Nanowarming and ice-free cryopreservation of large sized, intact porcine articular cartilage.

Successful organ or tissue long-term preservation would revolutionize biomedicine. Cartilage cryopreservation enables prolonged shelf life of articular cartilage, posing the prospect to broaden the implementation of promising osteochondral allograft (OCA) transplantation for cartilage repair. However, cryopreserved large sized cartilage cannot be successfully warmed with the conventional convection warming approach due to its limited warming rate, blocking its clinical potential. Here, we develope a nanowarming and ice-free cryopreservation method for large sized, intact articular cartilage preservation. Our method achieves a heating rate of 76.8 °C min-1, over one order of magnitude higher than convection warming (4.8 °C min-1). Using systematic cell and tissue level tests, we demonstrate the superior performance of our method in preserving large cartilage. A depth-dependent preservation manner is also observed and recapitulated through magnetic resonance imaging and computational modeling. Finally, we show that the delivery of nanoparticles to the OCA bone side could be a feasible direction for further optimization of our method. This study pioneers the application of nanowarming and ice-free cryopreservation for large articular cartilage and provides valuable insights for future technique development, paving the way for clinical applications of cryopreserved cartilage.

Ice Control during Cryopreservation of Heart Valves and Maintenance of Post-Warming Cell Viability.

Heart valve cryopreservation was employed as a model for the development of complex tissue preservation methods based upon vitrification and nanowarming. Porcine heart valves were loaded with cryoprotectant formulations step wise and vitrified in 1−30 mL cryoprotectant formulations ± Fe nanoparticles ± 0.6 M disaccharides, cooled to −100 °C, and stored at −135 °C. Nanowarming was performed in a single ~100 s step by inductive heating within a magnetic field. Controls consisted of fresh and convection-warmed vitrified heart valves without nanoparticles. After washing, cell viability was assessed by metabolic assay. The nanowarmed leaflets were well preserved, with a viability similar to untreated fresh leaflets over several days post warming. The convection-warmed leaflet viability was not significantly different than that of the nanowarmed leaflets immediately after rewarming; however, a significantly higher nanowarmed leaflet viability (p < 0.05) was observed over time in vitro. In contrast, the associated artery and fibrous cardiac muscle were at best 75% viable, and viability decreased over time in vitro. Supplementation of lower concentration cryoprotectant formulations with disaccharides promoted viability. Thicker tissues benefited from longer-duration cryoprotectant loading steps. The best outcomes included a post-warming incubation step with α-tocopherol and an apoptosis inhibitor, Q-VD-OPH. This work demonstrates progress in the control of ice formation and cytotoxicity hurdles for the preservation of complex tissues.

Development of a Vitrification Preservation Process for Bioengineered Epithelial Constructs.

The demand for human bioengineered tissue constructs is growing in response to the worldwide movement away from the use of animals for testing of new chemicals, drug screening and household products. Presently, constructs are manufactured and delivered just in time, resulting in delays and high costs of manufacturing. Cryopreservation and banking would speed up delivery times and permit cost reduction due to larger scale manufacturing. Our objective in these studies was development of ice-free vitrification formulations and protocols using human bioengineered epithelial constructs that could be scaled up from individual constructs to 24-well plates. Initial experiments using single EpiDerm constructs in vials demonstrated viability >80% of untreated control, significantly higher than our best freezing strategy. Further studies focused on optimization and evaluation of ice-free vitrification strategies. Vitrification experiments with 55% (VS55) and 70% (VS70) cryoprotectant (CPA) formulations produced constructs with good viability shortly after rewarming, but viability decreased in the next days, post-rewarming in vitro. Protocol changes contributed to improved outcomes over time in vitro. We then transitioned from using glass vials with 1 construct to deep-well plates holding up to 24 individual constructs. Construct viability was maintained at >80% post-warming viability and >70% viability on days 1−3 in vitro. Similar viability was demonstrated for other related tissue constructs. Furthermore, we demonstrated maintenance of viability after 2−7 months of storage below −135 °C.

Vitrification of Heart Valve Tissues.

Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples (1-3 mL total volume) where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, permits preservation of large samples (80-100 mL total volume) with several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at -80 °C; better matrix preservation than freezing with retention of material properties; very low cell viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is not used; and reduced manufacturing costs. More recently, we have developed Protocol 4 in which VS55 is supplemented with sugars resulting in reduced concerns regarding nucleation during cooling and warming. This method can be used for large samples resulting in retention of cell viability and permits short-term exposure to -80 °C with long-term storage preferred at or below -135 °C.

