CPA toxicity screening of cryoprotective solutions in rat hearts

Abstract

In clinical practice, donor hearts are transported on ice prior to transplant and discarded if cold ischemia time exceeds ∼5 h. Methods to extend these preservation times are critically needed, and ideally, this storage time would extend indefinitely, enabling improved donor-to-patient matching, organ utilization, and immune tolerance induction protocols. Previously, we demonstrated successful vitrification and rewarming of whole rat hearts without ice formation by perfusion-loading a cryoprotective agent (CPA) solution prior to vitrification. However, these hearts did not recover any beating even in controls with CPA loading/unloading alone, which points to the chemical toxicity of the cryoprotective solution (VS55 in Euro-Collins carrier solution) as the likely culprit. To address this, we compared the toxicity of another established CPA cocktail (VEG) to VS55 using ex situ rat heart perfusion. The CPA exposure time was 150 min, and the normothermic assessment time was 60 min. Using Celsior as the carrier, we observed partial recovery of function (atria-only beating) for both VS55 and VEG. Upon further analysis, we found that the VEG CPA cocktail resulted in 50 % lower LDH release than VS55 (N = 4, p = 0.017), suggesting VEG has lower toxicity than VS55. Celsior was a better carrier solution than alternatives such as UW, as CPA + Celsior-treated hearts spent less time in cardiac arrest (N = 4, p = 0.029). While we showed substantial improvement in cardiac function after exposure to vitrifiable concentrations of CPA by improving both the CPA and carrier solution formulation, further improvements will be required before we achieve healthy cryopreserved organs for transplant.

Keywords: CPA toxicity; Cryopreservation; Heart; Perfusion; Vitrification.

Fig. 1.

Compositions of CPA solutions tested. A: Compositions and concentrations of CPA cocktails (VS55 and VEG) with units of M for each component. B: Compositions and concentrations of carrier solutions (Celsior, UW, EC, and LM5) all with units of mM. C: Breakdown of each carrier by molar percent of each listed category in (B), with comparison to blood [25].

Fig. 2.

Overview of CPA perfusion setup and protocol in rat hearts. A: Schematic of hypothermic perfusion system. B: Perfusion concentration and temperature profiles. First, during hypothermic machine perfusion, rat hearts are loaded/unloaded with CPA. Second, during normothermic machine perfusion (NMP), rat hearts are assessed for recovery of mechanical function.

Fig. 3.

Rat heart TTC staining and functional recovery after CPA loading/unloading using Celsior as the carrier. A: TTC staining of rat hearts following CPA loading/unloading and normothermic perfusion. These images for VS55 and VEG correspond to the best-recovered heart for each treatment group as shown in (B). A dark red color indicates high cell viability whereas a pale color indicates low cell viability. The Live Control is given by a fresh-perfused control heart. The Dead Control is given by CPA perfusion (100 % VS55 in EC) at high temperature (10 °C). Note that some tissue is missing from the epicardial surface as the result of procurement of biopsies for later analysis. B: Recovery of mechanical function during normothermic perfusion. A CPA concentration value of 100 % indicates that the perfusate CPA concentration reached 55 w/v %. C: Time to recovery of atrial beating during normothermic perfusion. Celsior was used as the carrier solution in all cases. Each data point represents one heart. Error bars give the standard deviation. The difference between the two medians (1.3 min vs 1.0 min) was not statistically significant (p = 0.771, Mann Whitney test). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4.

LDH release during rat heart hypothermic and normothermic perfusion with VS55 and VEG in Celsior. A: LDH release rate during hypothermic perfusion. Each data point gives the average of N = 4 hearts. Shown above are the pairwise comparisons (T tests with pooled standard deviations) for VS55 versus VEG at the corresponding time points (p>.05 for time points below 165 min, p = 0.006 for 165 min using Holm-adjusted p-values). B: LDH release rate during normothermic perfusion. Each data point gives the average of N = 4 hearts. Shown above are the pairwise comparisons (T-tests with pooled standard deviations) for VS55 versus VEG at the corresponding time points (p = 1.0 for 10 min, p<.001 for 30 min, p = 0.052 for 50 min using Holm-adjusted p-values). C: Total LDH released during hypothermic perfusion and normothermic perfusion as calculated from (A) and (B). The data are normalized to wet weight before hypothermic perfusion. All error bars give standard deviations of N = 4 biological replicates. Each data point represents one heart. The difference between VS55 and VEG was statistically significant for normothermic perfusion (p = 0.017, Welch T-test) but not for hypothermic perfusion (p = 0.240, Welch T-test).

Fig. 5.

Rat heart cell viability and functional recovery based on perfusion with VEG and different carrier solutions. A: TTC staining of rat hearts following normothermic perfusion after CPA loading/unloading. These images for each carrier correspond to the best-recovered heart as shown in (B). A dark red color indicates high cell viability whereas a pale color indicates low cell viability. The Live Control is given by a fresh-perfused control heart. The Dead Control is given by CPA perfusion (100 % VS55 in EC) at high temperature (10 °C). Note that some tissue is missing from the epicardial surface as the result of procurement of biopsies for later analysis. B: Recovery of mechanical function during normothermic perfusion after CPA loading/unloading. A CPA concentration value of 100 % indicates that the perfusate CPA concentration reached 55 w/v %. C: Time to recovery of atrial beating during normothermic perfusion The difference between the two medians (1.0 min vs 3.0 min) was statistically significant (p = 0.029, Mann Whitney test). D: Time in cardiac arrest (no atrial or ventricular beating), including the time before the first observed contraction. The difference between the two medians (1.7 % vs 50 %) was statistically significant (p = 0.029, Mann Whitney test). VEG was used as the CPA cocktail in all cases. Each data point represents one heart. Error bars give the standard deviations. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)