A.M. Karow

The biophysics of organ cryopreservation

I Introduction.- Organ Transplantation: The Role of Preservation.- An Introduction to the Problems of Organ Cryopreservation.- II Perfusion of Organs with Cryoprotectants: Cooling and Freezing.- Biological Effects of Cryoprotectant Perfusion, Delivery and Removal to Nonfrozen Organs.- Mass Transfer of Liquids across Biological Barriers.- Cytoplasmic Organisation and the Properties of Cell Water: Speculations on Animal Cell Cryopreservation.- Heat Transfer during Cryopreservation.- Ice Crystals in Tissues and Organs.- III Freezing and Vitrification.- Aqueous Solutions: Crystallization, Vitrification and Liquefaction.- Crystallization and Vitrification in Cryoprotected Aqueous Systems.- Ice Crystal Growth in Aqueous Solutions.- Non-Equilibrium Formation of Ice in Aqueous Solutions: Efficiency of Polyalcohol Solutions for Vitrification.- Devitrification and Recrystallization of Glass Forming Aqueous Solutions.- Biological Effects of Vitrification and Devitrification.- IV Electromagnetic Heating.- Electromagnetic Thawing of Organs: A Multidisciplinary Challenge.- Electromagnetic Heating Techniques for Organ Rewarming.- Tissue Heating: Applicator Types and Analysis by Phantom Models.- The Design of an Electromagnetic Rewarming System for Cryopreserved Tissue.- Heat Control and Sensing during Electromagnetic Recovery of Cryopreserved Large Organs.- V Non-Invasive Methods for Studying Organs.- Freezing Patterns in Tissue by NMR Technology.- Nuclear Magnetic Resonance (NMR) Imaging of Perfusion.- Evaluation of Biochemical Processes by NMR.- Examination of Organ Physiology by Positron Emission Tomography.- Contributors.

Ultrastructure-function correlative studies for cardiac cryopreservation. V. Absence of a correlation between electrolyte toxicity and cryoinjury in the slowly frozen, cryoprotected rat heart☆

Abstract Hearts were frozen to −17 °C in the initial presence of 2.1 m DMSO. Attempts were made to prevent or minimize the consequences of an osmotic shock based on Lovelocks classical hypothesis of freezing injury. Substitution of mannitol or potassium for NaCl before freezing did not improve the results, nor did perfusion of thawed hearts with hyperosmotic perfusate. It was found that freezing and thawing resulted in a significant attenuation of coronary flow and that, as a result of this, DMSO was apparently retained within the heart after thawing. DMSO was also difficult to remove at 30 °C in the absence of prior freezing and caused a significant drop in coronary flow upon institution of DMSO washout with balanced salt solution. The blanching of freezing and thawing was also seen, in milder form, in nonfrozen hearts. For both frozen-thawed and nonfrozen hearts, the blanching was associated with DMSO washout with balanced salt solution. Flow was improved by perfusion with hyperosmotic perfusate in both nonfrozen and in frozen-thawed hearts, but the improvement was largely temporary. Evidence from earlier studies indicates that electrolyte concentrations during freezing cannot be correlated with cardiac cryoinjury, in support of the present findings. It is suggested instead that cryoprotectant toxicity may be the chief agent of injury under the conditions studied.

Factors influencing renal cryopreservation. II: Toxic effects of three cryoprotectants in combination with three vehicle solutions in nonfrozen rabbit cortical slices

The [K+]/[Na+] ratio of rabbit renal cortical slices was used to examine, at 25 degrees C, the effects on viability of three cryoprotectant agents (CPA) (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) in combination with three vehicle solutions (Krebs-Henseleit (K-H), solution A, and RPS-2). Viability assessment by [K+]/[Na+] for all test solutions was made after incubating the slices in modified Cross-Taggart solution (C-T). With K-H and solution A, all concentrations of ethylene glycol and glycerol resulted in lowered ratios, whereas with Me2SO, concentrations greater than 1.4 M are required to reduce [K+]/[Na+]. With RPS-2 no decrease in the ratios was found until concentrations greater than 2.8 M were reached for all three CPAs. Binding of Me2SO to albumin, studied using [14C]Me2SO, was inhibited by RPS-2 when compared to K-H. Introduction and removal of Me2SO at 10 degrees C allowed an improvement in viability, at higher Me2SO concentrations, as compared to 25 degrees C.

Factors influencing renal cryopreservation. I. Effects of three vehicle solutions and the permeation kinetics of three cryoprotectants assessed with rabbit cortical slices.

A renal cortical slice model was used to assess the effects on viability of three vehicle solutions-Krebs-Henseleit (K-H), solution A, and RPS-2--at 25 degrees C. After 120 min incubation no differences in [K+]/[Na+] ratios were found. Tracer techniques were used to study the osmotic effects and permeation kinetics at 25 degrees C of three cryoprotectants (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) and the effect of the vehicle solution (K-H or RPS-2) on Me2SO kinetics. It was found that Me2SO was most permeable and ethylene glycol least, and that ethylene glycol had unusual effects which suggest that it may not act as a simple solute. Differences were found when Me2SO was introduced in K-H and RPS-2 that are believed to be related to the binding properties of Me2SO to cell constituents.

Functional preservation of the mammalian kidney. III. Ultrastructural effects of perfusion with dimethylsulfoxide (DMSO).

