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Measurements of icefront kinetics and temperature fields in extended freezing containers
690
ABSTRACTS. 21st ANNUAL
moved across. Measurements of cell volume in fractionated human white blood cells (separated into lymphocytes, granulocytes and monocytes) after exposure to a non-permeating solute in the presence of various concentrations of DMSO were performed using a Coulter electronic particle counter interfaced to a microcomputer. A Simplex algorithm was used to find the best fit for the water permeability using independently determined values for the DMSO permeability and the reflection coefficient. There was no significant effect of DMSO, in concentrations of I, 3, 5 or 10% v/v, on the measured water permeability of the three cell types tested, although the rates of volume changes were significantly different. It is therefore necessary to consider the influence of permeant solutes on water movement, even when there is initially no concentration gradient of the permeant solute across the cell membrane. It is therefore necessary, when calculating the volume of cells during freezing at subzero temperatures, to estimate not only the water permeability and its temperature dependence, but also the permeability of the cryoprotectant. the reflection coefficient, and the dependence of these parameters on temperature. Since DMSO is known to have direct effects on the lipid portions of cell membranes, the observations imply that the lipid components of the membrane are not the determinants of water permeability. (Supported by grants from the Medical Research Council and the Alberta Heritage Savings Trust FundCancer.) 25. Measurements of Ic,efront Kinetics and Trmperuture Fields in Extended Freezing Contuiners. U. HARTMANN, AND G. RAU
CH. KORBER, M. W. SCHEIWE.*
(Helmholtz-Institut fur Biomedizinische Technik an der RWTH Aachen, Goethestrasse 27129, D-5100 Aachen, West Germany. and *Nordmark-Werke GmbH. Postfach 1244. D-2082 Uetersen. West Germany). The knowledge of temperature fields and icefront kinetics is important for successful design of cryopreservation processes. To measure these parameters, a special plate-like freezing container was constructed (i, d., I45 x 145 x 20 mm) with mainly unidirectional heat transfer. An array of miniature thermocouples (wire diameter, 75 urn) was built corresponding to the direction of the main heat flux. In addition the icefront kinetic was measured by means of ultrdsound-echotechnique. Test fluids were distilled water, 5 wt% saltwater solution, and a salt-HES-water solution. The container was cooled in a biofreezer as well as by immersion in liquid nitrogen with cooling rates (at the inner container walls) between 0.5 and 30”K/min. No significant difference between the icefront kinetics of the different test fluids was testified (specially at low cooling rates). For comparable cooling rates no drastic change of the icefront velocity could be observed when shaking the container. Only the temperature field in the fluid was altered by this forced convection.
MEETING
In the salt-containing test fluids no further freezing point depression with the advancing icefront could be detected either during normal cooling or during cooling with shaking the container. This can be interpreted as the icefront not being planar in both cases and the salt being incorporated in arising interdendritic spaces. 26. Repulsion and Encupsulation of Particles und Biological Cells by an Advancing Ice-Liquid Intecfacr. CH. KORBEK, E. G. CRAVALHO,* AND
M. D. COSMAN’ (Helmholtz-Institut fur Biomedizinische Technik an der RWTH Aachen, Goethestrasse 27129, D-5100 Aachen, West Germany, and *Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology. Cambridge, Massachusetts 02139). Suspended particles or cells can be encapsulated by an advancing ice-liquid interface if the phase front velocity is beyond a critical value determined by repulsive and attractive forces related to differences in surface free energy and viscous drag, respectively. This transition was analyzed by means of a special freezing stage [Cryobiology 21, 68 (1984)] which yields continuously increasing interface velocities in combination with thermal gradients high enough to maintain unidirectional planar freezing. Under these circumstances, four different phases can be distinguished: (I) initially, all particles are being repelled by the advancing ice front; (2) the particles are gradually left behind; (3) all particles are immediately encapsulated: (4) finally, nonplanar freezing leads to particle entrapment in channels of residual lipid. Critical interface velocities were determined as the mean value corresponding to the positions where the maximum number of particles were left behind during the second phase. Using spherical latex particles of various diameters tanging from I to I5 pm suspended in distilled water, it was found that, in accordance with theoretical predictions, the critical velocity decreases with increasing diameter. The experiments also revealed a considerable influence of the thermal gradients: the critical interface velocity decreases continuously if the transition point is shifted towards lower gradients, i.e., closer to the thermal midline of the system. In contrast, concentration gradients did not seem to have a major effect: the addition of solute (0.56 mol% NaMnO,) did not result in a significant change of the transition points as compared to the results obtained in pure water. Yeast cells were seen to be encapsulated at lower velocities than latex particles of corresponding diameter. 27. Chungrs in Cellular Light Stuttering Properties by Hypotonic Stress. L. Hooo, L. E. MCGANN,
