A Roadmap to Cardiac Tissue-Engineered Construct Preservation: Insights from Cells, Tissues, and Organs

Abstract

Worldwide, over 26 million patients suffer from heart failure (HF). One strategy aspiring to prevent or even to reverse HF is based on the transplantation of cardiac tissue-engineered (cTE) constructs. These patient-specific constructs aim to closely resemble the native myocardium and, upon implantation on the diseased tissue, support and restore cardiac function, thereby preventing the development of HF. However, cTE constructs off-the-shelf availability in the clinical arena critically depends on the development of efficient preservation methodologies. Short- and long-term preservation of cTE constructs would enable transportation and direct availability. Herein, currently available methods, from normothermic- to hypothermic- to cryopreservation, for the preservation of cardiomyocytes, whole-heart, and regenerative materials are reviewed. A theoretical foundation and recommendations for future research on developing cTE construct specific preservation methods are provided. Current research suggests that vitrification can be a promising procedure to ensure long-term cryopreservation of cTE constructs, despite the need of high doses of cytotoxic cryoprotective agents. Instead, short-term cTE construct preservation can be achieved at normothermic or hypothermic temperatures by administration of protective additives. With further tuning of these promising methods, it is anticipated that cTE construct therapy can be brought one step closer to the patient.

Keywords: antifreeze proteins; cardiac tissue engineering; cryopreservation; cryoprotective agents; heart failure; hypothermic and normothermic preservation; vitrification.

Figure 1

Compilation of the properties, advantages, and drawbacks of each preservation strategy. Preservation techniques can be subdivided depending on their working temperature (normothermic: ≈37 °C; hypothermic: 10 °C to subzero; vitrification: −150 to −160 °C; cryopreservation: −196 °C). At higher temperatures, samples are metabolically active and may experience ischemia/reperfusion (I/R) injury, with reactive oxygen species (ROS) formation, limiting preservation duration. Thermal‐hysteresis (TH) active antifreeze proteins (AFPs) can reduce the freezing point of the preservation solution and allow subzero storage. At lower temperatures, metabolism is halted, allowing for indefinite storage periods, but samples can experience ice‐induced damage. Cryoprotectants (CPAs) are used to prevent ice‐crystal formation and extreme dehydration. Ice‐recrystallization inhibition (IRI) active AFPs prevent ice crystal growth and mechanical damage. Created with BioRender.com.

Figure 2

A) In the work by van den Brink et al., hiPSC‐CMs at day 21 of differentiation were cryopreserved and thawed one week after freezing. Viability after dissociation (fresh) or thawing (cryo) was comparable (>95%), but replating efficiency of cryopreserved cells was found to be ≈50% of that of fresh cells. Six days after thawing, hiPSC‐CMs showed a normal cardiac signature (α‐Actinin⁺MHC⁺). In LUMC20 and two other hiPSC‐CM lines, a higher proportion of hiPSC‐CMs expressed the ventricular isoform of myosin light chain (MLC2v), which correlated with an increased duration of the action potentials. Reproduced under the terms of the CC BY license.^{[ 57 ]} Copyright 2020, The Authors. B) In the work by Zhang et al., hiPSC‐CMs were cryopreserved at differentiation day 23. After thawing, recovered hiPSC‐CMs showed normal cardiac signature (α‐Actinin⁺cTNT⁺). Yet, RNA‐seq analysis revealed transcriptomic alterations in the three analyzed hiPSC‐CM lines and functionally, cryopreserved hiPSC‐CMs showed reduced contraction velocity and shortened action potential duration. Reproduced under the terms of the CC BY‐NC‐ND license.^{[ 61 ]} Copyright 2020, The Authors. C) In the work by Ohkawara et al., scaffold‐free cell sheets containing human skeletal myoblasts were vitrified. Vitrification did not significantly affect the viability of the cell sheets nor the overall structure, preserving cell–cell adhesions and extracellular matrix composition. In addition, after transplantation onto the heart of a nude rat after surgical induction of MI, vitrified cell‐sheets contributed to an increment of ejection fraction and fractional shortening in the same extent as freshly produced cell sheets. Reproduced under the terms of the CC BY license.^{[ 62 ]} Copyright 2018, The Authors. D) The publication by Chiu‐Lam et al., reports the first successful vitrification of a rat heart. Briefly, hearts were excised and perfused with Custodiol HTK before being perfused with a magnetic cryopreservation agent (mCPA). After perfusion, the hearts were submerged into the mCPA, vitrified, and stored in liquid nitrogen. After one week of storage, hearts were thawed by nanowarming and subsequently perfused with Custodiol HTK to remove the remaining mCPA. Magnetic particle imaging revealed that mCPA was successfully loaded into the heart before vitrification and removed after thawing. Images on the right illustrate a successful example of rat heart vitrification. Reproduced under the terms of the CC BY‐NC license.^{[ 72 ]} Copyright 2021, The Authors.

