In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos.

In brief: In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species. Abstract: The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

Permeation of individual cryoprotectants and their different combinations into mouse liver tissue.

Development of successful tissue cryopreservation methods requires specific knowledge regarding tissue permeation of individual cryoprotective agents (CPAs) and their combinations. The present study assessed the permeation of dimethyl sulfoxide, ethylene glycol, and propylene glycol into liver tissue, and addressed whether the diffusion coefficient of individual CPAs changes when combining CPAs. To do this, mouse liver slices were exposed at room temperature to 3.5 mol/L concentrations of CPAs individually or in combination for 15, 30, 45, and 60 min. Subsequently, tissue CPA concentrations were determined using a gas chromatography/mass spectrometry (GC/MS) method. Our results show that (1) the GC/MS method allows measurement of multiple CPA concentrations in a single small tissue sample, (2) dimethyl sulfoxide has a higher diffusion coefficient than ethylene glycol and propylene glycol, and (3) the CPA diffusivity appears to decrease in mixtures with multiple CPAs. These findings may help the development of effective tissue cryopreservation methods.

Accelerating cryoprotectant delivery using vacuum infiltration.

The ability to cryopreserve bone marrow within the vertebral body (VB) would offer significant clinical and research benefits. However, cryopreservation of large structures, such as VBs, is challenging due to mass transport limitations that prevent the effective delivery of cryoprotectants into the tissue. To overcome this challenge, we examined the potential of vacuum infiltration, along with carbonation, to increase the penetration of cryoprotectants. In particular, we hypothesized that initial exposure to high-pressure carbon dioxide gas would introduce bubbles into the tissue and that subsequent vacuum cycling would cause expansion and contraction of the bubbles, thus enhancing the transport of cryoprotectant into the tissue. Experiments were carried out using colored dye and agarose gel as a model revealing that carbonation and vacuum cycling result in a 14% increase in dye penetration compared to the atmospheric controls. Experiments were also carried out by exposing VBs isolated from human vertebrae to 40% (v/v) DMSO solution. CT imaging showed the presence of gas bubbles within the tissue pores for carbonated VBs as well as control VBs. Vacuum cycling reduced the bubble volume by more than 50%, most likely resulting in replacement of this volume with DMSO solution. However, we were unable to detect a statistically significant increase in DMSO concentration within the VBs using CT imaging. This research suggests that there may be a modest benefit to carbonation and vacuum cycling for introduction of cryoprotectants into larger structures, like VBs.

Multiple cryoprotectant toxicity model for vitrification solution optimization.

Vitrification is a promising cryopreservation technique for complex specimens such as tissues and organs. However, it is challenging to identify mixtures of cryoprotectants (CPAs) that prevent ice formation without exerting excessive toxicity. In this work, we developed a multi-CPA toxicity model that predicts the toxicity kinetics of mixtures containing five of the most common CPAs used in the field (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide). The model accounts for specific toxicity, non-specific toxicity, and interactions between CPAs. The proposed model shows reasonable agreement with training data for single and binary CPA solutions, as well as ternary CPA solution validation data. Sloppy model analysis was used to examine the model parameters that were most important for predictions, providing clues about mechanisms of toxicity. This analysis revealed that the model terms for non-specific toxicity were particularly important, especially the non-specific toxicity of propylene glycol, as well as model terms for specific toxicity of formamide and interactions between formamide and glycerol. To demonstrate the potential for model-based design of vitrification methods, we paired the multi-CPA toxicity model with a published vitrification/devitrification model to identify vitrifiable CPA mixtures that are predicted to have minimal toxicity. The resulting optimized vitrification solution composition was a mixture of 7.4 molal glycerol, 1.4 molal DMSO, and 2.4 molal formamide. This demonstrates the potential for mathematical optimization of vitrification solution composition and sets the stage for future studies to optimize the complete vitrification process, including CPA mixture composition and CPA addition and removal methods.

General tissue mass transfer model for cryopreservation applications.

