Recent progress toward vitrification of kidneys

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present study, rabbit kidneys, permeated with 2 M glycerol using techniques previously described, were cooled to -80°C at four rates varying from 0.02 to 3”C/min and then rewarmed at four similar rates, giving 16 experimental treatments. After gradual deglycerolization at lO”C, each kidney was autografted and observed for 30 min. Assessment was by gross appearance, light and electron microscopy, tissue K+/Na+ ratio, and LDH release. The results indicate that the slowest cooling and rewarming rates are preferable; damage was predominantly to the glomeruli and the loops of Henle. Regional distribution of perfusate flow and residual glycerol concentration were measured. The distribution of ice at -80°C was assessed by freeze substitution and was observed to be located in the interstitial spaces and the glomerular capillaries. The mechanisms of cryoinjury affecting this system and possible means of circumventing them will be considered.

the introduction and removal of cryoprotectants, several additional features have been added to the perfused organ model of Levin (Cryobiology 18, 617 (1981)). A perfused organ is now modeled as a fourcompartment system consisting of a “welldistributed” capillary network with distinct arterial inlet and venous outlet ports, a luminal region, and an endothelial cell region; an interstitial space with a distinct lymphatic outlet port; and an intracellular region. The pertinent physiological media are modeled as four-component aqueous solutions consisting of water, a high-molecular-weight colloidal solute, a lowmolecular-weight electrolytic solute, and a cryoprotective agent. Each of the four compartments are and, unlike other assumed to be “well-mixed” analyses, “variable-in-size” such that their volumes depend upon the amount of water and solute present within the regions at a given instant of time. Furthermore, the amount of water and solute present within each of the regions are assumed to depend upon the time, varying hydrostatic and osmotic pressures within the spaces and the resistance to transfer of the water and the solutes between the various compartments. On the basis of these and other assumptions, the response of perfused organs is found to be strongly affected by the temporal variation in the perfusion pressure and the perfusate composition. In addition, the introduction and removal of highly permeable CPAs such as Me,SO is found to be “flow” rate-limited, while the introduction and removal of more slowly permeable CPAs such as glycerol is found to be “permeability” rate limited. Examples will be presented to illustrate the resulting transient variation in flow resistance, cell size, and overall organ weight for different CPA introduction/removal protocols.

61. Rabbit Kidney Function in vitro Following TwoStep Cooling. F. M. GUTTMAN, L. NHUYEN, AND N. B. SEGAL (Department of Surgery, Montreal Children’s Hospital, Montreal, Quebec, H3H lP3, Canada). Rabbit kidney function was assessed in vitro after various cryobiological manipulations under 3.0 M Me,SO protection. We have previously described the usefulness of the in vitro perfusion technique to assess rabbit kidney function following cooling with Me,SO infusion and hypertonic washout (N. B. Segal and F. M. Guttman, Cryobiology 19,50-60, 1982)). Using these techniques, we have tested kidneys cooled to 0°C for 1 hr; to -20°C for 1 hr; to -4 to -10°C for 1 hr;

TABLE-ABSTRACT CR.Cl (ml/mm/g) Control Control Cooling Cooling Cooling Cooling

(with Me,SO) 0°C (1 hr) -20,Y (1 hr) -4”3 to - 10°C (1 hr) 0°C (*/ hr), -20°C (1 hr)

0.3 0.26 0.24 0.23 0.29 0.33

MEETING

2 .19

k .lO f f f f

.07 .02 .08 0.23

61 Na Abs. (%) 36.2 45.6 15.4 18.34 24.6 10.9

2 17.2 2 24.3 2 13.7 -c2.71 k 16.7 f 6.35

Glut. Abs. (%) 84.9 91.01 65.75 30.42 56.6 18.2

+ f 2 k 2 f

18 8.3 17.93 5.04 21.9 4.0

and tinally a two-step technique to -0°C for half an 60. Effect of Cooling and Warming Rate on Glycerolized Rabbit Kidneys. I. A. JACOBSEN, hour and then to -20°C for 1 hr. The cooling rate was D. E. PEGG, H. STARKLINT, J. CHEMNITZ, C. HUNT, P. BARFORT,AND M. P. DIAPER(Laboratory of Nephrology, University of Odense, Denmark and MRC Medical Cryobiology Group, Department of Surgery, University of Cambridge, United Kingdom). Cooling and warming rates are known to be important determinants of viability for cryopreserved cells. Optimal rates for whole organs are not known. In the

uniform at l”C/min and thawing was by immersion in 37°C saline. The results are presented in the table above. Thus although tubular function is poor, the twostep technique to -20°C results in a fair general function with good CR.Cl.

62. Recent Progress toward

Vitrification

of Kidneys.

