Ice Control during Cryopreservation of Heart Valves and Maintenance of Post-Warming Cell Viability

Kelvin G.M. Brockbank; John C. Bischof; Zhenzhen Chen; Elizabeth D. Greene; Zhe Gao; Lia H. Campbell

doi:10.3390/cells11121856

Ice Control during Cryopreservation of Heart Valves and Maintenance of Post-Warming Cell Viability

Kelvin G.M. Brockbank, John C. Bischof, Zhenzhen Chen, Elizabeth D. Greene, Zhe Gao, Lia H. Campbell

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Abstract

Heart valve cryopreservation was employed as a model for the development of complex tissue preservation methods based upon vitrification and nanowarming. Porcine heart valves were loaded with cryoprotectant formulations step wise and vitrified in 1-30 mL cryoprotectant formulations ± Fe nanoparticles ± 0.6 M disaccharides, cooled to -100 °C, and stored at -135 °C. Nanowarming was performed in a single ~100 s step by inductive heating within a magnetic field. Controls consisted of fresh and convection-warmed vitrified heart valves without nanoparticles. After washing, cell viability was assessed by metabolic assay. The nanowarmed leaflets were well preserved, with a viability similar to untreated fresh leaflets over several days post warming. The convection-warmed leaflet viability was not significantly different than that of the nanowarmed leaflets immediately after rewarming; however, a significantly higher nanowarmed leaflet viability ( p < 0.05) was observed over time in vitro. In contrast, the associated artery and fibrous cardiac muscle were at best 75% viable, and viability decreased over time in vitro. Supplementation of lower concentration cryoprotectant formulations with disaccharides promoted viability. Thicker tissues benefited from longer-duration cryoprotectant loading steps. The best outcomes included a post-warming incubation step with α-tocopherol and an apoptosis inhibitor, Q-VD-OPH. This work demonstrates progress in the control of ice formation and cytotoxicity hurdles for the preservation of complex tissues.

Original language	English (US)
Article number	1856
Journal	Cells
Volume	11
Issue number	12
DOIs	https://doi.org/10.3390/cells11121856
State	Published - Jun 1 2022

Bibliographical note

Funding Information:
Funding: This work was funded by the U.S. Army Medical Research and Materiel Command (contract no. W81XWH-16-C-0074). The views, opinions, and findings contained in this report are those of the authors and should not be construed as an official Department of the Army position, policy, or decision unless so designated by other documentation. The commercial uses of the protocols disclosed in this work are subject to a U.S. patent [38] and a pending U.S. patent [39].

Publisher Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords

complex tissue preservation
cryopreservation
heart valve
nanowarming
vitrification
Disaccharides
Cell Survival
Heart Valves
Ice
Cryopreservation/methods
Animals
Swine
Cryoprotective Agents/pharmacology

PubMed: MeSH publication types

Research Support, U.S. Gov't, Non-P.H.S.
Journal Article

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title = "Ice Control during Cryopreservation of Heart Valves and Maintenance of Post-Warming Cell Viability",

abstract = "Heart valve cryopreservation was employed as a model for the development of complex tissue preservation methods based upon vitrification and nanowarming. Porcine heart valves were loaded with cryoprotectant formulations step wise and vitrified in 1-30 mL cryoprotectant formulations ± Fe nanoparticles ± 0.6 M disaccharides, cooled to -100 °C, and stored at -135 °C. Nanowarming was performed in a single ~100 s step by inductive heating within a magnetic field. Controls consisted of fresh and convection-warmed vitrified heart valves without nanoparticles. After washing, cell viability was assessed by metabolic assay. The nanowarmed leaflets were well preserved, with a viability similar to untreated fresh leaflets over several days post warming. The convection-warmed leaflet viability was not significantly different than that of the nanowarmed leaflets immediately after rewarming; however, a significantly higher nanowarmed leaflet viability ( p < 0.05) was observed over time in vitro. In contrast, the associated artery and fibrous cardiac muscle were at best 75% viable, and viability decreased over time in vitro. Supplementation of lower concentration cryoprotectant formulations with disaccharides promoted viability. Thicker tissues benefited from longer-duration cryoprotectant loading steps. The best outcomes included a post-warming incubation step with α-tocopherol and an apoptosis inhibitor, Q-VD-OPH. This work demonstrates progress in the control of ice formation and cytotoxicity hurdles for the preservation of complex tissues. ",

