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Comparison between the existing and the optimized VMP loading protocols. a, e The arterial concentration and pressure during perfusion loading. b, f The model-predicted and experimental perfusion resistances for the representative cases. R 2 values equal 0.987 and 0.983 for b and f, respectively. c, g The model-predicted CPA concentration inside the kidney tissue for the representative cases. d,
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Vitrification could enable long-term organ preservation, but only after loading high-concentration, potentially toxic cryoprotective agents (CPAs) by perfusion. In this paper, we combine a two-compartment Krogh cylinder model with a toxicity cost function to theoretically optimize the loading of CPA (VMP) in rat kidneys as a model system. First, ba...
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... proposed by Fahy et al. and modified by Han et al. into a reproducibly vitrifiable protocol using a combination of ramp and step loading, where VMP concentration was ramped from 0 to 5 M over 100 min (ramp rate = 50 mM/min), kept at 5 M for 10 min (plateau duration = 10 min), and then stepped to 8.4 M and held it for 25 min [13], as shown in Fig. 5a. The total duration for this loading protocol was 135 min. From the Krogh cylinder model, the final CPA concentration inside the kidney after loading is predicted to be 7.76 ...
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... numerically evaluated 3 parameters under this pattern: ramp rate, ramp duration, and plateau duration. The empirical ramp rates were evaluated between 40 and 70 mM/ min [6,15], which is justified as a reasonable range by the transport model, as shown in Figure S1. The empirical plateau concentration was set around 5 M, as shown in Fig. 5a [6]. Using our model, we were able to investigate increased plateau concentrations between 3 and 7 M, which allow accelerated CPA loading but must be balanced with an increase in the potential for osmotic injury. To mitigate this potential damage, we tested the ramp durations between 70 and 120 min. Additionally, we tested increased ...
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... of the kidney after loading is predicted to be exp(˗0.1383) = 87.1%. Compared to the existing protocol, the optimized protocol has a shorter loading time (110 min vs 135 min) and lower predicted toxicity parameter (0.1269 vs. 0.1383). Next, we tested the existing and optimized protocols experimentally and compared them to the model, as shown in Fig. 5. Both protocols include a 20-min flush with carrier solution (LM5-XZ) to stabilize the perfusion resistance. Also note that the perfusion pressures were increased to 60 mm Hg at the full-strength step to overcome the viscosity increase and maintain adequate flow rates to ensure adequate CPA transport. From Fig. 5b and f, one can tell ...
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... them to the model, as shown in Fig. 5. Both protocols include a 20-min flush with carrier solution (LM5-XZ) to stabilize the perfusion resistance. Also note that the perfusion pressures were increased to 60 mm Hg at the full-strength step to overcome the viscosity increase and maintain adequate flow rates to ensure adequate CPA transport. From Fig. 5b and f, one can tell that the experimental perfusion resistance (R p , normalized to baseline resistance established during the first 20-min of carrier solution perfusion) agrees with the model-predicted perfusion resistance very well for both loading protocols, which provides support for the validity of our model. As shown in Fig. 5c ...
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... From Fig. 5b and f, one can tell that the experimental perfusion resistance (R p , normalized to baseline resistance established during the first 20-min of carrier solution perfusion) agrees with the model-predicted perfusion resistance very well for both loading protocols, which provides support for the validity of our model. As shown in Fig. 5c and g, the final tissue concentrations (after loading) inside the kidney are both predicted to achieve 7.76 M; this is the criteria to determine the duration of the final full-strength step. The active volume plots, Fig. 5d and h, confirmed that the active volume for both protocols did not exceed the osmotic limit ...
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... perfusion resistance very well for both loading protocols, which provides support for the validity of our model. As shown in Fig. 5c and g, the final tissue concentrations (after loading) inside the kidney are both predicted to achieve 7.76 M; this is the criteria to determine the duration of the final full-strength step. The active volume plots, Fig. 5d and h, confirmed that the active volume for both protocols did not exceed the osmotic limit ...
Citations
... Hence, the minimum CPA concentration for vitrification would be ~62% w/w, which is slightly lower than M22 (~66%w/w which includes carrier solution), where we have shown successful vitrification at 3L. Higher concentrations of CPAs such as VS83 (83% w/w CPA) have even lower CCR and can be more easily vitrified but increase biological toxicity relative to the CPAs chosen here [34]. To remain at a lower concentration of CPA and still achieve vitrification at higher volumes without toxicity, future work can assess the impact of ice recrystallization inhibitors (IRIs), polymers (e.g., polyglycerol-PGL, polyvinyl alcohol-PVA, polyethylene glycol-PEG, x-1000, z-1000, etc.), or other novel cryoprotective agents [35,36]. ...
Organ banking by vitrification could revolutionize transplant medicine. However, vitrification and rewarming have never been demonstrated at the human organ scale. Using modeling and experimentation, we tested the ability to vitrify and rewarm 0.5–3 L volumes of three common cryoprotective agent (CPA) solutions: M22, VS55, and 40% EG+0.6M Sucrose. We first demonstrated our ability to avoid ice formation by convectively cooling faster than the critical cooling rates of these CPAs while also maintaining adequate uniformity to avoid cracking. Vitrification success was then verified by visual, thermometry, and x-ray μCT inspection. M22 and EG+sucrose were successfully vitrified in 0.5 L bags, but only M22 was vitrified at 3 L. VS55 did not vitrify at any tested volumes. As additional proof of principle, we successfully vitrified a porcine liver (~1L) after perfusion loading with 40% EG+0.6M Sucrose. Uniform volumetric rewarming was then achieved in up to 2 L volumes (M22 with ~5 mgFe/mL iron-oxide nanoparticles) using nanowarming, reaching a rate of ~88 °C/min with a newly developed 120 kW radiofrequency (RF) coil operating at 35kA/m and 360kHz. This work demonstrates that human organ scale vitrification and rewarming is physically achievable, thereby contributing to technology that enables human organ banking.
