- Email: [email protected]
Equilibrium and nonequilibrium phase determinations in the ternary system sucrose-sodium chloride-water
664
ABSTRACTS,
13TH
As reported elsewhere in these proceedings (Abstract No. 57), a solution of catalase in salt and buffer (27 mM NaCl + 10 mM KHPOI) exhibits, after freeze-thaw, an enzymatic recoverycooling rate curve with a clear optimum at 5”C/ min. This system has been used to compare the efficiency of various cryophylactants at supraoptimal and infraoptimal cooling rates and in the absence of salt as well. In addition to separation into high molecular weight ( > 1000) and low molecular weight ( < 1000) types, cryophylactants may be categorized by their reactive functional groups as aprotic (DMO, glyme, PVP), protic (alcohols and glycols ), and mixed ( cellosolves, HES ). Many of these agents develop aldehyde and peroxide impurities upon exposure to air, which may be largely responsible for their toxic effects (Bello, J., and Bello, H. R., Arch. Biochem. Biophys. 172, 608 ( 1976). Freshly distilled samples stored under nitrogen were used in the present study, and log dose-response curves were used to determine ED, values for cryoprotection. At present the following conclusions appear warranted: (1) The best high molecular weight agents, such as polyvinylpyrollidone (PVP), provide marked protection at levels of 0.1 pM (5 rg/ml), nearly stoichiometric with catalase subunits. (2) The best low molecular weight agents, such as cellosolve and diglyme, provide marked protection at IO-fold higher weight concentrations ( 1 mM), still far too low to be explained by colligative effects. (3) Potent cryophylaxis is usually manifest under all three freeze-thaw conditions: At supraoptimal as well as infraoptimal cooling rates and in the absence of salt. (4) Both protic and aprotic groups appear to contribute toward cryoprotective potency. (5) At certain levels, low molecular weight agents regularly exhibited a subzero damage zone in the range of -30 to 50°C. This work was supported in part by American Cancer Society Grant No. DT-42, under the auspices of Universities Associated for Research and Education in Pathology, Incorporated. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
59. A Heat and Mass Transfer Model of the Freezing of Biomaterials. M. G. O’CALLAGHAN,* E. G. CRAVALHO, AND C. E. HUGGINS. (Cryogenic Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Surgery, Harvard Medical School, Surgical Low Temperature Unit and Blood Bank Transfusion Service, Massachu-
ANNUAL
MEETING setts General setts 02114).
Hospital,
Boston,
Massachu-
In the optimization of thermal protocols for the freezing of biomaterials, a substantial number of experimental variables must be considered. The required experimental effort to study the influence of all these variables can be reduced through the utilization of computer models that describe the thermodynamic behavior of biomaterials under these conditions. Quantitative information obtained from these models permits researchers to focus on only the most promising protocols. Previous models of the freezing of biomaterials are generally either heat or mass transfer analysis, but not both. The present work extends these models to include the effects of coupled heat and mass transfer. To simplify the analysis many structural simplifications were made. The idealized model tissue consists of blocks of cubic cells, separated in discrete locations by channels. Ice was assumed to propagate only in these channels, causing dehydration of adjacent cells. The process was modeled by analyzing the one-dimensional solidification of an NaCl solution and then coupling the results to the mass transfer model of Levin (J. Heat Transfer Trans. ASME, in press). The temperature profiles were generated by a modified Karman-Pohlhausen integral technique, in which the form of the temperature profile is assumed and the “averaged” diffusion equation is solved with the full set of boundary conditions. The concentration profiles were assumed to be the pseudo-steadystate profiles which are known in closed form. The chemical potentials of the various species were determined from these profiles and were substituted into the Levin model to determine cell volume, degree of supercooling, and solute distribution as a function of time. By application of this model to an actual biological tissue, the destructive phenomena may be characterized and less injurious protocols found. Supported in part by the National Heart and Lung Institute, Grant No. HL-14322. 60. Equilibrium and terminations Sucrose-Sodium GAYLE, F. H. (Department and Materials Durham, North
Nonequilibrium Phase Dein the Ternary System Chloride-Water. F. W. COCKS, AND M. L. SHEPARD. of Mechanical Engineering Science, Duke University, Carolina 27706).
