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Cryobiology Symposia Cryobiology Symposium Abstracts C-1 concentrations of VFAs and relatively moderate rates of The Use of Engineering Modeling in Designing cooling and warming (reported in 1980s) turned attention back Cryopreservation Protocols for Articular Cartilage. J. A. W. to VF due it seemingly simple methodology. It however soon ELLIOTT1, A. Abazari2, N. Shardt2, L. McGann1, and N. became clear that E-VF had had its own substantial limitations Jomha3. 1University of Alberta, 7th Floor ECERF, Room 7- due to very high toxicity and osmotic damage produced by 026A, 9107 - 116 Street, Edmonton, AB T6G 2V4, CANADA; VFAs per se. As we pointed out in 2012, the wave has been 2Chemical and Materials Engineering, University of Alberta, turning back toward K-VF, especially in the fields of CP of Edmonton, AB T6G 2V4, CANADA; and 3Surgery, University reproductive and germ cells. In this presentation, we touch the of Alberta, Edmonton, AB T6G 2B7, CANADA. Email: basic distinctions between the 3 major ways of CP, with [email protected] specific emphasis to the means, methods and equipment, as well as pitfalls of the current approaches to K-VF, such as The transplantation of bone-bearing living articular cartilage is Leidenfrost effect (LFE) and restrictions of the sample size. A an effective treatment for joint injury and disease. Widespread short video that demonstrates an entirely new platform for access to this treatment, however, will require the ability to hyper-fast cooling (hundreds of thousands C/min) KrioBlast™ cryopreserve and bank this tissue. The cryopreservation of developed by CELLTRONIX, which allows to vitrify articular cartilage has been studied for more than 50 years, relatively large samples (up to 4,000 mcl at the day of with early unsuccessful attempts at freezing of cartilage slices reporting, can be further scaled up) practically without a need [R.C. Curran, T. Gibson, Proc. Royal Soc. London B 144 for potentially damaging vitrificants such as DMSO or (1956) 572] and the first successful cryopreservation of propylene glycol, traditionally used nowadays for VF. The isolated chondrocytes reported in 1965 [A.U. Smith, Nature system completely eliminates LFE and a need of potentially 205 (1965) 782]. Recently, our group developed a protocol to toxic and osmotically damaging permeating vitrificants such as cryopreserve human full-thickness articular cartilage attached DMSO or ethylene glycol used in the current methods of VF. to bone resulting in chondrocyte membrane integrity of 75.4 We have been able to vitrify12% and in som case as low as 7% +/– 12.1% with functionality of chondrocytes confirmed by a glycerol solutions as a cooling rate markers, which metabolic assay and the ability of extracted, pellet-cultured theoretically corresponds to the critical cooling rate up to chondrocytes to produce of collagen II and sulfated 1,600,000 C/min or higher. This can be considered as a step glycosaminoglycans at levels similar to healthy controls [N.M. toward the “Unified Cryopreservation Protocol”. preliminary Jomha et al., Biomaterials, 33 (2012) 6061.] This protocol, results that demonstrate excellent of survival of human aimed at vitrifying the tissue, requires the addition of four embryonic stem cells and sperm vitrified with KrioBlast™ are different mixtures of four different cryoprotectants added at also discussed. different temperatures for specific periods of time. To develop Disclosure: Author was or is employed by CELLTRONIX. this protocol, we relied heavily on engineering modeling. This presentation will review our research over the past 10 years on C-4 modeling of various key phenomena relevant to the Modulation of Heat and Mass Transfer During cryopreservation of articular cartilage including solution Freezing/Thawing of Cell Suspensions. GANG ZHAO1, thermodynamics; water transport; and the transport, toxicity, Xiaoming Zhou2, and Dayong Gao3. 1Department of Electronic and vitrifiability of cryoprotectants. The research being Science and Technology, University of Science and reviewed was funded primarily by the Canadian Institutes of Technology of China, Hefei 230027, CHINA; 2School of Health Research, the Natural Sciences and Engineering Mechanical, Electronic and Industrial Engineering, University Research Council of Canada, Alberta Innovates, and the of Electronic Science and Technology of China, Chengdu Edmonton Orthopaedic Research Committee. J. A. W. Elliott 611731, CHINA; and 3Department of Mechanical Engineering, holds a Canada Research Chair in Thermodynamics. University of Washington, Seattle, WA 98195. Email: [email protected] C-2 Kinetic Vitrification: Basic Thermodynamics, Methods and From the angel of the discipline of engineering thermophysics, Devices. IGOR I. KATKOV. CELLTRONIX, San