- Email: [email protected]
095 Hyperbaric theory of cryoinjury of microorganisms
424
Abstracts / Cryobiology 67 (2013) 398–442
Applications for induced pluripotent stem cells are extensive and include basic biology, drug discovery, toxicological assessment, tissue regeneration, and cancer therapy, among many others. However, reprogramming of somatic cells is an intensive and time consuming process and karyotypic abnormalities are known to occur after extended periods in culture. Therefore, the ability to cryopreserve iPSCs with a high survival rate would be advantageous. Although stem cells are most frequently cryopreserved as small cell clumps in suspension, colony dissociation is known to cause cell loss and increased differentiation. Thus cryopreservation in the adherent state would alleviate any need to dissociate stem cells from the culture surface and prevent any damage that is a result of dissociation. Vitrification methods are particularly promising for cryopreservation of adherent cells because damage due to extracellular ice is prevented. However, vitrification methods require the use of high CPA concentrations which increase the risk of osmotic and toxic damage. Recently, we developed a rational design algorithm for designing toxicity-minimized CPA addition and removal procedures. (J.D. Benson, A.J. Kearsley, A.Z. Higgins, Cryobiology 64 (2012) 144–151) To successfully apply these predicted procedures, accurate knowledge of cell biophysical parameters is required. The purpose of this study was to determine the necessary biophysical parameters for the rational design of CPA addition and removal procedures for vitrification of iPSCs. To determine membrane permeability parameters for human iPSCs, we adapted our calcein fluorescence quenching method (A.K. Fry, A.Z. Higgins, Cellular and Molecular Bioengineering 5 (2012) 287–298) for use with an automated plate reader. Permeability parameters were determined for dimethyl sulphoxide, ethylene glycol, glycerol, and propylene glycol at 4 °C, 21 °C, and 37 °C. Most notably, glycerol permeation was significantly slower than the other CPA types. To determine the osmotic tolerance limits of iPSCs, cells were exposed to test solutions, which included varying concentrations of hypotonic buffer and hypertonic sucrose, for 15 mins. Cell yield was assessed 24 h after cells were returned to culture using PrestoBlueÒ. Osmotic tolerance limits were determined for single cell suspensions and adherent iPSCs at 4 °C, 21 °C, and 37 °C. Using ANOVA analysis, the effect of the osmolality and temperature as well as the cross interaction between osmolality and temperature were significant for both hypotonic and hypertonic exposures. Also, the tolerable limits varied greatly depending on the test temperature. In particular, excessive cell volume changes were more damaging at 37 °C than 4 °C or 21 °C. The permeability parameters and osmotic tolerance limits presented in this study enable rational design of CPA addition and removal procedures. The information in this study is an important step toward development of successful vitrification strategies for adherent human iPSCs. Source of funding: This project was funded by a Collaborative Research Contract from Life Technologies with Oregon State University. Conflict of interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.098
093 Ultra rapid warming of cryo samples using an IR laser pulse. F.W. Kleinhans 1,2, Bo Jin 1, Estefania Paredes 1,3, Peter Mazur 1, 1 Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, USA, 2 Physics, IUPUI, Indianapolis, IN, USA, 3 Departamento de Ecoloxia e Bioloxia Animal, Universidad de Vigo, Estrada Colexio Universitario, Vigo, Galicia, Spain Our laboratory has been investigating the effect of warming rate on survival of mouse oocytes after very rapid cooling. Cooling and warming rates are affected by sample thermal mass, surface area, thermal impedance, and delta T. To maximize these rates we use 0.1 ul samples placed on Cryotops and plunged directly into LN2 for cooling and into room temperature buffer for warming. The maximum warming rate obtained is 100,000 C/min and it yields the highest survivals. However, extrapolation of the experimental data suggests faster warming would yield higher survivals. Here we report on the use of an IR laser pulse to achieve warming rates 3.5 to 100 times faster. Our objective was to choose a laser wavelength which would warm the cryo buffer while leaving the cells ‘untouched’, thus leading to indirect warming of the cells. Optical tweezer studies show that a wide variety of cell types can tolerate very high intensities of 1064 nm IR energy emitted by Nd:YAG lasers and so this was the type chosen. Calculations indicate the laser intensity for cryo melting to be significantly less than that used for optical tweezers. Most cryo buffers, including the EAFS we use are relatively transparent in the IR. The buffer IR absorption is easily increased by adding a little India ink. India ink consists primarily of carbon particles from 0.1 to 1 um in diameter, and at the concentration we use, 0.25% (V/V), is harmless to the oocytes. The concentration is carefully chosen to allow most of the IR energy to pass completely through the sample. This yields relatively even sample heating from front to back. We estimate inkfree samples absorb 9% of the incident energy, and the added ink absorbs 25% of the remaining energy, yielding a total absorption of 30%. We have developed a small cryo ‘jig’ which lifts the sample out of an LN2 bath, covers the bath, and fires a laser pulse, all in less than one sec. This assures that the sample is rapidly warmed by the laser pulse rather than by slower air warming. Thus, the samples are warmed