Imaging the distribution of iron oxide nanoparticles in hypothermic perfused tissues.

PURPOSE: Herein, we evaluate the use of MRI as a tool for assessing iron oxide nanoparticle (IONP) distribution within IONP perfused organs and vascularized composite allografts (VCAs) (i.e., hindlimbs) prepared for cryopreservation. METHODS: Magnetic resonance imaging was performed on room-temperature organs and VCAs perfused with IONPs and were assessed at 9.4 T. Quantitative T1 mapping and T2∗ -weighted images were acquired using sweep imaging with Fourier transformation and gradient-echo sequences, respectively. Verification of IONP localization was performed through histological assessment and microcomputer tomography. RESULTS: Quantitative imaging was achieved for organs and VCAs perfused with up to 642 mMFe (36 mgFe /mL), which is above previous demonstrations of upper limit detection in agarose (35.7mMFe [2 mgFe /mL]). The stability of IONPs in the perfusate had an effect on the quality of distribution and imaging within organs or VCA. Finally, MRI provided more accurate IONP localization than Prussian blue histological staining in this system, wherein IONPs remain primarily in the vasculature. CONCLUSION: Using MRI, we were able to assess the distribution of IONPs throughout organs and VCAs varying in complexity. Additional studies are necessary to better understand this system and validate the calibration between T1 measurements and IONP concentration.

The Impact of Heat Treatment on Porcine Heart Valve Leaflets.

The purpose of this study was to determine the impact of elevated temperature exposure in tissue banking on soft tissues. A secondary objective was to determine the relative ability of various assays to detect changes in soft tissues due to temperature deviations. Porcine pulmonary heart valve leaflets exposed to 37 °C were compared with those incubated at 52 and 67 °C for 10, 30 and 100 min. The analytical methods consisted of (1) viability assessment using the resazurin assay, (2) collagen content using the Sircol assay, and (3) permeability assessment using an electrical conductivity assay. Additionally, histology and two photon microscopy were used to reveal mechanisms of cell and tissue damage. Viability, collagen content, and permeability all decreased following heat treatment. In terms of statistical significance with respect to treatment temperature, cell viability was most affected (p < 0.0001), followed by permeability (p < 0.0001), and then collagen content (p = 0.13). After heat treatment, histology indicated increased apoptosis and two photon microscopy revealed a decrease in collagen fiber organization and an increase in elastin density. These results suggest that measures of cell viability would be best for assessing tissues where the cells are alive and that permeability may be best where cell viability is not intentionally maintained.

Best practices for cryopreserving, thawing, recovering, and assessing cells.

Long-term storage of cell stocks insures that cells are available for use whenever needed. Cryopreservation of cells is the method of choice for preservation of important or rare cell stocks. There are several factors to consider when establishing a protocol for freezing, thawing, and recovery of cells after storage. These parameters may include cell concentration, cryoprotectant choice and concentration, and thawing rate among others. Further, the assessment of cell viability and/or function prior to and following cryopreservation is imperative in order to accurately determine downstream utility as well for optimizing the cryopreservation process. This chapter is designed to provide guidance and insight into developing robust and successful protocols for preserving cells that will preserve cell stocks and provide optimal cell yield and viability.

Comparison and evaluation of biomechanical, electrical, and biological methods for assessment of damage to tissue collagen.

In regard to evaluating tissue banking methods used to preserve or otherwise treat (process) soft allograft tissue, current tests may not be sufficiently sensitive to detect potential damage inflicted before, during, and after processing. Using controlled parameters, we aim to examine the sensitivity of specific biomechanical, electrical, and biological tests in detecting mild damage to collagen. Fresh porcine pulmonary heart valves were treated with an enzyme, collagenase, and incubated using various times. Controls received no incubation. All valves were cryopreserved and stored at -135 °C until being rewarmed for evaluation using biomechanical, permeability, and cell viability tests. Statistically significant time dependent changes in leaflet ultimate stress, (p = 0.006), permeability (p = 0.01), and viability (p ≤ 0.02, four different days of culture) were found between heart valves subjected to 0-15 min of collagenase treatment (ANOVA). However, no statistical significance was found between the tensile modulus of treated and untreated valves (p = 0.07). Furthermore, the trends of decreasing and increasing ultimate stress and viability, respectively, were somewhat inconsistent across treatment times. These results suggest that permeability tests may offer a sensitive, quantitative assay to complement traditional biomechanical and viability tests in evaluating processing methods used for soft tissue allografts, or when making changes to current validated methods. Multiple test evaluation may also offer insight into the mechanism of potential tissue damage such as, as is the case here, reduced collagen content and increased tissue porosity.