Figure 3

A) In the work by Correia et al., hiPSC‐CMs in 2D monolayers or 3D aggregates were preserved at 4 °C to determine the effect of cell–cell and cell–matrix interactions on preservation success. Cells were preserved in hypothermic conditions for 3, 5, and 7 days (S3, S5, and S7, respectively). After preservation, cells were rewarmed and maintained in culture for up to 7 days. Preservation as 2D monolayers was only feasible up to 3 days, as metabolic activity recovery was significantly compromised for S5 and S7. In contrast, when preserved as 3D aggregates, differences in metabolic activity recovery were less pronounced (e.g., 3D aggregates preserved for 7 days recovered 70% of the metabolic activity 7 days post‐storage). Reproduced with permission.^{[ 32 ]} Copyright 2016, Wiley. B) In the work by Beckman et al., neonatal rat CMs were mixed with 10% Matrigel and 0.9 mg mL⁻¹ rat tail collagen type I in a silicon mold to generate a cardiac construct that was subsequently preserved for 1–7 days at 4 °C. Among the tested preservation solutions, ChillProtec could preserve mitochondrial function after 5 days of preservation (tetramethylrhodamine, methyl ester (TMRM) uptake and staining in active mitochondria, left picture) and cardiomyocyte structure (sarcomeric α‐actinin staining, right picture). In addition, myocardial contraction force was completely preserved after 1 day of preservation at 4 °C but gradually decreased after 5 days of preservation (light blue after 1 day of normothermia, dark blue after 5 days of normothermia). Reproduced with permission.^{[ 86 ]} Copyright 2018, IOP Publishing Ltd. C) In the work by Amir et al., whole rat hearts were preserved ex vivo at −1.3 °C in UW supplemented AFPIII for a maximum period of 24 h. After preservation, hearts were heterotopically transplanted in the abdomen of a recipient rat. 24 h after the transplant, hearts preserved in the presence of AFPIII showed a superior preservation of cardiomyocyte ultrastructure (membrane, nucleus, and mitochondria) and an improved fractional shortening, when compared to hearts preserved in UW at 4 °C. Reproduced with permission.^{[ 42 ]} Copyright 2005, Elsevier. Figure 3C top panel was created with BioRender.com.

Figure 4

A) In the work by Fischer et al., a new biomimetic culturing technique allowing the long‐term preservation of human myocardial tissue slices under physiological conditions was developed. This system allowed the continuous monitoring of myocardial tissue excitation and contraction, which could be stimulated or inhibited. Long‐term preserved tissue showed dense and well‐aligned myofibrils with preserved cross‐striations (α‐Actinin, red staining) and distinct localization of connexin‐43 (yellow staining). Reproduced under the terms of the CC BY license.^{[ 34 ]} Copyright 2019, The Authors. B) The publication by Koerner et al., reports a clinical study, where the clinical outcome of patients receiving a heart transplant preserved by normothermic ex vivo allograft blood perfusion (NEVABP) using the Organ Care System by Transmedics, USA, was compared to that of patients receiving a heart transplant preserved by static cold storage. Survival rates at 30 days, 1 year and 2 years after transplant were superior in the NEVABP group, which had a lower incidence of graft failure and severe acute rejection. Figure 4B left panel was created with BioRender.com.

Figure 5

Synopsis of strategies for preservation of cardiac cells, cTE constructs and hearts. *: Not required/optional. $: Considered useful but not experimentally tested/reported. #: Technically possible but less preferred compared to conventional cryopreservation. AFPs: antifreeze proteins. TH: thermal hysteresis. CPAs: cryoprotective agents. IRI: ice recrystallization inhibition. T _g: glass‐transition temperature. Created with BioRender.com.