from 180°C to 0°C by the laser pulse. We have computed the required laser energy for melting, but there are so many variables that we have found it preferable to empirically determine the required laser energy to just melt the samples for a given experiment. This is done by direct visual observation with a 15 x stereo microscope built into the laser system. We use a 40 joule welding laser (for long pulse durations!) with a beam diameter of 2 mm and a pulse length of 0.5 to 30 msec. This yields warming rates from 3.5 x 105 to 2 x 107 C/min. However, modeling indicates that rates higher than 1 x 107 C/min yield unacceptably large thermal gradients across the 70 um diameter oocytes and are not used. Preliminary experiments at a warming rate of 3.5 x 105 C/min yield survivals comparable to our traditional methods. Source of Funding: NIH Grant 8R01 OD 011201, Peter Mazur, PI. E. Paredes FPU Research Stays Fellowship from the Spanish Government, 1 November 2012-31 January 2013. Conflict of Interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.099
094 Application of the Kwei equation for predicting the Tg of stabilizing formulations containing sugars and salts. Lindong Weng 1, Ranganathan Vijayaraghavan 2, Douglas R. MacFarlane 2, Gloria D. Elliott 1, 1 Department of Mechanical Engineering and Engineering Sciences, University of North Carolina at Charlotte, Charlotte, NC, USA, 2 School of Chemistry, Monash University, Clayton, Victoria, Australia Sugars such as trehalose and sucrose have been used in numerous formulations to improve the shelf-life of products ranging from foods to pharmaceuticals and more recently they have also been used as a key protectant for preserving cellbased products. Disaccharides are known to have high glass transition temperatures and thus can immobilize molecules at high storage temperatures. Frequently these sugars are used in combination with salts for the purpose of buffering, oncotic control, or to maintain membrane potentials. The effect of salts on the glass-forming sugars has not been comprehensively studied. To better understand the effect of salts on the potential for the storage of glassy compositions we investigated various predictive models for sugar/salt formulations using data from the literature as well as new data generated on binary mixtures of trehalose and choline dihydrogen phosphate (CDHP). CDHP has recently been shown to have promise as a stabilizing agent for proteins and DNA, and thus may have utility in preserving certain biospecimens. For example, it can increase the thermal stability and shelf life of model proteins such as Cytochrome c, lysozyme and interleukin-2. In the current study we determined the glass transition temperature (Tg) of mixtures of CDHP and trehalose using a Q-100 differential scanning calorimeter (TA Instruments). The calorimetric results show that the relationship between Tg and composition exhibited a pronounced deviation from the conventional Gordon-Taylor prediction. Specifically, there is a ‘‘fall-and-rise” trend within the range of 70 90 wt% of trehalose. A general and accurate model for predicting the Tg can conserve experimental effort and lead to better insights about the interactions between sugars and salts. In this study we applied the Fox, Gordon-Taylor and Kwei equations to both literature data and new data acquired on trehalose/CDHP mixtures that are of interest in formulation science. The Kwei equation was found to have more generality and accuracy than the Fox and Gordon-Taylor equations. From a mathematical point of view, the curves of the Tg as a function of weight percentage modeled with the Kwei equation can be further grouped into three types (i.e., U, inverted U, and inverted S shapes) based on the values of its two parameters, k and q. It is believed that the deviations from the Gordon-Taylor prediction can be attributed to the strong interactions between the two components in a mixture as well as their structural and volumetric differences. Source of funding: This work was funded by grant #5RO1GM101796 from the National Institutes of Health. Conflict of interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.100
095 Hyperbaric theory of cryoinjury of microorganisms. Victor Bronshtein, Universal Stabilization Technologies, Inc., San Diego, CA, USA It has been shown that cells are captured by a growing ice crystal, forming inclusions, if the speed of crystallization front is higher than a critical speed, which usually does not exceed 10 lm/s. Further freezing of water leads to an increase of the hydrostatic pressure in the inclusions and to cell dehydration. Both of these processes depend on the rate of plastic relaxation of stress in the ice crystal near the