A. R. TUKNEK, AND J. M. TUKC (Department of Pathology. University of Alberta; Cross Cancer
ABSTRACTS. 21st ANNUAL
moved across. Measurements of cell volume in fractionated human white blood cells (separated into lymphocytes, granulocytes and monocytes) after exposure to a non-permeating solute in the presence of various concentrations of DMSO were performed using a Coulter electronic particle counter interfaced to a microcomputer. A Simplex algorithm was used to find the best fit for the water permeability using independently determined values for the DMSO permeability and the reflection coefficient. There was no significant effect of DMSO, in concentrations of I, 3, 5 or 10% v/v, on the measured water permeability of the three cell types tested, although the rates of volume changes were significantly different. It is therefore necessary to consider the influence of permeant solutes on water movement, even when there is initially no concentration gradient of the permeant solute across the cell membrane. It is therefore necessary, when calculating the volume of cells during freezing at subzero temperatures, to estimate not only the water permeability and its temperature dependence, but also the permeability of the cryoprotectant. the reflection coefficient, and the dependence of these parameters on temperature. Since DMSO is known to have direct effects on the lipid portions of cell membranes, the observations imply that the lipid components of the membrane are not the determinants of water permeability. (Supported by grants from the Medical Research Council and the Alberta Heritage Savings Trust FundCancer.) 25. Measurements of Ic,efront Kinetics and Trmperuture Fields in Extended Freezing Contuiners. U. HARTMANN, AND G. RAU
CH. KORBER, M. W. SCHEIWE.*
(Helmholtz-Institut fur Biomedizinische Technik an der RWTH Aachen, Goethestrasse 27129, D-5100 Aachen, West Germany. and *Nordmark-Werke GmbH. Postfach 1244. D-2082 Uetersen. West Germany). The knowledge of temperature fields and icefront kinetics is important for successful design of cryopreservation processes. To measure these parameters, a special plate-like freezing container was constructed (i, d., I45 x 145 x 20 mm) with mainly unidirectional heat transfer. An array of miniature thermocouples (wire diameter, 75 urn) was built corresponding to the direction of the main heat flux. In addition the icefront kinetic was measured by means of ultrdsound-echotechnique. Test fluids were distilled water, 5 wt% saltwater solution, and a salt-HES-water solution. The container was cooled in a biofreezer as well as by immersion in liquid nitrogen with cooling rates (at the inner container walls) between 0.5 and 30”K/min. No significant difference between the icefront kinetics of the different test fluids was testified (specially at low cooling rates). For comparable cooling rates no drastic change of the icefront velocity could be observed when shaking the container. Only the temperature field in the fluid was altered by this forced convection.
MEETING
In the salt-containing test fluids no further freezing point depression with the advancing icefront could be detected either during normal cooling or during cooling with shaking the container. This can be interpreted as the icefront not being planar in both cases and the salt being incorporated in arising interdendritic spaces. 26. Repulsion and Encupsulation of Particles und Biological Cells by an Advancing Ice-Liquid Intecfacr. CH. KORBEK, E. G. CRAVALHO,* AND
M. D. COSMAN’ (Helmholtz-Institut fur Biomedizinische Technik an der RWTH Aachen, Goethestrasse 27129, D-5100 Aachen, West Germany, and *Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology. Cambridge, Massachusetts 02139). Suspended particles or cells can be encapsulated by an advancing ice-liquid interface if the phase front velocity is beyond a critical value determined by repulsive and attractive forces related to differences in surface free energy and viscous drag, respectively. This transition was analyzed by means of a special freezing stage [Cryobiology 21, 68 (1984)] which yields continuously increasing interface velocities in combination with thermal gradients high enough to maintain unidirectional planar freezing. Under these circumstances, four different phases can be distinguished: (I) initially, all particles are being repelled by the advancing ice front; (2) the particles are gradually left behind; (3) all particles are immediately encapsulated: (4) finally, nonplanar freezing leads to particle entrapment in channels of residual lipid. Critical interface velocities were determined as the mean value corresponding to the positions where the maximum number of particles were left behind during the second phase. Using spherical latex particles of various diameters tanging from I to I5 pm suspended in distilled water, it was found that, in accordance with theoretical predictions, the critical velocity decreases with increasing diameter. The experiments also revealed a considerable influence of the thermal gradients: the critical interface velocity decreases continuously if the transition point is shifted towards lower gradients, i.e., closer to the thermal midline of the system. In contrast, concentration gradients did not seem to have a major effect: the addition of solute (0.56 mol% NaMnO,) did not result in a significant change of the transition points as compared to the results obtained in pure water. Yeast cells were seen to be encapsulated at lower velocities than latex particles of corresponding diameter. 27. Chungrs in Cellular Light Stuttering Properties by Hypotonic Stress. L. Hooo, L. E. MCGANN,
A. R. TUKNEK, AND J. M. TUKC (Department of Pathology. University of Alberta; Cross Cancer