Survey of Apoptosis After Hypothermic Storage of a Pancreatic ß-Cell Line.

Insulin-dependent diabetes mellitus is one of the leading causes of death world wide. Donor-derived pancreas and islet of Langerhans transplantation are potential cures, however, postmortem ischemia impacts islet quality. The murine ßt3 cell line was used as a model to study apoptosis after hypothermic storage by comparing Unisol™ with Belzer's machine perfusion solution (BMPS) and the University of Wisconsin (UW) solution. The objective was to determine which of these solutions provided the best support for ßt3 cells and which solution demonstrated the least amount of apoptotic activity. Several apoptosis markers were measured that included the translocation of phosphatidylserine, caspase activity, and the formation of DNA laddering. In addition, metabolic activity and membrane integrity were also measured. The results demonstrated that the three solutions behaved similarly during overnight cold storage at 4°C. However, Unisol was consistently better than UW solution and BMPS, demonstrating better cell viability and recovery, and lower levels of apoptotic activity when all three parameters were measured. These results demonstrated that apoptosis plays an important role in the survival of cells and tissues during cold storage. Development of solutions to help prevent or decrease the levels of apoptosis after cold storage will likely improve overall cell and tissue recovery and survival in a clinical setting.

Vitrification of heart valve tissues.

Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80 % cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, has several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at -80 °C; better matrix preservation than freezing with retention of material properties; very low cell viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is not used; and reduced manufacturing costs.

Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions.

Expanding cryopreservation methods to include a wider range of cell types, such as those sensitive to freezing, is needed for maintaining the viability of cell-based regenerative medicine products. Conventional cryopreservation protocols, which include use of cryoprotectants such as dimethylsulfoxide (Me2SO), have not prevented ice-induced damage to cell and tissue matrices during freezing. A family of antifreeze proteins (AFPs) produced in the larvae of the beetle, Dendroides canadensis allow this insect to survive subzero temperatures as low as -26°C. This study is an assessment of the effect of the four hemolymph D. canadensis AFPs (DAFPs) on the supercooling (nucleating) temperature, ice structure patterns and viability of the A10 cell line derived from the thoracic aorta of embryonic rat. Cryoprotectant solution cocktails containing combinations of DAFPs in concentrations ranging from 0 to 3mg/mL in Unisol base mixed with 1M Me2SO were first evaluated by cryomicroscopy. Combining multiple DAFPs demonstrated significant supercooling point depressing activity (∼9°C) when compared to single DAFPs and/or conventional 1M Me2SO control solutions. Concentrations of DAFPs as low as 1 µg/mL were sufficient to trigger this effect. In addition, significantly improved A10 smooth muscle cell viability was observed in cryopreservation experiments with low DAFP-6 and DAFP-2 concentrations in combination with Me2SO. No significant improvement in viability was observed with either DAFP-1 or DAFP-4. Low and effective DAFP concentrations are advantageous because they minimize concerns regarding cell cytotoxicity and manufacturing cost. These findings support the potential of incorporating DAFPs in solutions used to cryopreserve cells and tissues.

Development of pancreas storage solutions: Initial screening of cytoprotective supplements for ß-cell survival and metabolic status after hypothermic storage.

Insulin-dependent diabetes mellitus is one of the leading causes of death world-wide. Donor-derived pancreas and Islet of Langerhans transplantation are potential cures; however, postmortem ischemia impacts islet quality. The murine ßt3 cell line was employed as a model to study cell viability and proliferation after hypothermic storage by comparing Belzer's Machine Perfusion Solution with Unisol™ Solution. The objective was to determine which of these solutions provided the best base line support for ßt3 cells and to screen potential cytoprotective additives to the solutions. Initial ßt3 cell viability was similar in the two storage solutions; however, better proliferation was observed after storage in Unisol Solution. The caspase inhibitor, Q-VD-OPH, and α-tocopherol improved viability in both storage solutions, suggesting that apoptotic pathways may be responsible for cell death during hypothermic storage of ßt3 cells. Analysis of apoptosis markers, caspase activity, and DNA laddering showed a reduction in apoptosis when these additives were included. The effects of Q-VD-OPH and α-tocopherol were also synergistic when employed together during either hypothermic exposure, post-hypothermic physiologic incubation, or combinations of hypothermic exposure and physiologic incubation. These results suggest that both supplements should be included in pancreas hypothermic storage solutions and in islet culture media during post-isolation culture prior to transplantation.