Abstracts / Cryobiology 67 (2013) 398–442 inclusions. It is shown that the diffusion mechanism of plastic relaxation in ice is not sufficiently rapid to prevent the development of high (up to 108 Pa) pressures in inclusions at cooling rates greater than 3 °C/min. The average distance between dislocations in ice is much greater than the cell (inclusion) radius. So, there are no dislocations in the vicinity of inclusions, where the stress is large enough to create a plastic deformation. Hence, an expansion of the plastic relaxation region in an ice crystal containing small dilatating inclusions is possible only if the average tensile stress is more than the shear stress. The average tensile stress is equal to the product of twice the shear modulus, and the power and the dilatation center concentration. As shown by our theoretical analysis, the process of plastic relaxation of pressure in small dilatating inclusions in ice is percolating in character. Percolation begins when the volume concentration of cells is more than 0.1%. This unusual prediction agrees with the dependence of the survival rate of slowly frozen Streptococcus cremoris and Escherichia coli on the cell concentration. So, one can conclude that high hydrostatic pressure is a strong damaging factor for these microorganisms. The theory also suggests the possibility of cell protection from high pressure effects during freezing by adding neutral latex particles or by substantially decreasing the medium tonicity. This is also confirmed by experimental results. Source of funding: Universal Stabilization Technologies, Inc. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.101
096 Keynote presentation: 50 years of the Society for Cryobiology. Arthur Rowe, New York University School of Medicine, NY, USA During the past 50 years, the Society for Cryobiology with its journal Cryobiology, has been the primary leader in the field thanks to the many pioneers and their scientific colleagues and progeny who have contributed and disseminated research in low temperature biology and medicine. In 1940, B.J. Luyet published the treatise ‘‘Life and Death at Low Temperature” and is credited with being the Father of Cryobiology. The accidental discovery by Polge, Smith and Parkes, in 1949, of glycerol as an effective cryoprotectant for spermatozoa and later red blood cells stimulated great interest in freeze preservation. Research on freezing cells and other biological materials was rapidly initiated by many research laboratories in academia and industry in the early 1950s and 1960s. This increase in low temperature research became international and soon required venues for disseminating new findings. In the United States, the first organized assemblies of interested scientists were held at the large multi-disciplinary FASEB meetings in Atlantic City, New Jersey each year. One of the first large meetings devoted entirely to cryobiology took place in Buffalo in the early 1960’s and it soon became obvious to a group of attendees that there was sufficient interest to form a society. In 1964, the Society for Cryobiology was founded, with B. J. Luyet selected as its first President. Simultaneously, the Society initiated the publishing of Cryobiology, the International Journal of Low Temperature Biology and Medicine with T.I. Malinin as its first Editor-in-Chief. Interest and research in cryobiology expanded rapidly with many institutions being established in the United States, Canada, England, Switzerland, Ukraine, and Japan among others. Many smaller societies devoted to cryobiology, cryomedicine, cryosurgery, and cryogenic technology were spawned. In recent years, the Society has recognized those outstanding scientists who have had a scientific impact and contributed significantly to the science of cryobiology, generated outstanding scientific offspring, and contributed service to the Society, by establishing the category and distinguished honor of FELLOW of the Society for Cryobiology. This presentation will highlight those who were the early outstanding scientists instrumental in establishing the scientific foundation of a society devoted to cryobiology, and which is now celebrating 50 years of progress. Source of funding: None declared. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.102
097 Non-enzymatic cryo-isolation of viable Islets from juvenile porcine pancreas after storage for >6 months at 135 °C. Michael J. Taylor, Simona Baicu, Zhen Chen, Elizabeth Greene, Cell and Tissue Systems, N. Charleston, SC, USA Objective: Cryo-isolation of therapeutic cells such as pancreatic islets has been proposed as a novel new alternative to conventional enzymatic digestion of the pancreas (Taylor, Baicu., Transpl Proc. 43 (2011) 3181–3183. And Cell Transpl. 2013 (In Press)). Apart from avoiding the expense, inconsistencies and toxicity associated with the collagenase-digestion process, cryo-isolation embodies several inherent advantages including the convenience of biobanking for storage and ship-
425
