330 Abstracts / Cryobiology 63 (2011) 306–342 ated with each mechanism. Conversely, the conventional approach and the improved algorithm yielded comparable results if only a single mechanism of IIF was active in the cell population. Source of funding: National Science Foundation Grant CBET-0954587. Conflict of interest: None. doi:10.1016/j.cryobiol.2011.09.089 87. The triad of evidence that intracellular ice is the cause of death in COS-7 tissue culture cells rapidly cooled to 70 °C. (I): The observed occurrence of intracellular ice as a function of temperature and cooling rate. Shinsuke Seki 1, Diana B. Peckys 1,2, Peter Mazur * 1, 1 Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, USA, 2 Dept. Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA It was proposed nearly fifty years ago that the formation of intracellular ice (IIF) in cell interiors is a major cause of death and it is probably THE major cause in rapidly cooled cells). Although that hypothesis is now rather widely accepted, strong evidence for it exists for only a few cell types (mammalian oocytes and embryos, and yeast cells) and there is one case of evidence in sperm that argues against it. We have argued that the strong evidence is based on a triad that consists of (I) experimental observations showing the percentage of the cells that undergo IIF as s function of temperature and the cooling rate, (II) Physical–chemical computations describing the expected shrinkage of the cells as a function of the same two variables, and (III) the survival of cells as a function of cooling rate and temperature. This talk and abstract deal with aspect I. The succeeding talk deals with aspect II. After growth to confluence, COS-7 cells were put into suspension in Tyrde’s physiological salt solution containing 1 M ethylene glycol. Over the ensuing 15 min, a  1.5 ll droplet of the suspension was centered in the quartz sample container for a Linkam cryostage, and cooling initiated on that stage. Groups of the suspended cells were observed as they cooled to 70 °C at 5, 10, 15, 25, 50, and 100 °C/min. IIF is manifested as an abrupt darkening or ‘‘flashing.” At the three higher cooling rates, the mean flash temperature was 19.0 °C, and 94.6% of the cells had undergone flashing by 70 °C. At the lowest cooling rate of 5 °C/min, only 20.7% of the cells darkened, and the mean darkening temperature was 11.5 °C, just about the temperature at which extracellular ice formed. This coincidence suggests that at this cooling rate, the internal darkening was a consequence of prior damage to the membranes or that EIF itself damaged the membranes. The 79.3% of the slowly cooled cells that did not flash, were presumably prevented from doing so by cell dehydration. Another way to ascertain whether IIF has occurred is to determine whether heat is released during the cooling of a concentrated cell suspension and produces an exothermic peak at appropriate temperatures. COS-7 cells did not undergo flashing until cooled to 19 °C. That deep supercooling was a consequence of the intact cell membrane blocking intracellular ice nucleation. But once IIF occurs, it disrupts the membranes, so that in a second cooling of the same suspension, there will be no cell supercooling and no second exotherm. This is in fact what occurred. Research supported by NIH grant R01 RR018470. Conflict of interest: None declared. doi:10.1016/j.cryobiol.2011.09.090 88. The triad of evidence that intracellular ice is the cause of death in COS-7 tissue culture cells rapidly cooled to 70 °C. (II): Comparison between the computed occurrence of intracellular ice as a function of temperature and cooling rate and the observed relationship. Peter Mazur *, Shinsuke Seki, Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, USA It was proposed nearly 50 years ago that the formation of intracellular ice (IIF) in the interiors of rapidly cooled cells is the major cause of their death. The evidence for that conclusion in a given cell comes from a triad that consists of (I) experimental observations showing the percentage of the cells that undergo IIF as a function of the temperature and the cooling rate, (II) Physical–chemical computations describing the expected shrinkage of the cells as a function of the same two variables, and (III) the survival of cells as a function of cooling rate and temperature. The preceding talk dealt with Aspect I in COS-7 tissue cells. The current talk deals with Aspect II. Cells with normal intact membranes remain unfrozen (i.e., supercooled) to temperatures well below those producing external ice. Such cells are in thermodynamic disequilibrium with respect to that ice, and consequently they tend to dehydrate in an effort to reach equilibrium. The extent of dehydration and its rate depend on primarily on the cooling rate, the temperature, the permeability of the cell to water, the activation energy of that permeability, and the surface to volume ratio of the cell. If these values and parameters are known for a particular cell type, the resulting set of four differential equations can be solved numerically to generate curves of cell water volume vs. temperature and cooling rate for the given cell. One can overlay these curves on the experimental observations of % IIF vs. temperature. To convert the computed water content curves for a cell to the probability of IIF, one needs to know one additional fact; namely, the mean subzero temperature at which the cell undergoes IIF. We now have all of the parameters and values on hand to solve these equations for COS-7 cells and predict the probability of IIF as functions of cooling rate and subzero temperature. The predictions compare well with the experimental observations reported in the previous paper for these cells. To complete the triad, we now need to obtain data on the survival of the COS-7 cells as a function of these same variables; i.e., cooling rate and temperature. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.091 89. Toxicity-minimized cryoprotectant addition and removal procedures for human oocytes. Allyson K. Fry 1, James D. Benson 2, Adam Z. Higgins * 1, 1 School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR 97331-2702, USA, 2 Applied and Computational Mathematics Division, National Institute of Standards and Technology, Gaithersburg, MD 20879, USA Vitrification methods show promise for cryopreservation of several types of cells and tissues, including human oocytes. These methods require the use of high cryoprotectant (CPA) concentrations to prevent ice formation. However, high CPA concentrations can be cytotoxic. Despite its importance, toxicity is not explicitly addressed in the traditional multistep strategy for designing CPA addition and removal procedures. In this study, we present a new strategy for designing multistep procedures with minimal toxicity, and we use this strategy to predict toxicity-minimized CPA addition and removal procedures for human oocytes. Our strategy for optimizing CPA addition and removal procedures is based on minimization of a toxicity cost functional, a quantity that represents the damage incurred due to toxicity. We defined the cost functional as the time integral of a concentration-dependent toxicity rate, and we estimated the toxicity rate using published viability data for fibroblasts and chondrocytes during exposure to Me2SO. We previously minimized this cost functional in an optimization algorithm to determine the time-varying extracellular solution composition that would achieve a goal state (i.e., a specific intracellular CPA content and cell volume) while keeping the cell volume within the osmotic tolerance limits. Because the resulting toxicity-minimized procedures involve a continuously changing extracellular solution composition, they are difficult to implement experimentally. In this study we restrict our analysis to procedures with step changes in solution composition, which are relatively easy to implement. Published permeability data for human oocytes were used to design toxicity-minimized procedures for addition and removal of ethylene glycol and dimethyl sulfoxide, CPAs that are commonly used for oocyte vitrification. To improve the speed of the mathematical optimization process, we took advantage of the fact that the two parameter membrane transport model can be reparameterized to obtain an analytical solution in terms of a transformed time. We reparametized the toxicity cost functional as well, enabling evaluation and minimization of the cost functional in transformed time using the analytical solution. This obviated the need for numerical integration and greatly improved convergence speed. The resulting addition procedures involve an initial step in which cells are induced to swell by exposure to a CPA solution with a hypotonic concentration of nonpermeating solutes. The cells are held in this solution until they swell to the maximum volume limit. In the final step of the addition procedure, the target intracellular CPA concentration is achieved by exposure to a concentrated CPA solution, which causes the cells to shrink to the minimum volume limit. Removal of CPA is achieved by exposure to CPA-free solutions that contain nonpermeating solutes to prevent excessive swelling. Funding for this research was provided by a National Research Council/National Institute of Standards and Technology Postdoctoral Associateship (to JDB) and a grant from the Medical Research Foundation of Oregon (to AZH). Conflict of interest: None declared. doi:10.1016/j.cryobiol.2011.09.092 90. Characterization of heart valves cryopreserved with 83% cryoprotectants and stored at 80 °C. Agnes J. Huber * 1, Kelvin G.M. Brockbank 2,3, Alexandra Bayrak 4,5, Timo Aberle 1, Martina Schleicher 1, Hans-Peter Wendel 6, Martina Seifert 4,5, Ulrich A. Stock 1, 1 Dept. Thoracic, Cardiac and Vascular Surgery, University Hospital Tübingen, Germany, 2 Cell & Tissue Systems, Inc., North Charleston, SC, USA, 3 Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA, 4 Institute of Medical Immunology, Charité Universitätsmedizin Berlin, Germany, 5 Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Germany, 6 Dept. Paediatric Cardiosurgery, University Hospital Tübingen, Germany Controlled rate freezing is state of the art for preserving cardiovascular tissues. Our group has previously described a novel ice-free cryopreservation (IFC) method Abstracts / Cryobiology 63 (2011) 306–342 as a simpler less expensive alternative for heart valve preservation. In a sheep model IFC heart valves performed significantly