Sucrose has been suggested as a cryoprotective agent for human red blood cells. As part of a study of the relationship between protective action and low temperature phase relationships, the sucrosesodium chloride-water ternary system has been investigated to determine equilibrium and meta-
ABSTRACTS,
13TH
stable phase transformations in the primary ice phase field. Metastable glass formation is essential in preventing ice crystallization, while both stable and metastable phase transformations determine the extent to which solutes concentrate on cooling. The liquidus surface has been determined by standard differential thermal analysis techniques. It was found that sucrose is not as effective as is dimethyl sulfoxide or glycerol in depressing the freezing point and suppressing salt concentrations in water-based ternary solutions. The line of twofold saturation from the H,O-NaCI binary system which determines the limit of the primary ice phase field has been determined, as well as the approximate location (30 w/o HZO, 10 w/o NaCI, 60 w/o sucrose) and temperature (-35*C) of a ternary eutectic point. The sucrose-sodium chloride-water system tends to form stable glasses near the ternary eutectic point. At higher or lower salt concentrations an unstable glass is formed on cooling, and over the range of heating rates investigated, 5 to 2O”C/min, this glass devitrifies. The support of this work by the American Cancer Society under Grant No. PDT-42A is gratefully acknowledged. 61. Equillibrium and Nonequilibrium Therm& Behavior of Aqueous Ternary Solutions Based on Complex Physiologic Support Media Containing NaCl and Dimethyl Sulfoxide or Glycerol. R. I. POZNER, M. L. SHEPARD, of MechanAND F. H. COCKS. (Department ical Engineering and Materials Science, Duke University, Durham, North Carolina 27706). Precipitation of ice with concomitant solute enrichment of the residential liquid and the formation and stability of glassy phase developed during freezing have profound effects on postfreeze cell viability. The present studies have utilized standard differential thermal analysis (DTA) techniques to establish the equilibrium liquidus surfaces of the primary ice phase fields of ternary systems containing NaCl, a cryoprotective agent (either glycerol or dimethyl sulfoxide (DMSO)), and an aqueous physiologic support medium. The media employed in these studies were Eagle’s minimum essential medium (MEM ) and MEM supplemented with 20 ~01% fetal calf serum ( FCS). Additionally, qualitative evaluations of the glassforming tendencies in the systems MEM-NaClglycerol, MEM-NaCI-DMSO, and (MEM + FCS )-NaCI-DMSO were established. Both equilibrium and nonequilibrium thermal behavior, in the three systems investigated, exhibited only minor variations relative to analogous Ha-based systems previously investigated. Results obtained in these studies may provide insight into the
ANNUAL
MEETING
665
physicochemical events which occur when cells suspended in such solutions are exposed to cryogenic temperatures. The support of this work by the American Cancer Society under Grant No. PDT-42A is gratefully acknowledged. 62. Preliminady tration JOHN G. partment Houston,
Studies on the Differential Peneof Cryoprotectants through Ice. BAUST AND MARK DONVITO.’ (Deof Biology, University of Houston, Texas 77004 ) .
Polyhydric alcohols and disaccharides demonstrate differential rates of penetration through type I ice. Depth of penetration is a function of time at a given temperature, ambient pressure, and molecular diameter. Ice samples prepared from degassed, distilled (3x) water were frozen in 6-mm-diameter cylinders for 1.5 hr at -35°C followed by topical application of 0°C cryoprotectant-saturated filter paper discs. Temperature was maintained at -35°C for l-96 hr while ambient pressure was varied up to 100 atm. Absolute rates of penetration for glycerol exceeded 3600 rmol/hr (1 ATA) and 8200 /Imol/hr (100 ATA) while sorbitol rates exceeded 1500 pmoF/hr (1 ATA) and 3450 pmol/hr (100 ATA). Depth of penetration of disaccharides was comparatively reduced. A procedure is described for cryoprotectant supplementation in or “removal” (dilution) from prefrozen tissue samples. 63. A Simple One-Step Procedure Tissue for Light Microscopy. (Department of Anatomy, Medical College and Hospital, Pennsylvania 19102).