ping. This study investigates the feasibility of obtaining viable islets from juvenile porcine pancreas after storage at 135 °C for >6 months using the cryo-isolation technique. Methods: Pancreases were procured from juvenile pigs using approved procedures and processed using the cryo-isolation protocol. This involved: (1) Infiltrating islets in situ preferentially with 2M dimethyl sulfoxide as the cryoprotectant (CPA) via antegrade perfusion of the major arteries; (2) retrograde ductal infusion of water, and then (3) the entire pancreas was frozen solid to 160 °C and stored in the vapor phase of liquid nitrogen. After storage at 135 °C for 1 day to 8 months, pancreases were further processed as follows: The frozen pancreas was mechanically crushed and pulverized into small fragments which were then 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 with Syto13/Propidium iodide (PI) for fluorescence viability testing and assessed for function using a conventional glucose-stimulated insulin secretion (GSIS) assay. Results: The cryo-isolate from pancreata stored for >6 months contained pancreatic fragments comprising discrete dithizone-positive islets embedded in a shell of dead amorphous material. The cryo-isolate product was similar to that obtained from frozen pancreases stored for 1 day in that there was an abundance of largely intact and viable (>90%) islets. Moreover, the cryo-isolated islets were typically larger (range 50– 500 lm) than their counterparts isolated from juvenile pigs using conventional collagenase digestion techniques and demonstrated GSIS indices (>3), which was not significantly different from controls (fresh islets from collagenase digestion). Conclusion: An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a new and novel method that avoids the problems associated with conventional collagenase digestion methods and permits long-term biobanking of pancreases as a ready source of islets. Source of funding: This work was funded in part by a grant from NIH/ NIDDK#1R43DK096773. Conflict of interest: The authors are, or have been, employed by Cell and Tissue Systems. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.103
098 Developing an Intelligent tutoring system for prostate cryosurgery. Anjali Sehrawat, Robert Keelan, Kenji Shimada, Yoed Rabin, Carnegie Mellon University, Pittsburgh, PA, USA The objective of this study is to develop a proof-of-concept for a cryosurgery Intelligent Tutoring System (ITS), aimed at shortening the clinician’s learning curve, by integrating computer-generated cryosurgery planning, abnormal growth of the prostate gland, and established criteria for clinical success. Cryosurgery is the destruction of undesired tissue by freezing. Minimally invasive cryosurgery is performed by strategically placing an array of cryoprobes within a prespecified target region. In prostate cryosurgery, the target region for destruction can be a portion of or the entire gland. A key to cryosurgery success is optimal selection and layout of cryoprobes, which maximizes destruction to the target region while minimizing cryoinjury to surrounding healthy tissues. Currently, selection of cryoprobe type, layout, sequence of operation, and procedure duration is based on the cryosurgeon’s personal experience and accepted practices. Suboptimal cryosurgery protocols may leave untreated areas in the target region, lead to cryoinjury of healthy surrounding tissues, increase the duration of the surgical procedure, and increase the likelihood of post-cryosurgery complications—all of which affect the quality and cost of medical treatment. An ITS guides the student through a problem-solving process by providing instruction and tailored feedback promptly. The cryosurgery ITS presents problems, strategically broken down into smaller steps, that are consistent with clinical practice and pedagogical rational. The ITS proposed in the current study focuses on determining cryoprobe layout, given an overall number of cryoprobes and their operational parameters. Optimal (expert) planning is determined by previously developed planning algorithms, with the trainee attempting to generate a layout that (1) matches the expert layout, with a predefined tolerance, or (2) generates a defect value lower than the expert defect; a defect is defined as either external tissue to the target area that was simulated to be cryoinjured, or internal tissue that was simulated to be uninjured. Key to ITS success is a set of rules and constraints used to evaluate a student’s solution, such as: (1) minimum distance from the urethra; (2) minimum distance from the prostate surface; (3) active cooling surface of a probe included within the gland; and (4) minimum probe layout defect value. ITS effectiveness is measured by evaluating students’ learning gains with increased practice. This study demonstrates a proof-of-concept for a fully-functioning cryosurgery ITS. The cryosurgery ITS prototype is composed of four primary components: a domain model, a student model, a tutor, and a problem interface. In order to establish an expert solution and to evaluate the training progress, the domain model uses: cryoprobe planning algorithms, the simulator, and a set of cryosurgery rules and con-