better in vivo. Explant analyses revealed reduced inflammation, particularly CD3+ T-cells, and less leaflet thickening than implanted conventionally frozen cryopreserved (FC) tissues (Lisy et al., Biomaterials, 2010). The aim of the present study was to further characterize IFC heart valve viability, hemocompatibility and immunogenicity using in vitro techniques in anticipation of human trials. The IFC valve leaflets were compared with leaflets from FC and fresh control valves. All porcine pulmonary valves employed in this study were decontaminated using antibiotic treatment. IFC valves were infiltrated with 83% cryoprotectant solution (one-step addition, 4.65 M each Me2SO/formamide and 3.31 M propane-1,2diol in EuroCollins solution), rapidly cooled in a pre-cooled methylbutane bath and stored at 80 °C. FC valves were control rate frozen at 1 °C/min in Dulbecco’s minimum essential medium with 10% Me2SO and either 10% fetal bovine serum or human serum albumin to 80 °C and stored in vapor phase nitrogen. Cryopreserved leaflets were compared with fresh antibiotic treated valve leaflets after rewarming using a battery of coagulation protein assays after exposure to human blood, human peripheral blood mononuclear cell (PBMC) immune responses with low dose anti-CD3 costimulation, the alamarBlue assay for assessment of metabolic activity, cell membrane integrity using fluorescent dyes (hoechst33342 and exclusion dye propidium iodide), apoptosis using TUNEL stain and endothelial cell retention using the von Willebrand Factor stain. The proliferation profiles of PBMC immune cell subpopulations were analysed after co-culturing with all tissue types by flow cytometry. The viability of leaflets was significantly decreased after ice-free cryopreservation, p < 0.05, compared with both FC and untreated control leaflets. Von Willebrand Factor staining indicated that most of the endothelium was lost. TUNEL staining showed that IFC did not induce significant amounts of apoptosis, suggesting that necrosis is the predominant cell death pathway. Hemocompatibility, employing thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, b-thromboglobulin and terminal complement complex SC5b-9, was preserved compared with both fresh and frozen tissues. IFC leaflets induced the lowest PBMC proliferation response compared with fresh leaflets, which showed the strongest proliferation, and FC leaflets with moderate proliferation. The main differences in leaflets of valves preserved with the new IFC method compared with the conventional FC method were decreased viability and immunogenicity. It is hypothesized that the reduction of cell viability may be responsible for the reduced immunogenicity. The hemocompatibility results also support the establishment of IFC as a simplified preservation method, since no statistically significant differences between the preservation methods and fresh untreated controls were detected. These results combined with the prior in vivo sheep study suggest that IFC valves are ready for clinical studies. German Research Foundation. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.093 Cell Preservation Protocols II 91. Cryo-isolation: A novel new method for enzyme-free isolation of pancreatic islets involving in situ cryopreservation of islets and selective destruction of acinar tissue. Michael J. Taylor, Simona C. Baicu, Cell and Tissue Systems, N. Charleston, SC 29406, USA Transplantation of pancreatic islets offers a potential means for clinical treatment of Type I diabetes but the technique is critically dependent upon the availability of sufficient high quality islets. Currently the field relies totally upon enzymatic digestion processes that destroy the extracellular matrix of the donor tissue releasing the entrapped islets for further processing and purification. This procedure has recognized pit falls due principally to the difficulty of controlling the digestive process to yield an optimum quantity of viable cells. Moreover, the process is harsh and even toxic causing inevitable loss of valuable cells. Furthermore, the process relying upon the purest forms of the enzymes are very expensive and are subject to batch variations that have led to frustrating variability and inconsistency in attempts to optimize and standardize these processes. A totally new approach is proposed here that avoids, or reduces the need for enzymatic digestion of the pancreas and instead relies upon the known susceptibilities of cells to freezing injury to affect the separation of endocrine tissue from exocrine tissue by virtue of a facilitated differential freezing and cryopreservation technique. In essence, we attempted to pre-treat the pancreas by differential perfusion of the endocrine and exocrine tissue in a way designed to maximize the destruction of the exocrine tissue at the same time as preserving the islets (Cryo-isolation). Pancreases were procured from juvenile pigs using approved procedures. The concept of cryo-isolation is based on differential processing of the pancreas in 5 stages: (1) Infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries under controlled conditions of temperature and pressure. (2) Retrograde ductal infusion of water (or saline) until the gland was fully distended. (3) The entire