GREGORYM. FAHY (Cryobiology Lab, ARC

ABSTRACTS,

19th ANNUAL

Blood Services Labs, 9312 Old Georgetown Rd., Bethesda, Maryland 20814) AND DOUGLAS R. MACFARLANE AND C. A. ANGELL (Department of Chemistry, Purdue University, West Lafayette, Indiana 47907). The following recent observations have been made which improve the prospects for eventual kidney preservation by vitrification. (1) Perfusate-colloids such as polyvinylpyrrolidone (PVP) and hydroxyethyl starch (HES), when present in normal concentrations (668% w/v), reduce by 6-8% w/v the concentration of penetrating cryoprotectant (PCP) required for vitrification (see also Cryo-letters 2, 353-358 (1981)). At 1000atm, only 42.5% w/v dimethylsulfoxide-acetamide (DA) or 2% w/v DA + 10% w/v propylene glycol (PG) is required. (2) Cells in the renal cortex, medulla, and papilla will vitrify in these polymer-PCP mixtures, provided polymer is perfused into the kidney to allow distribution of polymer into Bowman’s space. Thus, renal cell types all behave as though they contain at least 6% w/v PVP. (3) Mixtures of 45% DA + 6% PVP or 18% acetamide + 12% Me,SO + 12% PG and 6% PVP devitrify only slightly or not at all when warmed at only 16OYYmin at atmospheric pressure. (4) DA toxicity can be suppressed substantially by increasing the rates of addition and removal of this PCP, while leaving the exposure time at the peak concentration constant. Apparently the toxicity depends as much on total exposure time as on the peak concentration. Toxicity can now be completely suppressed at a concentration of 40% w/v DA. However, 42% DA + 6% PVP still causes substantial damage. (Supported in part by NIH Grants GM17959 and RR05737.)

63. The Limiting Effects of Heat and Mass Transfer on the Osmotic Behavior of Cells during Freezing and Thawing. RONALD L. LEVIN

(Biomedical Engineering and Instrumentation Branch, National Institutes of Health, Bethesda, Maryland 20205). During the past twenty years, numerous models have been proposed to describe the osmotic behavior of cells during freezing and thawing. Although these studies have pinpointed the important parameters governing the volumetric response of cells (e.g., solution composition, cell surface area to volume ratio, cell water, and solute permeability) and have greatly aided in the understanding of the physical-chemical events occurring during freezing and thawing, from the practical point of view they all have one serious drawback. Namely, all of the current models deal with a single, isolated cell suspended in an infinite amount of solution which is being cooled or warmed uniformly at a constant rate. No provision is made for those common situations where (1) the volume of cells is comparable to the volume of the suspending solution or (2) the cellular system is cooled or warmed in a nonuniform

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manner with time due to the large amounts of latent heat liberated during freezing or absorbed during thawing. The purpose of the present study was therefore to analytically investigate cellular osmotic behavior under nonideal, but typical, freeze-thaw conditions. Our results indicate that the cytocrit of the cell suspension begins to significantly affect cellular volumetric behavior at levels above 10%. This is especially true for prefrozen suspensions which are being warmed at very high rates, in which case the cells are exposed to strongly hypotonic conditions just after the complete melting of the extracellular ice. Our results also indicate that most of the cell water loss during freezing and gain during thawing may occur during the long temperature-time plateaus which usually occur just after the initial formation of ice during cooling and just before the final melting of the ice during warming, rather than at lower or higher temperatures.

SYMPOSIUM AND

IV-WATER

ADAPTATION:

COMMON

ARE

THERE

DENOMINATORS?

Systems. PAULA T. BEALL (Department of Physiology, Baylor College of Medicine, Houston, Texas 77030).

64. States of Water in Biological

Water makes up 70-90% of the mass of most living cells, and its many roles in biological processes have long been a subject of study. Early investigations measured the amount of water in cells, or the ratio of water to dry solids, as a sensitive indicator of diverse physiological conditions and disease states. Today however, by utilizing a battery of biophysical techniques, one is able to study more than just the water content of cells; we may seek to examine the physical properties of water inside the complex milieu of cells and tissues, or in appropriate macromolecular model systems. Techniques which look at the properties of water molecules on a very short time scale often suggest that the molecular properties of water in cells are very similar to those in pure bulk water. However, techniques such as calorimetry, NMR, and dielectric relaxation that measure an average over all molecules can be interpreted to show a real reduction in the mobility of water in cells. The question under investigation is whether water in cells behaves exactly as ifit were in a dilute solution or whether the surfaces, charges, and charge distributions of the cellular matrix influence the physical properties of water in a discernible manner. The extent of these influences, their origins, and tinally their physiological significance remain difficult questions to answer. This presentation will review data from numerous techniques which suggest that at least a portion of water molecules in living cells are found in environments where they display altered physical properties. Specific correlations between cytoskeletal structural organization and water proton relaxation times in cultured cell systems will be dis-