M.P. Diaper

Effect of cooling and warming rate on glycerolized rabbit kidneys

Cooling and warming rates are known to be important determinants of viability for cryopreserved cells, but optimal rates have not previously been determined for any whole organ. In this study, rabbit kidneys, permeated with 2 M glycerol were cooled to -80 degrees C at four rates varying from 1 degrees C/hr to 3.1 degrees C/min and then rewarmed at four rates from 1 degrees C/hr to 4.2 degrees C/min, giving 16 experimental treatments. After gradual deglycerolization at 10 degrees C, each kidney was autografted and observed for 30 min. Assessment was by measurement of vascular resistance, immediate post-thaw lactate dehydrogenase (LDH) release, gross appearance, light- and electron microscopy, and tissue K+/Na+ ratio 30 min after transplantation. The best results were obtained after cooling at 1 degrees C/hr; warming rate had little apparent influence on the criteria used to assess function with the exception of LDH release, which indicated a preferred warming rate around 1 degrees C/min. Histological studies revealed extensive vascular damage, notably to the glomerular capillaries, that was minimized by very slow cooling. Freeze substitution, carried out on samples removed at -80 degrees C, demonstrated extensive ice formation in the interstitial space and, at the faster cooling rates, in the glomerular capillaries. Intracapillary ice formation was reduced in the kidneys cooled at 1 degrees C/hr.

Introduction and removal of cryoprotective agents with rabbit kidneys: Assessment by transplantation

Rabbit kidneys were perfused with up to 4 M glycerol or propane-1,2-diol (propylene glycol, PG) in three vehicle solutions: one normokalemic and made hypertonic with mannitol (HP5), one hyperkalemic but without mannitol (HP6), and one hyperkalemic and with mannitol (HP7). Subsequent function was assessed by autotransplantation. Up to 3 M glycerol in HP5 was well tolerated but not in HP6 or HP7. Conversely, up to 3 M PG in HP7 was compatible with excellent post-transplant function, but the same concentration in HP5 was severely damaging. PG (4 M) in either solution was severely injurious and no kidneys survived perfusion with this concentration. Vascular resistance was well controlled by the vehicle solutions with mannitol, but it was generally higher during perfusion with the hyperkalemic HP7 compared with the normokalemic HP5. No kidneys perfused with 3 M solutions of either of the cryoprotective agents and cooled briefly to -6 degrees C without freezing had any post-transplant function, and neither did kidneys perfused with 3 M PG or 4 M glycerol tolerate slow cooling to -80 degrees C and warming. The need to optimize perfusate composition for the CPA being used is clear, and the dramatic increase in toxicity of PG when the concentration exceeds 3 M supports the suggestion that mixtures of PG and glycerol should be considered. The observation of damage at high subzero temperatures, before freezing has occurred, requires further detailed study.

Perfusion of rabbit kidneys with solutions containing propane-1,2-diol.

Propane-1,2-diol (propylene glycol, PG) permeates more rapidly than glycerol, has a strong glass-forming tendency, and appears to have a low toxicity. It is therefore attractive as a potential cryoprotectant for renal preservation. In this paper we compared the effect on subsequent function, of exposing rabbit renal cortical slices to 1 M PG or glycerol in a range of vehicle solutions and we demonstrated a remarkably low toxicity of PG at this concentration. Rabbit kidneys were then perfused with solutions containing PG up to a maximum concentration of 3 M, after which the cryoprotectant was removed and the function of cortical slices prepared from the perfused kidneys was assessed. Marked differences in perfusion characteristics were found between PG and glycerol and between different vehicle solutions for PG, but the two most suitable perfusates, both containing about 100 mM mannitol, permitted normal function in slices prepared after removal of PG. These results indicate that, with an appropriate vehicle perfusate, exposure to PG up to a concentration of 3 mol/liter has remarkably little effect upon vascular resistance and the renal cortical functions measured.