Successful cryopreservation of complex specimens, such as tissues and organs, would greatly benefit both the medical and scientific research fields. Vitrification is one of the most promising techniques for complex specimen cryopreservation, but toxicity remains a major challenge because of the high concentration of cryoprotectants (CPAs) needed to vitrify. Our group has approached this problem using mathematical optimization to design less toxic CPA equilibration methods for cells. To extend this approach to tissues, an appropriate mass transfer model is required. Fick's law is commonly used, but this simple modeling framework does not account for the complexity of mass transfer in tissues, such as the effects of fixed charges, tissue size changes, and the interplay between cell membrane transport and transport through the extracellular fluid. Here, we propose a general model for mass transfer in tissues that accounts for all of these phenomena. To create this model, we augmented a previously published acellular model of mass transfer in articular cartilage to account for the effects of cells. We show that the model can accurately predict changes in CPA concentration and tissue size for both articular cartilage and pancreatic islets, tissue types with vastly different properties.

Human induced pluripotent stem cell derived kidney organoids as a model system for studying cryopreservation.

The ability to cryopreserve organs would have an enormous impact in transplantation medicine. To investigate organ cryopreservation strategies, experiments are typically done on whole organs, or on cells in 2D culture. Whole organs are not amenable to high throughput investigation, while conventional 2D culture is limited to a single cell type and lacks the complexity of the whole organ. In this study, we examine kidney organoids as a model system for studying cryopreservation. Consistent with previous studies, we show that kidney organoids comprised of multiple cell types can be generated in 96-well plates, with an average of about 8 organoids per well. We present a live/dead staining and image analysis method for quantifying organoid viability and show that this method can be used for assessing cryoprotectant toxicity. Our results highlight the potential for using organoids for high throughput investigation of cryopreservation approaches.

Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method.

Cryopreservation in a vitrified state has vast potential for long-term storage of tissues and organs that may be damaged by ice formation. However, the toxicity imparted by the high concentration of cryoprotectants (CPAs) required to vitrify these specimens remains a hurdle. To address this challenge, we previously developed a mathematical approach to design less toxic CPA equilibration methods based on the minimization of a toxicity cost function. This approach was used to design improved methods for equilibration of bovine pulmonary artery endothelial cells (BPAEC) with glycerol. To fully capitalize on the toxicity cost function approach, it is critical to describe the toxicity kinetics of additional CPAs, including multi-CPA mixtures that are commonly used for vitrification. In this work, we used automated liquid handling to characterize the toxicity kinetics of five of the most common CPAs (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide), along with their binary and ternary mixtures for BPAEC. In doing so, we developed experimental methods that can be used to determine toxicity kinetics more quickly and accurately. Our results highlight some common CPA toxicity trends, including the relatively low toxicity of ethylene glycol and a general increase in toxicity as the CPA concentration increases. Our results also suggest potential new approaches to reduce toxicity, including a surprising toxicity neutralization effect of glycerol on formamide. In the future, this dataset will serve as the basis to expand our CPA toxicity model, enabling application of the toxicity cost function approach to vitrification solutions containing multiple CPAs.

Implications of variability in cell membrane permeability for design of methods to remove glycerol from frozen-thawed erythrocytes.

In North America, red blood cells (RBCs) are currently cryopreserved in a solution of 40% glycerol. While glycerol is not inherently toxic to humans, it must be removed prior to transfusion to prevent intravascular osmotic hemolysis. The current deglycerolization procedure requires about 45 min per RBC unit. We previously presented predictions suggesting that glycerol could be safely removed from RBCs in less than 1 min. However, experimental evaluation of these methods resulted in much higher hemolysis than expected. Here we extend our previous study by considering both concentration-dependence of permeability and variability in permeability values in the mathematical optimization algorithm. To establish a model for the concentration dependence of glycerol permeability, we combined literature data with new measurements of permeability in the presence of 40% glycerol. To account for cell-dependent variability we scaled the concentration-dependent permeability model to define a permeability range for optimization. Methods designed using a range extending to 50% of the model-predicted glycerol permeability had a duration of less than 3 min and resulted in hemolysis ranging from 34% to 83%; hemolysis values were highly dependent on the blood donor. Extending the permeability range to 5% of the model-predicted value yielded a 30 min method that resulted in an average hemolysis of 12%. Our results suggest high variability in the glycerol permeability between donors and within a population of cells from the same donor. Such variability has broad implications for design of methods for equilibration of cells with cryoprotectants.

Simultaneous monitoring of different vitrification solution components permeating into tissues.