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note = "Funding Information: Funding: This work was funded by the U.S. Army Medical Research and Materiel Command (contract no. W81XWH-16-C-0074). The views, opinions, and findings contained in this report are those of the authors and should not be construed as an official Department of the Army position, policy, or decision unless so designated by other documentation. The commercial uses of the protocols disclosed in this work are subject to a U.S. patent [38] and a pending U.S. patent [39]. Publisher Copyright: {\textcopyright} 2022 by the authors. Licensee MDPI, Basel, Switzerland.",

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AU - Gao, Zhe

AU - Campbell, Lia H.

N1 - Funding Information: Funding: This work was funded by the U.S. Army Medical Research and Materiel Command (contract no. W81XWH-16-C-0074). The views, opinions, and findings contained in this report are those of the authors and should not be construed as an official Department of the Army position, policy, or decision unless so designated by other documentation. The commercial uses of the protocols disclosed in this work are subject to a U.S. patent [38] and a pending U.S. patent [39]. Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2022/6/1

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N2 - Heart valve cryopreservation was employed as a model for the development of complex tissue preservation methods based upon vitrification and nanowarming. Porcine heart valves were loaded with cryoprotectant formulations step wise and vitrified in 1-30 mL cryoprotectant formulations ± Fe nanoparticles ± 0.6 M disaccharides, cooled to -100 °C, and stored at -135 °C. Nanowarming was performed in a single ~100 s step by inductive heating within a magnetic field. Controls consisted of fresh and convection-warmed vitrified heart valves without nanoparticles. After washing, cell viability was assessed by metabolic assay. The nanowarmed leaflets were well preserved, with a viability similar to untreated fresh leaflets over several days post warming. The convection-warmed leaflet viability was not significantly different than that of the nanowarmed leaflets immediately after rewarming; however, a significantly higher nanowarmed leaflet viability ( p < 0.05) was observed over time in vitro. In contrast, the associated artery and fibrous cardiac muscle were at best 75% viable, and viability decreased over time in vitro. Supplementation of lower concentration cryoprotectant formulations with disaccharides promoted viability. Thicker tissues benefited from longer-duration cryoprotectant loading steps. The best outcomes included a post-warming incubation step with α-tocopherol and an apoptosis inhibitor, Q-VD-OPH. This work demonstrates progress in the control of ice formation and cytotoxicity hurdles for the preservation of complex tissues.

AB - Heart valve cryopreservation was employed as a model for the development of complex tissue preservation methods based upon vitrification and nanowarming. Porcine heart valves were loaded with cryoprotectant formulations step wise and vitrified in 1-30 mL cryoprotectant formulations ± Fe nanoparticles ± 0.6 M disaccharides, cooled to -100 °C, and stored at -135 °C. Nanowarming was performed in a single ~100 s step by inductive heating within a magnetic field. Controls consisted of fresh and convection-warmed vitrified heart valves without nanoparticles. After washing, cell viability was assessed by metabolic assay. The nanowarmed leaflets were well preserved, with a viability similar to untreated fresh leaflets over several days post warming. The convection-warmed leaflet viability was not significantly different than that of the nanowarmed leaflets immediately after rewarming; however, a significantly higher nanowarmed leaflet viability ( p < 0.05) was observed over time in vitro. In contrast, the associated artery and fibrous cardiac muscle were at best 75% viable, and viability decreased over time in vitro. Supplementation of lower concentration cryoprotectant formulations with disaccharides promoted viability. Thicker tissues benefited from longer-duration cryoprotectant loading steps. The best outcomes included a post-warming incubation step with α-tocopherol and an apoptosis inhibitor, Q-VD-OPH. This work demonstrates progress in the control of ice formation and cytotoxicity hurdles for the preservation of complex tissues.

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KW - Animals

KW - Swine

KW - Cryoprotective Agents/pharmacology

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SN - 2073-4409

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ER -

Ice Control during Cryopreservation of Heart Valves and Maintenance of Post-Warming Cell Viability

Abstract

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Keywords

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