Px was frozen solid to 160 °C and stored in the vapor phase of liquid nitrogen. (4) The frozen pancreas was mechani- 331 cally crushed and pulverized into small fragments. (5) The frozen fragments were thawed, filtered and washed with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets and samples were taken for static glucose-stimulated insulin release assessment. As predicted the cryo-isolate contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact embedded islets. The degree of cleavage of the cryoprotected islets from the freezedestroyed exocrine cells was variable. Islets were typically larger than their counterparts isolated from juvenile pigs using conventional enzyme-digestion techniques. Functionally, the islets from replicate cryo-isolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7 (n = 3). Conclusion: an enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freezedestruction of acinar tissue is feasible and proposed as a new and novel method that avoids the problems associated with conventional collagenase digestion methods. Acknowledgement: This pilot study was supported by funds from Cell and Tissue Systems with whom the authors are employed. We thank Dr. David E. Pegg for insightful discussions during the conceptual stage of this work. Conflict of interest: None declared. doi:10.1016/j.cryobiol.2011.09.094 92. Investigation into hepatocellular stress response activation following non-frozen storage. William L. Corwin 1,2,3, John M. Baust 1,3, Robert G. Van Buskirk 1,2,3, John G. Baust * 1,2, 1 Institute of Biomedical Technology, Binghamton University, Binghamton, NY 13902, USA, 2 Department of Biological Sciences, Binghamton University, Binghamton, NY 13902, USA, 3 CPSI BioTech, 2 Court St., Owego, NY 13827, USA Optimal human hepatocyte processing and storage is critical given the diverse uses for liver cells from in vitro assessments of metabolism, drug–drug interactions and hepatotoxicity to implications for whole organ transplantation. It is well established that a molecular based response, namely apoptosis, contributes significantly to cell system failure following processing and storage stresses, however, it remains unclear whether the specific molecular pathways activated promoting cellular demise are universal or cell specific. The use of hepatocyte derivatives, such as the C3A cell line in bioartificial liver support systems presents a distinctly different cell system which may require alternative processing and storage protocols. We hypothesized that cell type specific stress pathways may be activated under different processing or storage conditions. To this end, a comparison of C3A and normal hepatocytes was conducted examining responses to both hypothermic and anoxic normothermic storage. Normal hepatocytes and C3A were stored at 4 °C in either their respective growth media or ViaSpan (UW solution) for 18 h to 3 d. These cells were also held at anoxic normothermic (37 °C) conditions for 1–14 d in either their respective growth media or HBSS (Hank’s Balanced Salt Solution). The additions of salubrinal and resveratrol were made to evaluate cell stress response alterations. Salubrinal is a specific inhibitor of ER stress activated pathway and resveratrol has been shown to have life-extending properties through activation of genes that mimic caloric restriction. Viability was assessed at 0, 24 and 48 h post-storage. Apoptotic and necrotic involvement were assessed via flow cytometry and fluorescent microscopy at 1, 4, 8 and 24 h post-storage. Further, immunoblotting was performed to evaluate alterations in ER stress pathway proteins and classical apoptotic associated proteins. Initial viability data demonstrated a stark contrast between the two cell systems employed. Hypothermic storage of normal hepatocytes resulted in significant viability loss for growth media and ViaSpan storage with both remaining 30% viable after 18 h. The addition of resveratrol significantly improved viability for both solutions with growth media improvement of 30% and ViaSpan improvement of  70%. The incorporation of salubrinal in these storage solutions showed no appreciable effects. Conversely, hypothermic storage of C3A cells demonstrated the opposite pattern with resveratrol and salubrinal additions. The addition of resveratrol to media demonstrated a profound loss in C3A viability as compared to the uninhibited counterpart (2% vs. 30%, respectively). However, the addition of salubrinal has a marked beneficial effect of 30%. Interestingly, normothermic storage produced opposite results from hypothermia. Both salubrinal and resveratrol additions had a negative effect on C3A storage while hepatocytes stored with salubrinal addition had a beneficial effect and resveratrol had a negative impact. These data demonstrate that the cell systems do have a differential response to storage stress that appears to be dependent upon storage temperature, solution and inhibitor additions. This in an important consideration for processing and storage of cells and tissue because it demonstrates that universal protocols may not be effective for all cell types and conditions, and more cell type specific approaches may be necessary for optimal efficacy. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.095