MitoQ blunts mitochondrial and renal damage during cold preservation of porcine kidneys.

Cold preservation has greatly facilitated the use of cadaveric kidneys for transplantation but damage occurs during the preservation episode. It is well established that oxidant production increases during cold renal preservation and mitochondria are a key target for injury. Our laboratory has demonstrated that cold storage of renal cells and rat kidneys leads to increased mitochondrial superoxide levels and mitochondrial electron transport chain damage, and that addition of Mitoquinone (MitoQ) to the preservation solutions blunted this injury. In order to better translate animal studies, the inclusion of large animal models is necessary to develop safe preclinical protocols. Therefore, we tested the hypothesis that addition of MitoQ to cold storage solution preserves mitochondrial function by decreasing oxidative stress, leading to less renal tubular damage during cold preservation of porcine kidneys employing a standard criteria donor model. Results showed that cold storage significantly induced oxidative stress (nitrotyrosine), renal tubular damage, and cell death. Using High Resolution Respirometry and fresh porcine kidney biopsies to assess mitochondrial function we showed that MitoQ significantly improved complex II/III respiration of the electron transport chain following 24 hours of cold storage. In addition, MitoQ blunted oxidative stress, renal tubular damage, and cell death after 48 hours. These results suggested that MitoQ decreased oxidative stress, tubular damage and cell death by improving mitochondrial function during cold storage. Therefore this compound should be considered as an integral part of organ preservation solution prior to transplantation.

Culturing with trehalose produces viable endothelial cells after cryopreservation.

Dimethylsulfoxide, the most commonly employed cryoprotectant for cells, has well documented cytotoxic effects in patients. Among the compounds available that may provide protection to cells and tissues during preservation with less cytotoxicity is trehalose. Some animals, such as brine shrimp and tardigrades, accumulate trehalose during periods of extreme environmental stress. In this study, experiments were performed to evaluate the effects of culturing a bovine endothelial cell line (ATCC #CCL-209) in the presence of trehalose prior to preservation by freezing. A number of factors were shown to contribute to cell retention of metabolic activity and proliferative potential including cell culture time with trehalose and the solution conditions during cryopreservation. Using an optimized protocol consisting of 24 h of cell culture with 0.2 M trehalose followed by cryopreservation with 0.2-0.4 M trehalose in sodium bicarbonate buffered Eagles minimum essential medium at pH 7.4 resulted in 87±4% post-preservation cell metabolic activity expressed as relative fluorescence based upon reduction of resazurin to resorufin. This new method provides an alternative preservation strategy to the more classical preservation methods employing dimethylsulfoxide available for cells and tissues.

Lessons from nature for preservation of mammalian cells, tissues, and organs.

The study of mechanisms by which animals tolerate environmental extremes may provide strategies for preservation of living mammalian materials. Animals employ a variety of compounds to enhance their survival, including production of disaccharides, glycerol, and antifreeze compounds. The cryoprotectant glycerol was discovered before its role in amphibian survival. In the last decade, trehalose has made an impact on freezing and drying methods for mammalian cells. Investigation of disaccharides was stimulated by the variety of organisms that tolerate dehydration stress by accumulation of disaccharides. Several methods have been developed for the loading of trehalose into mammalian cells, including inducing membrane lipid-phase transitions, genetically engineered pores, endocytosis, and prolonged cell culture with trehalose. In contrast, the many antifreeze proteins (AFPs) identified in a variety of organisms have had little impact. The first AFPs to be discovered were found in cold water fish; their AFPs have not found a medical application. Insect AFPs function by similar mechanisms, but they are more active and recombinant AFPs may offer the best opportunity for success in medical applications. For example, in contrast to fish AFPs, transgenic organisms expressing insect AFPs exhibit reduced ice nucleation. However, we must remember that nature's survival strategies may include production of AFPs, antifreeze glycolipids, ice nucleators, polyols, disaccharides, depletion of ice nucleators, and partial desiccation in synchrony with the onset of winter. We anticipate that it is only by combining several natural low temperature survival strategies that the full potential benefits for mammalian cell survival and medical applications can be achieved.

Comparison of electroporation and Chariot™ for delivery of ß-galactosidase into mammalian cells: strategies to use trehalose in cell preservation.

