storage preferred at or below À135
C.
Key words Heart valves, Vitrification, Tissue banking
1 Introduction
Three types of heart valves are employed for replacement of defective valves in patients: mechanical, xenogeneic tissue, and allogeneic
human valves derived from donors’ postmortem. Most patients
receive either xenogeneic tissue or mechanical valves; however,
the use of cryopreserved human valve allografts became established
during the 1970s and 1980s for certain patient subsets. Two
tri-leaflet valves, the aortic and pulmonary, with associated arterial
conduit and cardiac muscle band, are dissected from cadaver donor
hearts for patient use. Following cryopreservation, aortic and pulmonary valve allografts have typically been used to reconstruct the
left or right ventricular outflow tract for repair of diverse congenital
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_31, © Springer Science+Business Media, LLC, part of Springer Nature 2021
593