Abstracts / Cryobiology 67 (2013) 398–442
Applications for induced pluripotent stem cells are extensive and include basic biology, drug discovery, toxicological assessment, tissue regeneration, and cancer therapy, among many others. However, reprogramming of somatic cells is an intensive and time consuming process and karyotypic abnormalities are known to occur after extended periods in culture. Therefore, the ability to cryopreserve iPSCs with a high survival rate would be advantageous. Although stem cells are most frequently cryopreserved as small cell clumps in suspension, colony dissociation is known to cause cell loss and increased differentiation. Thus cryopreservation in the adherent state would alleviate any need to dissociate stem cells from the culture surface and prevent any damage that is a result of dissociation. Vitrification methods are particularly promising for cryopreservation of adherent cells because damage due to extracellular ice is prevented. However, vitrification methods require the use of high CPA concentrations which increase the risk of osmotic and toxic damage. Recently, we developed a rational design algorithm for designing toxicity-minimized CPA addition and removal procedures. (J.D. Benson, A.J. Kearsley, A.Z. Higgins, Cryobiology 64 (2012) 144–151) To successfully apply these predicted procedures, accurate knowledge of cell biophysical parameters is required. The purpose of this study was to determine the necessary biophysical parameters for the rational design of CPA addition and removal procedures for vitrification of iPSCs. To determine membrane permeability parameters for human iPSCs, we adapted our calcein fluorescence quenching method (A.K. Fry, A.Z. Higgins, Cellular and Molecular Bioengineering 5 (2012) 287–298) for use with an automated plate reader. Permeability parameters were determined for dimethyl sulphoxide, ethylene glycol, glycerol, and propylene glycol at 4 °C, 21 °C, and 37 °C. Most notably, glycerol permeation was significantly slower than the other CPA types. To determine the osmotic tolerance limits of iPSCs, cells were exposed to test solutions, which included varying concentrations of hypotonic buffer and hypertonic sucrose, for 15 mins. Cell yield was assessed 24 h after cells were returned to culture using PrestoBlueÒ. Osmotic tolerance limits were determined for single cell suspensions and adherent iPSCs at 4 °C, 21 °C, and 37 °C. Using ANOVA analysis, the effect of the osmolality and temperature as well as the cross interaction between osmolality and temperature were significant for both hypotonic and hypertonic exposures. Also, the tolerable limits varied greatly depending on the test temperature. In particular, excessive cell volume changes were more damaging at 37 °C than 4 °C or 21 °C. The permeability parameters and osmotic tolerance limits presented in this study enable rational design of CPA addition and removal procedures. The information in this study is an important step toward development of successful vitrification strategies for adherent human iPSCs. Source of funding: This project was funded by a Collaborative Research Contract from Life Technologies with Oregon State University. Conflict of interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.098
093 Ultra rapid warming of cryo samples using an IR laser pulse. F.W. Kleinhans 1,2, Bo Jin 1, Estefania Paredes 1,3, Peter Mazur 1, 1 Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, USA, 2 Physics, IUPUI, Indianapolis, IN, USA, 3 Departamento de Ecoloxia e Bioloxia Animal, Universidad de Vigo, Estrada Colexio Universitario, Vigo, Galicia, Spain Our laboratory has been investigating the effect of warming rate on survival of mouse oocytes after very rapid cooling. Cooling and warming rates are affected by sample thermal mass, surface area, thermal impedance, and delta T. To maximize these rates we use 0.1 ul samples placed on Cryotops and plunged directly into LN2 for cooling and into room temperature buffer for warming. The maximum warming rate obtained is 100,000 C/min and it yields the highest survivals. However, extrapolation of the experimental data suggests faster warming would yield higher survivals. Here we report on the use of an IR laser pulse to achieve warming rates 3.5 to 100 times faster. Our objective was to choose a laser wavelength which would warm the cryo buffer while leaving the cells ‘untouched’, thus leading to indirect warming of the cells. Optical tweezer studies show that a wide variety of cell types can tolerate very high intensities of 1064 nm IR energy emitted by Nd:YAG lasers and so this was the type chosen. Calculations indicate the laser intensity for cryo melting to be significantly less than that used for optical tweezers. Most cryo buffers, including the EAFS we use are relatively transparent in the IR. The buffer IR absorption is easily increased by adding a little India ink. India ink consists primarily of carbon particles from 0.1 to 1 um in diameter, and at the concentration we use, 0.25% (V/V), is harmless to the oocytes. The concentration is carefully chosen to allow most of the IR energy to pass completely through the sample. This yields relatively even sample heating from front to back. We estimate inkfree samples absorb 9% of the incident energy, and the added ink absorbs 25% of the remaining energy, yielding a total absorption of 30%. We have developed a small cryo ‘jig’ which lifts the sample out of an LN2 bath, covers the bath, and fires a laser pulse, all in less than one sec. This assures that the sample is rapidly warmed by the laser pulse rather than by slower air warming. Thus, the samples are warmed