Diego, CA cryopreservation involves typical heat and mass transfer, and 92126. Email: [email protected] thermodynamic processes, e.g., i) freezing and thawing of both extra and intracellular solutions, ii) transport of water and Cryopreservation (CP) of cells by ultra-fast (tens of thousands cryoprotective agents across cell membrane, and iii) ice C/min) kinetic vitrification (K-VF) of small samples without nucleation and the growth of ice crystals in these solutions. To additional thickeners and ice-blocking vitrification agents achieve the highest cell survival rate after a freeze-thaw cycle, (VFAs) had been the first method of cryopreservation back in efforts on optimization of these processes are indispensable. the 1940s. The mainstream of CP later shifted towards slow Recent studies have revealed that the introduction of freezing (SF). Equilibrium vitrification (E-VF) with very high nanoparticles into cell suspensions may affect the thermal 26 Cryobiology Symposium Abstracts properties of both the extra and intracellular solutions, the MICHAEL J. TAYLOR. Cell and Tissue Systems, N. nucleation and the growth of ice crystals in them, and the cell Charleston, SC and Dept. Mechanical Engineering, Carnegie membrane permeability, and thus it may be used to change the Mellon University, Pittsburgh, PA. Email: mtaylor@ heat and mass transfer, and the thermodynamic processes. celltissuesystems.com Besides, the superparamagnetic nanoparticles could be used to further enhance the rewarming efficiency of frozen cell The application of hypothermia for in vivo and ex vivo organ suspensions in the external electromagnetic field usually preservation is based upon the protective effect of reduced excited in a single mode cavity. This presentation will temperatures against the deleterious effects of ischemia and highlight recent work on the mode of action by nanoparticles hypoxia that ensues from blood loss, or organ procurement for influencing heat transfer of the extra and intracellular transplantation. However, low temperatures have a multitude solutions, mass transfer across cell membrane, and ice of effects, not all of which are beneficial. Special conditions nucleation and the growth of ice crystals during have been developed to harness the protective properties of freezing/thawing. cold for effective organ preservation and the development of specialized solutions is the primary factor in the evolution of C-5 hypothermic organ preservation technology. Even in pre- A Versatile and Robust MEMS-based Microdevice for High clinical research models, extreme hemodilution by blood Accuracy Thermal Conductivity Measurements of Semi-rigid replacement with synthetic, acellular crystalloid/colloid Biomaterials and Solutions. XIN M. LIANG1, 2, Praveen K. solutions has proved efficacious for avoiding Sekar2, Xiaoming Zhou3, Gang Zhao1, Weiping Ding1, Jingru ischemia/reperfusion in vivo after several hours of circulatory Yi1, and Dayong Gao2. 1Centre for Biomedical Engineering, arrest under ultraprofound hypothermia (<10°C). The modern Department of Electronic Science and Technology, University era of organ transplantation, which increasingly uses organs of Science and Technology of China, Hefei, Anhui 230027, from marginal donors including those compromised by prior CHINA; 2Department of Mechanical Engineering, University warm ischemia, is reliant on preservation technology to of Washington, Seattle, WA 98195; and 3School of stabilize and even resuscitate the donor organ. Ischemic insults Mechanical, Electronic, and Industrial Engineering, University (warm and cold) are incurred during each stage of the of Electronic Science and Technology of China, Chengdu, transplantation process from procurement, through Sichuan 611731, CHINA. Email: [email protected], preservation for storage and shipping, to re-implantation and [email protected] re-perfusion. Each stage demands optimized conditions for the best outcome and hypothermia enhanced by appropriately Accurate thermal property measurements of biological designed preservation solutions is not only the basis for current materials and various solutions (i.e. CPAs with different standard practices, but offers the path towards improved concentrations) are critical in the development of techniques in the future. The most promising approach for cryopreservation- and cryotherapy-related applications. further improvement of hypothermic preservation technology Although a number of approaches for obtaining the thermal is the selection of cytoprotective and resuscitative agents as conductivity of semi-rigid materials and solutions have been supplements to the baseline perfusates to counteract developed and characterized, they all exhibit deficiencies in ischemia/reperfusion injury. Specific targets
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