There are many compounds that can and have been used as cryoprotectants including disaccharides such as trehalose. Many organisms in nature use trehalose to help protect themselves at colder temperatures. Trehalose has also been used to a limited extent for the preservation of mammalian cells and tissues, but mainly as a supplement to other cryoprotectants like dimethyl sulfoxide. Recently, the use of trehalose as the primary cryoprotectant has gained much interest because of its low-potential cytotoxicity. Trehalose does not readily pass through mammalian cells membranes and research has shown that it is most effective when present on both sides of the cell membrane prior to preservation. Different strategies for introducing disaccharide sugars into cells have been investigated with limited success. In this study, two separate strategies are investigated for the introduction of disaccharide sugars into cells. Electroporation using an electric pulse to create temporary holes in the membrane so that molecules could pass through and a transport peptide (Chariot™) that covalently binds to the molecule of interest and then moves it across the membrane. Both strategies have the potential to load disaccharide sugars into cells at concentrations that would provide ample protection during preservation. In preparation for cryopreservation studies, smooth muscle cells that are difficult to cryopreserve using conventional preservation protocols were used to evaluate and compare the translocation potential of these two strategies using ß-galactosidase. Assessment of each loading strategy was done by measuring viability and the presence of ß-galactosidase inside the cells. The results indicate that both methods appear feasible as potential delivery systems and that treatment cytotoxicity can be minimized. The next step is definition of the best loading strategy to introduce trehalose into cells followed by preservation by freezing.

Serum-free solutions for cryopreservation of cells.

With the development of cell-based assays and therapies, the purity of reagents used to grow and maintain cells has become much more important. In particular, the use of fetal calf serum for culturing cells presents a direct path for potential contamination of cell cultures. In recent years, much research has focused on the development of serum-free culturing systems, not only to alleviate difficulties due to availability and cost of fetal calf serum but also to prevent the transmission of potentially fatal diseases to human patients. Additionally, methods need to be developed for long-term storage of cell stocks that also reduce the risk of exposure to harmful diseases. As most methods employ fetal calf serum in their freezing formulations, solutions that avoid the use of fetal calf serum while providing equivalent or better recovery of cells upon thawing would be ideal. In this study, two vascular cell lines have been cryopreserved as adherent cell populations in two widely used cryoprotectants, dimethyl sulfoxide and 1,2-propanediol, and two vehicle solutions, Euro-Collins and Unisol-cryoprotectant vehicle specifically formulated for the maintenance of cell homeostasis at temperatures below 37 degrees C. The addition of serum to these formulations was also evaluated to determine if its presence provided any additional benefit to the cells during cryopreservation. The results demonstrated that using vehicle solutions designed for lower temperatures produced viable cells that retained cell population viability values up to 75% of unfrozen controls. These results also demonstrated that including serum in the formulation provided no additional benefit to the cells and in some cases actually produced lower cell viability after cryopreservation. In conclusion, the development of solutions designed for low-temperature storage of cells provides a viable alternative to more conventional cryopreservation protocols and eliminates the necessity of including serum in these formulations.

Interstitial fluid analysis for assessment of organ function.

Evaluation methods are required for non-heart-beating donor (NHBD) kidneys to ensure the success of transplantation. In this study, the microdialysis technique was employed for the ex-vivo assessment of hypothermically preserved NHBD kidney function. Microdialysis probes were placed in the renal cortex of 2 h warm ischaemic porcine kidneys to monitor interstitial pyruvate dynamics during hypothermic machine perfusion with perfusate containing 29.4 mM fructose-1,6-diphosphate (FDP). The presence of exogenous FDP in the perfusate induced no changes in the renal flow rate and vascular resistance, renal artery effluent biochemistry, or pyruvate concentration relative to untreated control kidneys. Significant increases in pyruvate production (P < 0.05), however, were observed after 12 h of perfusion in the interstitial fluid of FDP-treated kidneys relative to control kidneys. After 24 h of perfusion, interstitial fluid concentrations of pyruvate were 149.1 +/- 58.4 vs. 55.6 +/- 17.9 micro M (P < 0.05) in the FDP and control group, respectively. The microdialysis probe collected the interstitial fluid directly from the cellular sites of metabolic and synthetic activity, where perfusate dilution was minimal. Consequently, the biochemical changes induced by the organ metabolic activity were detected only at the interstitial level, in the microdialysates. Interstitial fluid pyruvate may be a good indicator of kidney function. The addition of FDP to the perfusion solution during ischaemic kidney preservation resulted in enhanced pyruvate production in the extracellular space, indirectly reflecting an increase in anaerobic ATP production. The pyruvate will be transformed during organ reperfusion into acetyl Co-A enzyme allowing an immediate start of aerobic metabolism. This in turn can increase the amount of ATP available to the cells and may help prevent reperfusion injury upon transplantation.