from 180°C to 0°C by the laser pulse. We have computed the required laser energy for melting, but there are so many variables that we have found it preferable to empirically determine the required laser energy to just melt the samples for a given experiment. This is done by direct visual observation with a 15 x stereo microscope built into the laser system. We use a 40 joule welding laser (for long pulse durations!) with a beam diameter of 2 mm and a pulse length of 0.5 to 30 msec. This yields warming rates from 3.5 x 105 to 2 x 107 C/min. However, modeling indicates that rates higher than 1 x 107 C/min yield unacceptably large thermal gradients across the 70 um diameter oocytes and are not used. Preliminary experiments at a warming rate of 3.5 x 105 C/min yield survivals comparable to our traditional methods. Source of Funding: NIH Grant 8R01 OD 011201, Peter Mazur, PI. E. Paredes FPU Research Stays Fellowship from the Spanish Government, 1 November 2012-31 January 2013. Conflict of Interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.099
094 Application of the Kwei equation for predicting the Tg of stabilizing formulations containing sugars and salts. Lindong Weng 1, Ranganathan Vijayaraghavan 2, Douglas R. MacFarlane 2, Gloria D. Elliott 1, 1 Department of Mechanical Engineering and Engineering Sciences, University of North Carolina at Charlotte, Charlotte, NC, USA, 2 School of Chemistry, Monash University, Clayton, Victoria, Australia Sugars such as trehalose and sucrose have been used in numerous formulations to improve the shelf-life of products ranging from foods to pharmaceuticals and more recently they have also been used as a key protectant for preserving cellbased products. Disaccharides are known to have high glass transition temperatures and thus can immobilize molecules at high storage temperatures. Frequently these sugars are used in combination with salts for the purpose of buffering, oncotic control, or to maintain membrane potentials. The effect of salts on the glass-forming sugars has not been comprehensively studied. To better understand the effect of salts on the potential for the storage of glassy compositions we investigated various predictive models for sugar/salt formulations using data from the literature as well as new data generated on binary mixtures of trehalose and choline dihydrogen phosphate (CDHP). CDHP has recently been shown to have promise as a stabilizing agent for proteins and DNA, and thus may have utility in preserving certain biospecimens. For example, it can increase the thermal stability and shelf life of model proteins such as Cytochrome c, lysozyme and interleukin-2. In the current study we determined the glass transition temperature (Tg) of mixtures of CDHP and trehalose using a Q-100 differential scanning calorimeter (TA Instruments). The calorimetric results show that the relationship between Tg and composition exhibited a pronounced deviation from the conventional Gordon-Taylor prediction. Specifically, there is a ‘‘fall-and-rise” trend within the range of 70 90 wt% of trehalose. A general and accurate model for predicting the Tg can conserve experimental effort and lead to better insights about the interactions between sugars and salts. In this study we applied the Fox, Gordon-Taylor and Kwei equations to both literature data and new data acquired on trehalose/CDHP mixtures that are of interest in formulation science. The Kwei equation was found to have more generality and accuracy than the Fox and Gordon-Taylor equations. From a mathematical point of view, the curves of the Tg as a function of weight percentage modeled with the Kwei equation can be further grouped into three types (i.e., U, inverted U, and inverted S shapes) based on the values of its two parameters, k and q. It is believed that the deviations from the Gordon-Taylor prediction can be attributed to the strong interactions between the two components in a mixture as well as their structural and volumetric differences. Source of funding: This work was funded by grant #5RO1GM101796 from the National Institutes of Health. Conflict of interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.100
095 Hyperbaric theory of cryoinjury of microorganisms. Victor Bronshtein, Universal Stabilization Technologies, Inc., San Diego, CA, USA It has been shown that cells are captured by a growing ice crystal, forming inclusions, if the speed of crystallization front is higher than a critical speed, which usually does not exceed 10 lm/s. Further freezing of water leads to an increase of the hydrostatic pressure in the inclusions and to cell dehydration. Both of these processes depend on the rate of plastic relaxation of stress in the ice crystal near the