Cryopreservation can be used for long-term preservation of tissues and organs. It relies on using complex mixtures of cryoprotective agents (CPAs) to reduce the damaging effects of freezing, but care should be taken to avoid toxic effects of CPAs themselves. In order to rationally design cryopreservation strategies for tissues, it is important to precisely determine permeation kinetics of the protectants that are used to ensure maximum permeation, while minimizing the exposure time and toxicity effects. This is particularly challenging with protectant solutions consisting of multiple components each with different physical properties and diffusing at a different rate. In this study, we show that an attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) setup can be used to simultaneously monitor diffusion of multiple components in a mixture into tissues in real time. Diffusion studies were done with decellularized heart valves using a sucrose-DMSO mixture as well as vitrification solution VS83. To assess diffusion kinetics of different solutes in mixtures, the increase in specific infrared absorbance bands was monitored during diffusion through the tissue. Solute specific wavenumber ranges were selected, and the calculated area was assumed to be proportional to the CPA concentration in the tissue. A diffusion equation based on Fick's second law of diffusion fitted the experimental data quite well, and clear differences in permeation rates were observed among the different mixture components dependent on molecular size and physical properties.

Concentration dependence of the cell membrane permeability to cryoprotectant and water and implications for design of methods for post-thaw washing of human erythrocytes.

For more than fifty years the human red blood cell (RBC) has been a widely studied model for transmembrane mass transport. Existing literature spans myriad experimental designs with varying results and physiologic interpretations. In this review, we examine the kinetics and mechanisms of membrane transport in the context of RBC cryopreservation. We include a discussion of the pathways for water and glycerol permeation through the cell membrane and the implications for mathematical modeling of the membrane transport process. In particular, we examine the concentration dependence of water and glycerol transport and provide equations for estimating permeability parameters as a function of concentration based on a synthesis of literature data. This concentration-dependent transport model may allow for design of improved methods for post-thaw removal of glycerol from cryopreserved blood. More broadly, the consideration of the concentration dependence of membrane permeability parameters may be important for other cell types as well, especially for design of methods for equilibration with the highly concentrated solutions used for vitrification.

A toxicity cost function approach to optimal CPA equilibration in tissues.

There is growing need for cryopreserved tissue samples that can be used in transplantation and regenerative medicine. While a number of specific tissue types have been successfully cryopreserved, this success is not general, and there is not a uniform approach to cryopreservation of arbitrary tissues. Additionally, while there are a number of long-established approaches towards optimizing cryoprotocols in single cell suspensions, and even plated cell monolayers, computational approaches in tissue cryopreservation have classically been limited to explanatory models. Here we develop a numerical approach to adapt cell-based CPA equilibration damage models for use in a classical tissue mass transport model. To implement this with real-world parameters, we measured CPA diffusivity in three human-sourced tissue types, skin, fibroid and myometrium, yielding propylene glycol diffusivities of 0.6 × 10-6 cm2/s, 1.2 × 10-6 cm2/s and 1.3 × 10-6 cm2/s, respectively. Based on these results, we numerically predict and compare optimal multistep equilibration protocols that minimize the cell-based cumulative toxicity cost function and the damage due to excessive osmotic gradients at the tissue boundary. Our numerical results show that there are fundamental differences between protocols designed to minimize total CPA exposure time in tissues and protocols designed to minimize accumulated CPA toxicity, and that "one size fits all" stepwise approaches are predicted to be more toxic and take considerably longer than needed.

Investigating the potential for cryopreservation of human granulocytes with concentrated glycerol.

The purpose of this study was to investigate the potential for cryopreservation of granulocytes using 30% glycerol. Recently reported permeability data was used to design two different methods for addition and removal of glycerol: a fast method that is predicted to keep cell volumes between 80% and 150% of the isotonic volume and a slow method that is predicted to keep cell volumes between 80% and 115% of the isotonic volume. The fast method resulted in cell recoveries of 31% ± 9% and 11% ± 3% before and after freezing, respectively, whereas the slow method resulted in even lower cell recoveries of 5% ± 2% and 4% ± 2%. The reduced cell recovery for the slow method is consistent with an increase in damage as a result of glycerol toxicity. Our results suggest that cryopreservation of granulocytes in concentrated glycerol is not feasible.

Mathematically optimized cryoprotectant equilibration procedures for cryopreservation of human oocytes.