... Joule heating has also been proposed for kidneys, but it requires separation into thin kidney slices and high CPA concentrations for effective heat diffusion [101]. Therefore, vitrification is the most promising method [19,99,100,[102][103][104]. ...
... Machine perfusion technology enhances CPA delivery and washing, crucial for vascularized tissues and organs. It plays a vital role in preconditioning, preservation, and recovery stages, making it pivotal for innovative cryopreservation approaches [38,99,100,102,112]. ...
Recent years have witnessed significant advancements in the cryopreservation of various tissues and cells, yet several challenges persist. This review evaluates the current state of cryopreservation, focusing on contemporary methods, notable achievements, and ongoing difficulties. Techniques such as slow freezing and vitrification have enabled the successful preservation of diverse biological materials, including embryos and ovarian tissue, marking substantial progress in reproductive medicine and regenerative therapies. These achievements highlight improved post-thaw survival and functionality of cryopreserved samples. However, there are remaining challenges such as ice crystal formation, which can lead to cell damage, and the cryopreservation of larger, more complex tissues and organs. This review also explores the role of cryoprotectants and the importance of optimizing both cooling and warming rates to enhance preservation outcomes. Future research priorities include developing new cryoprotective agents, elucidating the mechanisms of cryoinjury, and refining protocols for preserving complex tissues and organs. This comprehensive overview underscores the transformative potential of cryopreservation in biomedicine, while emphasizing the necessity for ongoing innovation to address existing challenges.
... The thicknesses of 3% alginate or 5% gelatin-methacryloyl hydrogel encapsulation optimally preserved mouse testicular tissues by inhibiting ice crystal formation, minimizing basement membrane contraction, improving cell morphology, and augmenting mitochondrial activity (14). Vitrification is the rapid cooling of an organ to a stable, ice-free, glassy state, preventing solid ice injury to tissues; however, vitrification necessitates the application of highly concentrated and possibly hazardous CPAs (17)(18)(19). High-quality CPAs should possess excellent water solubility, membrane permeability, and little harmful effects at lower temperatures and higher concentrations (20,21). Using higher concentrations of cryoprotectants, reducing solution volumes to lessen toxicity and osmotic stresses, and implementing innovative technologies like radiofrequency heating or nano-heating to fast and evenly rewarm are all likely to minimize freezing injury further (17,22,23). ...
... The vitrification techniques enabled the cryopreservation of rat kidneys for a duration of up to 100 days, with the ability for the gradual restoration of kidney function following nano-heated thawing and allografting (17). The same technique was applied to freeze and revive rat liver, successfully retaining the liver tissue architecture and vascular endothelial cells, enabling the liver to absorb indocyanine green and generate bile during reperfusion (19). The unsatisfactory results of vitrification-based cryopreservation for the heart may be attributed to the loss of myocardial tone caused by uneven freezing and rewarming, which inhibits the myocardium's ability to pump blood. ...
Despite the annual rise in patients with end-stage diseases necessitating organ transplantation, the scarcity of high-quality grafts constrains the further development of transplantation. The primary causes of the graft shortage are the scarcity of standard criteria donors, unsatisfactory organ preservation strategies, and mismatching issues. Organ preservation strategies are intimately related to pre-transplant graft viability and the incidence of adverse clinical outcomes. Static cold storage (SCS) is the current standard practice of organ preservation, characterized by its cost-effectiveness, ease of transport, and excellent clinical outcomes. However, cold-induced injury during static cold preservation, toxicity of organ preservation solution components, and post-transplantation reperfusion injury could further exacerbate graft damage. Long-term ex vivo dynamic machine perfusion (MP) preserves grafts in a near-physiological condition, evaluates graft viability, and cures damage to grafts, hence enhancing the usage and survival rates of marginal organs. With the increased use of extended criteria donors (ECD) and advancements in machine perfusion technology, static cold storage is being gradually replaced by machine perfusion. This review encapsulates the latest developments in cryopreservation, subzero non-freezing storage, static cold storage, and machine perfusion. The emphasis is on the injury mechanisms linked to static cold storage and optimization strategies, which may serve as references for the optimization of machine perfusion techniques.
Mesenchymal stem cells (MSCs) are a type of cell capable of regulating the immune system, as well as exhibiting self-renewal and multi-lineage differentiation potential. Mesenchymal stem cells have emerged as an essential source of seed cells for therapeutic cell therapy. It is crucial to cryopreserve MSCs in liquid nitrogen prior to clinical application while preserving their functionality. Furthermore, efficient cryopreservation greatly enhances MSCs’ potential in a range of biological domains. Nevertheless, there are several limits on the MSC cryopreservation methods now in use, necessitating thorough biosafety assessments before utilizing cryopreserved MSCs. Therefore, in order to improve the effectiveness of cryopreserved MSCs in clinical stem cell treatment procedures, new technological techniques must be developed immediately. The study offers an exhaustive analysis of the state-of-the-art MSC cryopreservation techniques, their effects on MSCs, and the difficulties encountered when using cryopreserved MSCs in clinical applications.
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.