Abstracts / Cryobiology 67 (2013) 398–442 inclusions. It is shown that the diffusion mechanism of plastic relaxation in ice is not sufficiently rapid to prevent the development of high (up to 108 Pa) pressures in inclusions at cooling rates greater than 3 °C/min. The average distance between dislocations in ice is much greater than the cell (inclusion) radius. So, there are no dislocations in the vicinity of inclusions, where the stress is large enough to create a plastic deformation. Hence, an expansion of the plastic relaxation region in an ice crystal containing small dilatating inclusions is possible only if the average tensile stress is more than the shear stress. The average tensile stress is equal to the product of twice the shear modulus, and the power and the dilatation center concentration. As shown by our theoretical analysis, the process of plastic relaxation of pressure in small dilatating inclusions in ice is percolating in character. Percolation begins when the volume concentration of cells is more than 0.1%. This unusual prediction agrees with the dependence of the survival rate of slowly frozen Streptococcus cremoris and Escherichia coli on the cell concentration. So, one can conclude that high hydrostatic pressure is a strong damaging factor for these microorganisms. The theory also suggests the possibility of cell protection from high pressure effects during freezing by adding neutral latex particles or by substantially decreasing the medium tonicity. This is also confirmed by experimental results. Source of funding: Universal Stabilization Technologies, Inc. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.101
096 Keynote presentation: 50 years of the Society for Cryobiology. Arthur Rowe, New York University School of Medicine, NY, USA During the past 50 years, the Society for Cryobiology with its journal Cryobiology, has been the primary leader in the field thanks to the many pioneers and their scientific colleagues and progeny who have contributed and disseminated research in low temperature biology and medicine. In 1940, B.J. Luyet published the treatise ‘‘Life and Death at Low Temperature” and is credited with being the Father of Cryobiology. The accidental discovery by Polge, Smith and Parkes, in 1949, of glycerol as an effective cryoprotectant for spermatozoa and later red blood cells stimulated great interest in freeze preservation. Research on freezing cells and other biological materials was rapidly initiated by many research laboratories in academia and industry in the early 1950s and 1960s. This increase in low temperature research became international and soon required venues for disseminating new findings. In the United States, the first organized assemblies of interested scientists were held at the large multi-disciplinary FASEB meetings in Atlantic City, New Jersey each year. One of the first large meetings devoted entirely to cryobiology took place in Buffalo in the early 1960’s and it soon became obvious to a group of attendees that there was sufficient interest to form a society. In 1964, the Society for Cryobiology was founded, with B. J. Luyet selected as its first President. Simultaneously, the Society initiated the publishing of Cryobiology, the International Journal of Low Temperature Biology and Medicine with T.I. Malinin as its first Editor-in-Chief. Interest and research in cryobiology expanded rapidly with many institutions being established in the United States, Canada, England, Switzerland, Ukraine, and Japan among others. Many smaller societies devoted to cryobiology, cryomedicine, cryosurgery, and cryogenic technology were spawned. In recent years, the Society has recognized those outstanding scientists who have had a scientific impact and contributed significantly to the science of cryobiology, generated outstanding scientific offspring, and contributed service to the Society, by establishing the category and distinguished honor of FELLOW of the Society for Cryobiology. This presentation will highlight those who were the early outstanding scientists instrumental in establishing the scientific foundation of a society devoted to cryobiology, and which is now celebrating 50 years of progress. Source of funding: None declared. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.102
097 Non-enzymatic cryo-isolation of viable Islets from juvenile porcine pancreas after storage for >6 months at 135 °C. Michael J. Taylor, Simona Baicu, Zhen Chen, Elizabeth Greene, Cell and Tissue Systems, N. Charleston, SC, USA Objective: Cryo-isolation of therapeutic cells such as pancreatic islets has been proposed as a novel new alternative to conventional enzymatic digestion of the pancreas (Taylor, Baicu., Transpl Proc. 43 (2011) 3181–3183. And Cell Transpl. 2013 (In Press)). Apart from avoiding the expense, inconsistencies and toxicity associated with the collagenase-digestion process, cryo-isolation embodies several inherent advantages including the convenience of biobanking for storage and ship-