Abstract Isolated rabbit kidneys were perfused at 37 ° C with a cell-free solution containing 0.0, 1.4, 2.1, and 2.8 m DMSO for 60 min. Electron microscopic examination of cortical structures revealed relatively high sensitivity of proximal tubular cells to DMSO-induced alterations, which included cytoplasmic clarification and disruption of microvilli. Glomeruli exhibited proliferation of rough endoplasmic reticulum, but were otherwise well preserved after perfusion with and without DMSO. Vascular endothelial cells were also intact after perfusion with DMSO, suggesting that DMSO-induced resistance changes are not necessarily associated with degeneration of capillary cells. Perfusion without DMSO resulted in good preservation of all cortical structures. These studies suggest that ultrastructural effects of DMSO must be considered in assessment of damage done in whole kidneys during freeze-preservation studies.

Effects of temperature, potassium concentration, and sugar on human spermatozoa motility : a cell preservation model from reproductive medicine

Two experiments were performed in vitro with human sperm from a panel of 11 donors. In both experiments, washed sperm from each donor were divided into four equivolume samples, three of which were resuspended in various formulations of Tyrodes solution and the fourth was resuspended in its own seminal plasma. Each of the four samples was then stored at 37 or 3 degrees C for fixed intervals of 0, 2, 6, 12, and 24 h. Motility was assessed at 37 degrees C for all samples at the end of each interval. The results indicate that sperm survival in vitro at 3 degrees C was significantly enhanced by 20 mM K+ in Tyrodes solution relative to Tyrodes with less K+. A metabolizable sugar such as glucose was essential to maintaining sperm viability in K(+)-free media. The addition of raffinose to media containing glucose improved motility of sperm stored at 3 degrees C for 6 h.

Kidney model for study of electromagnetic thawing

Abstract The rationale for using electromagnetic heating techniques, such as in the microwave frequency range (2450 MHz), to thaw large organs after cryopreservation is that this technique, in contrast to numerous others, has been shown to yield functioning dog kidneys which had been frozen to −20 °C and even to −80 °C. The development of equipment specifically designed to thaw dog and human kidneys must be based upon knowledge of the fundamental interaction of the biomaterial (volume and shape), the radiation frequency, and the electrical properties (dielectric constant and loss tangent) of the biomaterial. The electrical properties of the organ will be partially determined by the choice of cryoprotectant and its concentration. The electrical properties will change with temperature and with the ratio of liquid to solid water. A model which predicts the influence of these parameters is given. The values of the electrical properties of tissue generally were up to an order of magnitude greater in the thawed state than in the frozen state.

PHARMACOLOGICAL EFFECTS OF DIMETHYL SULFOXIDE ON THE MAMMALIAN MYOCARDIUM

The broad spectrum of the chemical and biological properties of dimethylsulfoxide (DMSO) has been reviewed and in this monograph. Although it is a dipolar aprotic compound, DMSO shares many of the biological properties of low-molecular-weight polyhydroxy compounds such as glycerol, ethylene glycol, and other agents that can protect various cellular systems from cryoinjury. In addition to its ability to serve as an effective cryoprotectant in these various systems, several specific properties of DMSO stand out as being of special relevance to its actions on the heart. During the perfusion of whole hearts with DMSO, or the incubation of smaller samples of myocardium in DMSO, modifications of function of the cardiac contractile apparatus and intrinsic pacemakers occur. Often these are accompanied by electrophysiological changes and alterations of the myocardial cell ultrastructure. Additionally, DMSO is able to modify the responses of various effector cells in the myocardium to specific drugs that act on them, and to ions that regulate cardiac function.

Functional preservation of the mammalian kidney. I. Normothermia, low-flow perfusion.

Isolated rabbit kidneys were perfused at normothermia and at low flow rates with perfusates of various compositions. K+- and Mg2+-rich perfusates appeared to ameliorate loss of renal function encountered with salt solutions of extracellular composition (Tyrodes). A vascular mechanism for the initial vasoconstriction observed during renal perfusion is suggested, and addition of isoxsuprine, a beta-adrenergic agonist, blocked the initial vasoconstriction. Normothermic, low-flow perfusion is able to maintain functional viability of the rabbit kidney for periods up to 90 min, after which some deterioration of function is noted. Na and K reabsorption, creatinine clearance, oxygen consumption, and urine acidification are, furthermore, observable in the isolated, normothermically perfused rabbit kidney at low flows. Normothermic perfusion for periods up to 90 min is a suitable means of administering cryoprotectant drugs in preparing whole kidneys for freezing studies.

Microwave thawing of frozen kidneys: A theoretically based experimentally-effective design

Equipment was designed and fabricated for uniformly thawing frozen canine kidneys using single-frequency electromagnetic radiation. Complete and uniform warming of frozen kidneys from −70 to +14 °C over periods ranging from 1.5 to 4.5 min was achieved without “cooking” or experiencing thermal runaway. Dielectric measurements of renal slices (medulla and cortex) were performed as a function of temperature at a frequency of 918 MHz for a Me2SO cryoprotectant concentration of 5% (0.7 M). Results of these measurements were then employed as an input to analytical computer models which were used to predict the internal field intensities and power distribution results for both frozen and thawed kidneys. From these predictions, a 918-MHz EM illuminator for thawing canine kidneys was designed and fabricated. Twenty-seven kidneys were thawed using this illumination system. Of these, excellent uniformity of thawing was achieved for 17 kidneys, good uniformity for 8 kidneys, and for only 2 kidneys was thawing uniformity fair to poor.