for Freezing L. TERRACIO. Hahnemam Philadelphia,
Many histochemical and autoradiographic techniques require the use of frozen-dried tissue. Commonly used methods for freezing tissue such as a supercoobd metal surface 1 or quenching fluid have the drawback of adding preparatory steps to an already lengthy procedure. These additional steps can be eliminated for light microscopic procedures by using the following method. Slices of fresh tissue were placed on an aluminum foil-covered spatula and the tissue was frozen by introducing it into a rapidly running stream of liquid nitrogen. This is easily obtained from a standard storage tank under maximum flow. The frozen tissue was dissected into small pieces and placed in a cooled copper specimen block equipped with a thermometer and heating coil. The specimen block was then transferred to a glass freeze-drier and the tissue was dried by increasing the temperature of the block lO”C/hr until it reached room temperature.’ The dry tissue
ABSTRACTS,
13TH
As reported elsewhere in these proceedings (Abstract No. 57), a solution of catalase in salt and buffer (27 mM NaCl + 10 mM KHPOI) exhibits, after freeze-thaw, an enzymatic recoverycooling rate curve with a clear optimum at 5”C/ min. This system has been used to compare the efficiency of various cryophylactants at supraoptimal and infraoptimal cooling rates and in the absence of salt as well. In addition to separation into high molecular weight ( > 1000) and low molecular weight ( < 1000) types, cryophylactants may be categorized by their reactive functional groups as aprotic (DMO, glyme, PVP), protic (alcohols and glycols ), and mixed ( cellosolves, HES ). Many of these agents develop aldehyde and peroxide impurities upon exposure to air, which may be largely responsible for their toxic effects (Bello, J., and Bello, H. R., Arch. Biochem. Biophys. 172, 608 ( 1976). Freshly distilled samples stored under nitrogen were used in the present study, and log dose-response curves were used to determine ED, values for cryoprotection. At present the following conclusions appear warranted: (1) The best high molecular weight agents, such as polyvinylpyrollidone (PVP), provide marked protection at levels of 0.1 pM (5 rg/ml), nearly stoichiometric with catalase subunits. (2) The best low molecular weight agents, such as cellosolve and diglyme, provide marked protection at IO-fold higher weight concentrations ( 1 mM), still far too low to be explained by colligative effects. (3) Potent cryophylaxis is usually manifest under all three freeze-thaw conditions: At supraoptimal as well as infraoptimal cooling rates and in the absence of salt. (4) Both protic and aprotic groups appear to contribute toward cryoprotective potency. (5) At certain levels, low molecular weight agents regularly exhibited a subzero damage zone in the range of -30 to 50°C. This work was supported in part by American Cancer Society Grant No. DT-42, under the auspices of Universities Associated for Research and Education in Pathology, Incorporated. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
59. A Heat and Mass Transfer Model of the Freezing of Biomaterials. M. G. O’CALLAGHAN,* E. G. CRAVALHO, AND C. E. HUGGINS. (Cryogenic Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Surgery, Harvard Medical School, Surgical Low Temperature Unit and Blood Bank Transfusion Service, Massachu-
ANNUAL
MEETING setts General setts 02114).
Hospital,
Boston,
Massachu-
In the optimization of thermal protocols for the freezing of biomaterials, a substantial number of experimental variables must be considered. The required experimental effort to study the influence of all these variables can be reduced through the utilization of computer models that describe the thermodynamic behavior of biomaterials under these conditions. Quantitative information obtained from these models permits researchers to focus on only the most promising protocols. Previous models of the freezing of biomaterials are generally either heat or mass transfer analysis, but not both. The present work extends these models to include the effects of coupled heat and mass transfer. To simplify the analysis many structural simplifications were made. The idealized model tissue consists of blocks of cubic cells, separated in discrete locations by channels. Ice was assumed to propagate only in these channels, causing dehydration of adjacent cells. The process was modeled by analyzing the one-dimensional solidification of an NaCl solution and then coupling the results to the mass transfer model of Levin (J. Heat Transfer Trans. ASME, in press). The temperature profiles were generated by a modified Karman-Pohlhausen integral technique, in which the form of the temperature profile is assumed and the “averaged” diffusion equation is solved with the full set of boundary conditions. The concentration profiles were assumed to be the pseudo-steadystate profiles which are known in closed form. The chemical potentials of the various species were determined from these profiles and were substituted into the Levin model to determine cell volume, degree of supercooling, and solute distribution as a function of time. By application of this model to an actual biological tissue, the destructive phenomena may be characterized and less injurious protocols found. Supported in part by the National Heart and Lung Institute, Grant No. HL-14322. 60. Equilibrium and terminations Sucrose-Sodium GAYLE, F. H. (Department and Materials Durham, North
Nonequilibrium Phase Dein the Ternary System Chloride-Water. F. W. COCKS, AND M. L. SHEPARD. of Mechanical Engineering Science, Duke University, Carolina 27706).