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ping. This study investigates the feasibility of obtaining viable islets from juvenile porcine pancreas after storage at 135 °C for >6 months using the cryo-isolation technique. Methods: Pancreases were procured from juvenile pigs using approved procedures and processed using the cryo-isolation protocol. This involved: (1) Infiltrating islets in situ preferentially with 2M dimethyl sulfoxide as the cryoprotectant (CPA) via antegrade perfusion of the major arteries; (2) retrograde ductal infusion of water, and then (3) the entire pancreas was frozen solid to 160 °C and stored in the vapor phase of liquid nitrogen. After storage at 135 °C for 1 day to 8 months, pancreases were further processed as follows: The frozen pancreas was mechanically crushed and pulverized into small fragments which were then 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 with Syto13/Propidium iodide (PI) for fluorescence viability testing and assessed for function using a conventional glucose-stimulated insulin secretion (GSIS) assay. Results: The cryo-isolate from pancreata stored for >6 months contained pancreatic fragments comprising discrete dithizone-positive islets embedded in a shell of dead amorphous material. The cryo-isolate product was similar to that obtained from frozen pancreases stored for 1 day in that there was an abundance of largely intact and viable (>90%) islets. Moreover, the cryo-isolated islets were typically larger (range 50– 500 lm) than their counterparts isolated from juvenile pigs using conventional collagenase digestion techniques and demonstrated GSIS indices (>3), which was not significantly different from controls (fresh islets from collagenase digestion). Conclusion: An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a new and novel method that avoids the problems associated with conventional collagenase digestion methods and permits long-term biobanking of pancreases as a ready source of islets. Source of funding: This work was funded in part by a grant from NIH/ NIDDK#1R43DK096773. Conflict of interest: The authors are, or have been, employed by Cell and Tissue Systems. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.103
098 Developing an Intelligent tutoring system for prostate cryosurgery. Anjali Sehrawat, Robert Keelan, Kenji Shimada, Yoed Rabin, Carnegie Mellon University, Pittsburgh, PA, USA The objective of this study is to develop a proof-of-concept for a cryosurgery Intelligent Tutoring System (ITS), aimed at shortening the clinician’s learning curve, by integrating computer-generated cryosurgery planning, abnormal growth of the prostate gland, and established criteria for clinical success. Cryosurgery is the destruction of undesired tissue by freezing. Minimally invasive cryosurgery is performed by strategically placing an array of cryoprobes within a prespecified target region. In prostate cryosurgery, the target region for destruction can be a portion of or the entire gland. A key to cryosurgery success is optimal selection and layout of cryoprobes, which maximizes destruction to the target region while minimizing cryoinjury to surrounding healthy tissues. Currently, selection of cryoprobe type, layout, sequence of operation, and procedure duration is based on the cryosurgeon’s personal experience and accepted practices. Suboptimal cryosurgery protocols may leave untreated areas in the target region, lead to cryoinjury of healthy surrounding tissues, increase the duration of the surgical procedure, and increase the likelihood of post-cryosurgery complications—all of which affect the quality and cost of medical treatment. An ITS guides the student through a problem-solving process by providing instruction and tailored feedback promptly. The cryosurgery ITS presents problems, strategically broken down into smaller steps, that are consistent with clinical practice and pedagogical rational. The ITS proposed in the current study focuses on determining cryoprobe layout, given an overall number of cryoprobes and their operational parameters. Optimal (expert) planning is determined by previously developed planning algorithms, with the trainee attempting to generate a layout that (1) matches the expert layout, with a predefined tolerance, or (2) generates a defect value lower than the expert defect; a defect is defined as either external tissue to the target area that was simulated to be cryoinjured, or internal tissue that was simulated to be uninjured. Key to ITS success is a set of rules and constraints used to evaluate a student’s solution, such as: (1) minimum distance from the urethra; (2) minimum distance from the prostate surface; (3) active cooling surface of a probe included within the gland; and (4) minimum probe layout defect value. ITS effectiveness is measured by evaluating students’ learning gains with increased practice. This study demonstrates a proof-of-concept for a fully-functioning cryosurgery ITS. The cryosurgery ITS prototype is composed of four primary components: a domain model, a student model, a tutor, and a problem interface. In order to establish an expert solution and to evaluate the training progress, the domain model uses: cryoprobe planning algorithms, the simulator, and a set of cryosurgery rules and con-