Osmotic response during kidney perfusion with cryoprotectant in isotonic or hypotonic vehicle solution

Organ cryopreservation would revolutionize transplantation by overcoming the shelf-life limitations of conventional organ storage. To prepare an organ for cryopreservation, it is first perfused with cryoprotectants (CPAs). These chemicals can enable vitrification during cooling, preventing ice damage. However, CPAs can also cause toxicity and osmotic damage. It is a major challenge to find the optimal balance between protecting the cells from ice and avoiding CPA-induced damage. In this study, we examined the organ perfusion process to shed light on phenomena relevant to cryopreservation protocol design, including changes in organ size and vascular resistance. In particular, we compared perfusion of kidneys (porcine and human) with CPA in either hypotonic or isotonic vehicle solution. Our results demonstrate that CPA perfusion causes kidney mass changes consistent with the shrink-swell response observed in cells. This response was observed when the kidneys were relatively fresh, but disappeared after prolonged warm and/or cold ischemia. Perfusion with CPA in a hypotonic vehicle solution led to a significant increase in vascular resistance, suggesting reduced capillary diameter due to cell swelling. This could be reversed by switching to perfusion with CPA in isotonic vehicle solution. Hypotonic vehicle solution did not cause notable osmotic damage, as evidenced by low levels of lactate dehydrogenase (LDH) in the effluent, and it did not have a statistically significant effect on the delivery of CPA into the kidney, as assessed by computed tomography (CT). Overall, our results show that CPA vehicle solution tonicity affects organ size and vascular resistance, which may have important implications for cryopreservation protocol design.


Analysis of CT images
In one case, the orientation of the kidney in the CT scanner did not yield clear images in the coronal plane.In this case, images slices in an oblique plane were analyzed.Cortical and medullary regions were identified as illustrated in Figure S3.

Figure S3
. Representative image showing segmentation of a kidney in an oblique plane.Within the red medullary border, it can be seen that there is perhaps some pelvis within the plane at the very center of the image, but this region was deemed insignificant to the analysis.The darker region in the blue cortical border was identified as an abnormality and was segmented out according to the smaller blue border.

Effect of Cold Ischemia Time on Cell Death
To examine the effects of cold ischemia on cell death, we compared the LDH released during the 30 min isotonic vehicle solution equilibration period of all available experiments.The results are presented in Figure S4.LDH release increases substantially as cold ischemia time increases.The kidneys had an approximate warm ischemia time of 20 min and a cold ischemia time of 3-5 hr.Resistance was calculated as in Pegg et al [7], using either the influent (dashed lines) or effluent (solid lines) flowrates (see Results for details).At t = 0 that average vascular resistance was 3324 ± 503 mm -3 .

Pitfalls of Slaughterhouse Kidneys
To obtain kidneys from the slaughterhouse, it was necessary to use methods that only minimally disrupted the slaughterhouse workflow.Kidneys were typically removed by slaughterhouse personnel individually, rather than en bloc, and in some cases, this resulted in kidneys with only a short segment of the renal artery remaining.This made cannulation difficult, and may have resulted

Figure S1 .
Figure S1.Standard curves between Me2SO concentration and the grayscale value in CT images.(A) Grayscale values for Me2SO in isotonic vehicle solution, hypotonic vehicle solution, or water.Each solution was tested in triplicate.A representative image is included, showing wells containing Me2SO in water at concentrations from 0% to 25% m/v.(B) Combined measurements for Me2SO in all three solutions, obtained by subtracting off the background attenuation in the absence of Me2SO.The best-fit line is shown.Error bars represent the standard deviation.
Figure S2 compares Me2SO concentration estimates using two different assumed values for the solids volume fraction: xs = 0.30 and xs = 0.15.The assumed value of xs does not affect the qualitative trends, but it does affect the magnitude of the Me2SO concentration estimates.Higher concentrations are predicted when the assumed value of xs is increased.Thus, a limitation of our method is that it relies on an accurate estimate of the solids volume fraction -uncertainty about the value of xs causes uncertainty in the Me2SO concentration estimates.

Figure S2 .
Figure S2.Effect of the assumed value of the kidney solids volume fraction on Me2SO concentration estimates.Porcine kidneys were perfused with 15% m/v Me2SO in either isotonic (A, C) or hypotonic (B, D) vehicle solution.

Figure S4 .
Figure S4.LDH released as a function of cold ischemia time.All kidneys have a warm ischemia time of 25-40 min.The cold ischemia time groups are less than or equal to 8 hours (n = 10), approximately 20 hours (n = 4), and approximately 5 days (n = 6).

Figure
Figure S5 shows vascular resistance values for the kidney group with the lowest warm ischemia time.The vascular resistance was approximately constant after perfusion with CPA in isotonic vehicle solution, but the vascular resistance increased 10-fold within 10 minutes after perfusion with CPA in hypotonic vehicle solution.

Figure S5 .
Figure S5.Normalized vascular resistance for porcine kidneys perfused with 10% m/v ethylene glycol in either isotonic (red squares, n = 3) or hypotonic vehicle solution (blue circles, n = 3).The kidneys had an approximate warm ischemia time of 20 min and a cold ischemia time of 3-5 hr.Resistance was calculated as in Pegg et al[7], using either the influent (dashed lines) or in insertion of the cannula past the first arterial bifurcation in some cases (FigureS6, left).The lack of control over the kidney resection process also made it difficult to prevent introduction of air bubbles into the kidney vasculature (FigureS6, right).

Figure S6 .
Figure S6.CT image of two kidneys before a perfusion took place.The left kidney shows a cannula position that appears to be past the first arterial bifurcation.The right kidney shows some pockets of air trapped in the vasculature.

Table S1. Mass fraction of solids in kidneys from various species.
Table S1 summarizes values of the kidney solids mass fraction reported in the literature.
paper.If we assume that the solids mass fraction is approximately equal to the solids volume fraction, the value we used in this paper (xs = 30%) is on the high end of values reported in the literature.