Optimization of a vehicle solution for the introduction and removal of glycerol with rabbit kidneys

Previous studies with rabbit kidneys in our laboratories have used a plasma-like solution as the vehicle for the introduction and removal of glycerol. Other workers have usually employed high-potassium solutions. In this study we have assayed the function of rabbit renal cortical slices after incubation in a range of solutions, each of which contained 1 M glycerol, for 4 hr, followed by stepwise removal of the cryoprotectant. The functions measured were endogenous oxygen consumption, p-aminohippurate uptake, and the ability of the slices to accumulate potassium. Exposure to glycerol produced a considerable reduction of slice function, but, in the presence of glycerol, elevation of the potassium concentration was beneficial, whereas high concentrations of magnesium were detrimental. The optimum potassium concentration was 70-100 mM. Replacement of chloride by a range of anions of higher molecular weight was either without benefit (glycerophosphate) or detrimental (sulfate, citrate, and gluconate). Elevation of total osmolality from 300 to 400 mosmolal with glucose, mannitol, glycerophosphate, or Pipes reduced slice function, but when the same osmolality was achieved by raising the concentration of all the components of the solution in the same ratio, there was no significant loss of function. There was a weak optimum pH at ca. 7.0. These experiments led to the formulation of a bicarbonate-buffered perfusate containing 80 mM potassium and 17.5 g Haemaccel per liter, having a pH of 7.0 with 5% CO2 at 10 degrees C, and an osmolality of 400 mosmol/kg. This solution was used to preserve rabbit kidneys for 20 hr at 10 degrees C, by continuous perfusion, and was compared with our previous Haemaccel perfusate, HP5, which contained 4 mM K+, 111 mM mannitol, and had a pH of 7.4. The two solutions were equally effective.

The effect of cooling and warming rate on cortical cell function of glycerolized rabbit kidneys

Experiments previously reported (I.A. Jacobsen, D.E. Pegg, H. Starklint, J. Chemnitz, C.J. Hunt, P. Barfort, and M.P. Diaper, Cryobiology 19, 668, 1982) suggested that rabbit kidneys permeated with 2 M glycerol are least damaged during freezing and thawing if they are cooled very slowly (1 degree C/hr). Using similar techniques of glycerolization, cooling, storage at -80 degrees C, rewarming, and deglycerolization, active cell function in cortical tissue slices prepared from such kidneys has now been studied. Oxygen uptake, tissue K+/Na+ ratio after incubation, and slice/medium PAH ratio after incubation were measured. Kidneys cooled at 3.1 degree C/min and warmed at 4.2 degrees C/min gave poor results in the previous studies and the lowest levels of cell function in the present experiments. Kidneys cooled at 1 degree C/hr exhibited degrees of slice function that were dependent on warming rate: warming at 1 degree C/min was better than warming at either 1 degree C/hr or c.20 degrees C/min. These results refine the previously drawn conclusions, (loc cit) and indicate optimal cooling and warming rates for rabbit kidneys containing 2 M glycerol, in the region of 1 degree C/hr cooling and 1 degree C/min warming. These rates are much lower than have hitherto been used by others for any system. Some implications of these findings are discussed.

Droplet freezing of antibody-linked indicator red cells of sheep, ox, and human origin

Abstract A droplet freezing technique for the cryopreservation of indicator red cells is described. Recovery was crucially dependent on the composition of the solution in which the cells were suspended. Preliminary experiments to determine the relative importance of sucrose, glucose, sodium chloride and hydroxyethyl starch (HES) in determining the survival of trypsin-treated sheep red cells showed that the addition of sucrose or HES or both to isotonic sodium chloride solution increased recovery, whereas the additional inclusion of glucose was detrimental. It was shown that glucose penetrated the cells whereas sucrose did not. The optimum combination of sucrose and sodium chloride concentration, in the presence of 6 g/dl HES, was 7 g/dl sucrose plus 0.3 g/dl sodium chloride. Recovery was increased by increasing the concentration of HES, and maximal recovery was obtained by thawing the frozen droplets in phosphate-buffered saline at 40 °C. Trypsintreated ox and human cells gave much lower recovery than sheep cells when HES was used in the freezing mixture but the substitution of dextran (10 g/dl) for HES gave greater than 80% recovery with all three species. Ten different antibody-coupled reagent cells all gave >83% recovery. The effects of hematocrit, incubation time, and storage temperature are described. The preservation technique described is simple and convenient, and will make it possible to extend the use of immunoassay procedures using antibody-coupled red cells.