BACKGROUND: Simple and effective cryopreservation of human oocytes would have an enormous impact on the financial and ethical constraints of human assisted reproduction. Recently, studies have demonstrated the potential for cryopreservation in an ice-free glassy state by equilibrating oocytes with high concentrations of cryoprotectants (CPAs) and rapidly cooling to liquid nitrogen temperatures. A major difficulty with this approach is that the high concentrations required for the avoidance of crystal formation (vitrification) also increase the risk of osmotic and toxic damage. We recently described a mathematical optimization approach for designing CPA equilibration procedures that avoid osmotic damage and minimize toxicity, and we presented optimized procedures for human oocytes involving continuous changes in solution composition. METHODS: Here we adapt and refine our previous algorithm to predict piecewise-constant changes in extracellular solution concentrations in order to make the predicted procedures easier to implement. Importantly, we investigate the effects of using alternate equilibration endpoints on predicted protocol toxicity. Finally, we compare the resulting procedures to previously described experimental methods, as well as mathematically optimized procedures involving continuous changes in solution composition. RESULTS: For equilibration with CPA, our algorithm predicts an optimal first step consisting of exposure to a solution containing only water and CPA. This is predicted to cause the cells to initially shrink and then swell to the maximum cell volume limit. To reach the target intracellular CPA concentration, the cells are then induced to shrink to the minimum cell volume limit by exposure to a high CPA concentration. For post-thaw equilibration to remove CPA, the optimal procedures involve exposure to CPA-free solutions that are predicted to cause swelling to the maximum volume limit. The toxicity associated with these procedures is predicted to be much less than that of conventional procedures and comparable to that of the corresponding procedures with continuous changes in solution composition. CONCLUSIONS: The piecewise-constant procedures described in this study are experimentally facile and are predicted to be less toxic than conventional procedures for human oocyte cryopreservation. Moreover, the mathematical optimization approach described here will facilitate the design of cryopreservation procedures for other cell types.

Membrane permeability of the human granulocyte to water, dimethyl sulfoxide, glycerol, propylene glycol and ethylene glycol.

Granulocytes are currently transfused as soon as possible after collection because they rapidly deteriorate after being removed from the body. This short shelf life complicates the logistics of granulocyte collection, banking, and safety testing. Cryopreservation has the potential to significantly increase shelf life; however, cryopreservation of granulocytes has proven to be difficult. In this study, we investigate the membrane permeability properties of human granulocytes, with the ultimate goal of using membrane transport modeling to facilitate development of improved cryopreservation methods. We first measured the equilibrium volume of human granulocytes in a range of hypo- and hypertonic solutions and fit the resulting data using a Boyle-van't Hoff model. This yielded an isotonic cell volume of 378 µm(3) and an osmotically inactive volume of 165 µm(3). To determine the permeability of the granulocyte membrane to water and cryoprotectant (CPA), cells were injected into well-mixed CPA solution while collecting volume measurements using a Coulter Counter. These experiments were performed at temperatures ranging from 4 to 37°C for exposure to dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol. The best-fit water permeability was similar in the presence of all of the CPAs, with an average value at 21°C of 0.18 µmatm(-1)min(-1). The activation energy for water transport ranged from 41 to 61 kJ/mol. The CPA permeability at 21°C was 6.4, 1.0, 8.4, and 4.0 µm/min for dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol, respectively, and the activation energy for CPA transport ranged between 59 and 68 kJ/mol.

Osmotic tolerance limits of red blood cells from umbilical cord blood.

Effective methods for long-term preservation of cord red blood cells (RBCs) are needed to ensure a readily available supply of RBCs to treat fetal and neonatal anemia. Cryopreservation is a potential long-term storage strategy for maintaining the quality of cord RBCs for the use in intrauterine and neonatal transfusion. However, during cryopreservation, cells are subjected to damaging osmotic stresses during cryoprotectant addition and removal and freezing and thawing that require knowledge of osmotic tolerance limits in order to optimize the preservation process. The objective of this study was to characterize the osmotic tolerance limits of cord RBCs in conditions relevant to cryopreservation, and compare the results to the osmotic tolerance limits of adult RBCs. Osmotic tolerance limits were determined by exposing RBCs to solutions of different concentrations to induce a range of osmotic volume changes. Three treatment groups of adult and cord RBCs were tested: (1) isotonic saline, (2) 40% w/v glycerol, and (3) frozen-thawed RBCs in 40% w/v glycerol. We show that cord RBCs are more sensitive to shrinkage and swelling than adult RBCs, indicating that osmotic tolerance limits should be considered when adding and removing cryoprotectants. In addition, freezing and thawing resulted in both cord and adult RBCs becoming more sensitive to post-thaw swelling requiring that glycerol removal procedures for both cell types ensure that cell volume excursions are maintained below 1.7 times the isotonic osmotically active volume to attain good post-wash cell recovery. Our results will help inform the development of optimized cryopreservation protocol for cord RBCs.