Sucrose has been suggested as a cryoprotective agent for human red blood cells. As part of a study of the relationship between protective action and low temperature phase relationships, the sucrosesodium chloride-water ternary system has been investigated to determine equilibrium and meta-
ABSTRACTS,
13TH
stable phase transformations in the primary ice phase field. Metastable glass formation is essential in preventing ice crystallization, while both stable and metastable phase transformations determine the extent to which solutes concentrate on cooling. The liquidus surface has been determined by standard differential thermal analysis techniques. It was found that sucrose is not as effective as is dimethyl sulfoxide or glycerol in depressing the freezing point and suppressing salt concentrations in water-based ternary solutions. The line of twofold saturation from the H,O-NaCI binary system which determines the limit of the primary ice phase field has been determined, as well as the approximate location (30 w/o HZO, 10 w/o NaCI, 60 w/o sucrose) and temperature (-35*C) of a ternary eutectic point. The sucrose-sodium chloride-water system tends to form stable glasses near the ternary eutectic point. At higher or lower salt concentrations an unstable glass is formed on cooling, and over the range of heating rates investigated, 5 to 2O”C/min, this glass devitrifies. The support of this work by the American Cancer Society under Grant No. PDT-42A is gratefully acknowledged. 61. Equillibrium and Nonequilibrium Therm& Behavior of Aqueous Ternary Solutions Based on Complex Physiologic Support Media Containing NaCl and Dimethyl Sulfoxide or Glycerol. R. I. POZNER, M. L. SHEPARD, of MechanAND F. H. COCKS. (Department ical Engineering and Materials Science, Duke University, Durham, North Carolina 27706). Precipitation of ice with concomitant solute enrichment of the residential liquid and the formation and stability of glassy phase developed during freezing have profound effects on postfreeze cell viability. The present studies have utilized standard differential thermal analysis (DTA) techniques to establish the equilibrium liquidus surfaces of the primary ice phase fields of ternary systems containing NaCl, a cryoprotective agent (either glycerol or dimethyl sulfoxide (DMSO)), and an aqueous physiologic support medium. The media employed in these studies were Eagle’s minimum essential medium (MEM ) and MEM supplemented with 20 ~01% fetal calf serum ( FCS). Additionally, qualitative evaluations of the glassforming tendencies in the systems MEM-NaClglycerol, MEM-NaCI-DMSO, and (MEM + FCS )-NaCI-DMSO were established. Both equilibrium and nonequilibrium thermal behavior, in the three systems investigated, exhibited only minor variations relative to analogous Ha-based systems previously investigated. Results obtained in these studies may provide insight into the
ANNUAL
MEETING
665
physicochemical events which occur when cells suspended in such solutions are exposed to cryogenic temperatures. The support of this work by the American Cancer Society under Grant No. PDT-42A is gratefully acknowledged. 62. Preliminady tration JOHN G. partment Houston,
Studies on the Differential Peneof Cryoprotectants through Ice. BAUST AND MARK DONVITO.’ (Deof Biology, University of Houston, Texas 77004 ) .
Polyhydric alcohols and disaccharides demonstrate differential rates of penetration through type I ice. Depth of penetration is a function of time at a given temperature, ambient pressure, and molecular diameter. Ice samples prepared from degassed, distilled (3x) water were frozen in 6-mm-diameter cylinders for 1.5 hr at -35°C followed by topical application of 0°C cryoprotectant-saturated filter paper discs. Temperature was maintained at -35°C for l-96 hr while ambient pressure was varied up to 100 atm. Absolute rates of penetration for glycerol exceeded 3600 rmol/hr (1 ATA) and 8200 /Imol/hr (100 ATA) while sorbitol rates exceeded 1500 pmoF/hr (1 ATA) and 3450 pmol/hr (100 ATA). Depth of penetration of disaccharides was comparatively reduced. A procedure is described for cryoprotectant supplementation in or “removal” (dilution) from prefrozen tissue samples. 63. A Simple One-Step Procedure Tissue for Light Microscopy. (Department of Anatomy, Medical College and Hospital, Pennsylvania 19102).
for Freezing L. TERRACIO. Hahnemam Philadelphia,
Many histochemical and autoradiographic techniques require the use of frozen-dried tissue. Commonly used methods for freezing tissue such as a supercoobd metal surface 1 or quenching fluid have the drawback of adding preparatory steps to an already lengthy procedure. These additional steps can be eliminated for light microscopic procedures by using the following method. Slices of fresh tissue were placed on an aluminum foil-covered spatula and the tissue was frozen by introducing it into a rapidly running stream of liquid nitrogen. This is easily obtained from a standard storage tank under maximum flow. The frozen tissue was dissected into small pieces and placed in a cooled copper specimen block equipped with a thermometer and heating coil. The specimen block was then transferred to a glass freeze-drier and the tissue was dried by increasing the temperature of the block lO”C/hr until it reached room temperature.’ The dry tissue