Optimization of cryoprotectant loading into murine and human oocytes.

Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethyl sulfoxide (Me(2)SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me(2)SO exposure time, revealing that neither shrinkage nor Me(2)SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me(2)SO addition appears to result from interactions between the effects of Me(2)SO toxicity and osmotic stress. We also investigated Me(2)SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me(2)SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me(2)SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach.

Effects of intercellular junction protein expression on intracellular ice formation in mouse insulinoma cells.

The development of cryopreservation procedures for tissues has proven to be difficult in part because cells within tissue are more susceptible to intracellular ice formation (IIF) than are isolated cells. In particular, previous studies suggest that cell-cell interactions increase the likelihood of IIF by enabling propagation of ice between neighboring cells, a process thought to be mediated by gap junction channels. In this study, we investigated the effects of cell-cell interactions on IIF using three genetically modified strains of the mouse insulinoma cell line MIN6, each of which expressed key intercellular junction proteins (connexin-36, E-cadherin, and occludin) at different levels. High-speed video cryomicroscopy was used to visualize the freezing process in pairs of adherent cells, revealing that the initial IIF event in a given cell pair was correlated with a hitherto unrecognized precursor phenomenon: penetration of extracellular ice into paracellular spaces at the cell-cell interface. Such paracellular ice penetration occurred in the majority of cell pairs observed, and typically preceded and colocalized with the IIF initiation events. Paracellular ice penetration was generally not observed at temperatures >-5.65°C, which is consistent with a penetration mechanism via defects in tight-junction barriers at the cell-cell interface. Although the maximum temperature of paracellular penetration was similar for all four cell strains, genetically modified cells exhibited a significantly higher frequency of ice penetration and a higher mean IIF temperature than did wild-type cells. A four-state Markov chain model was used to quantify the rate constants of the paracellular ice penetration process, the penetration-associated IIF initiation process, and the intercellular ice propagation process. In the initial stages of freezing (>-15°C), junction protein expression appeared to only have a modest effect on the kinetics of propagative IIF, and even cell strains lacking the gap junction protein connexin-36 exhibited nonnegligible ice propagation rates.

Detection of volume changes in calcein-stained cells using confocal microscopy.

Calcein is an intracellular fluorescent probe that has been used as an indicator of cell volume in several previous studies. These studies have reported two different fluorescence responses depending on the optical setup used to collect the data: wide-field microscopy has resulted in a decrease in fluorescence upon cell shrinkage, whereas confocal microscopy has been shown to yield the opposite result. In this short communication, we have investigated the effect of optical setup on detection of cell volume changes in calcein-stained endothelial cells. A confocal microscope was used to collect the fluorescence data, and the pinhole diameter was varied in order to examine the effects of optical section thickness on fluorescence response. For large pinhole diameters - which correspond to relatively thick optical sections - fluorescence intensity decreased when cells were induced to shrink. In contrast, for small pinhole diameters the fluorescence intensity increased with cell shrinkage. The transition between these two types of fluorescence responses occurred when using a pinhole diameter of 285 µm, which corresponds with an optical section thickness slightly less than the height of the cells. Our results have implications for the design and interpretation of experiments involving the use of calcein as a cell volume indicator.

Effect of intercellular junction protein expression on water transport during freezing of MIN6 cells.

A mouse insulinoma (MIN6) strain in which connexin expression has been inhibited by antisense technology holds promise as an experimental model system for investigating the role of gap junctions in intercellular ice propagation. However, to properly interpret measurements of intracellular ice formation kinetics, the effects of cell dehydration on cytoplasmic supercooling must be determined. Thus, the cell membrane water permeability in monolayer cultures of the antisense-transfected MIN6 strain was measured using a fluorescence quenching method. By repeating the experiments at 4°C, 12°C, 21°C, and 37°C, the activation energy for water transport was determined to be E(a) = 51 ± 3 k J/mol. Although differences between membrane permeability measurements in theantisense and wild-type strains were not statistically significant, simulation of water transport during rapid freezing (130°C/min) predicted that intracellular supercooling in the genetically modified MIN6 strain may become significantly larger than the supercooling in wild-type cells at temperatures below -15°C.

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.