Mechanism of hemolysis of erythrocytes by freezing at near-zero temperatures

Nei, Tokio

doi:10.1016/s0011-2240(68)80128-2

Cited by 28 publications

(6 citation statements)

References 3 publications

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“…The decrease in the volume of unfrozen channels during freezing not only increases the chance of interactions between cells and the ice crystals that border the channels, it also increases the chances of cell-to-cell interactions. Indeed, Pegg (1981) has recently reported that the hemolysis of human erythrocytes that accompanies a given slow freezing treatment is increased greatly when the erythrocytes are frozen at a hematocrit above 60%, and Nei (1968) has found that it is decreased substantially when cells are frozen at a hematocrit of only 4%. Although this cell-crowding effect could well have a similar genesis to the effects reported here, cell-cell interactions are unlikely to explain our results, since the hematocrit of our cell suspensions was only 2%.…”

Section: Mazur Et Al Survival Ofslowly Frozen Human Erythrocytesmentioning

confidence: 99%

Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes

Mazur¹,

Rall²,

Rigopoulos³

1981

Biophysical Journal

144

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As suspensions of cells freeze, the electrolytes and other solutes in the external solution concentrate progressively, and the cells undergo osmotic dehydration if cooling is slow. The progressive concentration of solute comes about as increasing amounts of pure ice precipitate out of solution and cause the liquid-filled channels in which the cells are sequestered to dwindle in size. The consensus has been that slow freezing injury is related to the composition of the solution in these channels and not to the amount of residual liquid. The purpose of the research reported here was to test this assumption on human erythrocytes. Ordinarily, solute concentration and the amount of liquid in the unfrozen channels are inversely coupled. To vary them independently, one must vary the initial solute concentration. Two solutes were used here: NaCl and the permeating protective additive glycerol. To vary the total initial solute concentration while holding the mass ratio of glycerol to NaCl constant, we had to allow the NaCl tonicity to depart from isotonic. Specifically, human red cells were suspended in solutions with weight ratios of glycerol to NaCl of either 5.42 or 11.26, where the concentrations of NaCl were 0.6, 0.75, 1.0, 2.0, 3.0, or 4.0 times isotonic. Samples were then frozen to various subzero temperatures, which were chosen to produce various molalities of NaCl (0.24-3.30) while holding the fraction of unfrozen water constant, or conversely to produce various unfrozen fractions (0.03-0.5) while holding the molality of salt constant. (Not all combinations of these values were possible). The following general findings emerged: (a) few cells survived the freezing of greater than 90% of the extracellular water regardless of the salt concentration in the residual unfrozen portion. (b) When the fraction of frozen water was less than 75% the majority of the cells survived even when the salt concentration in the unfrozen portion exceeded 2 molal. (c) Salt concentration affected survival significantly only when the frozen fraction lay between 75 and 90%. To find a major effect on survival of the fraction of water that remains unfrozen was unexpected. It may require major modifications in how cryobiologists view solution-effect injury and its prevention.

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Section: Mazur Et Al Survival Ofslowly Frozen Human Erythrocytesmentioning

confidence: 99%

Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes

Mazur¹,

Rall²,

Rigopoulos³

1981

Biophysical Journal

144

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“…For sufficiently low cooling rates, thermodynamic equilibrium with the extracellular solution is achieved by dehydration. Damage at low cooling rates may be associated with the excessive dehydration (1 9,27), the attainment of high intracellular concentration (16,23), or mechanical effects (22,18). It is clear that in order to avoid or at least minimize the freezing injury, the cooling rate should be high enough to minimize exposure of the cells to high concentrations but low enough to avoid IIF.…”

Section: Introductionmentioning

confidence: 99%

Intracellular Ice Formation during the Freezing of Hepatocytes Cultured in a Double Collagen Gel

Hubel

Toner

Cravalho

et al. 1991

Biotechnology Progress

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During freezing, intracellular ice formation (IIF) has been correlated with loss in viability for a wide variety of biological systems. Hence, determination of IIF characteristics is essential in the development of an efficient methodology for cryopreservation. In this study, IIF characteristics of hepatocytes cultured in a collagen matrix were determined using cryomicroscopy. Four factors influenced the IIF behavior of the hepatocytes in the matrix: cooling rate, final cooling temperature, concentration of Me2SO, and time in culture prior to freezing. The maximum cumulative fraction of cells with IIF increased with increasing cooling rate. For cultured cells frozen in Dulbecco's modified Eagle's medium (DMEM), the cooling rate for which 50% of the cells formed ice (B50) was 70 degrees C/min for cells frozen after 1 day in culture and decreased to 15 degrees C/min for cells frozen after 7 days in culture. When cells were frozen in a 0.5 M Me2SO + DMEM solution, the value of B50 decreased from 70 to 50 degrees C/min for cells in culture for 1 day and from 15 to 10 degrees C/min for cells in culture for 7 days. The value of the average temperature for IIF (TIIF) for cultured cells was only slightly depressed by the addition of Me2SO when compared to the IIF behavior of other cell types. The results of this study indicate that the presence of the collagen matrix alters significantly the IIF characteristics of hepatocytes. Thus freezing studies using hepatocytes in suspension are not useful in predicting the freezing behavior of hepatocytes cultured in a collagen matrix. Furthermore, the weak effect of Me2SO on IIF characteristics implies that lower concentrations of Me2SO (0.5 M) may be just as effective in preserving viability. Finally, the value of B50 measured in this study indicates that cooling rates nearly an order of magnitude faster than those previously investigated could be used for cryopreservation of the hepatocytes in a collagen gel.

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“…Despite several reports suggesting that mechanical and physical factors play an important role in cryodamage (Meryman, 1956;Nei, 1967Nei, , 1968Takamatsu and Rubinsky, 1999), this phenomenon has been largely ignored. If we consider each of the components of the two-factor theory separately, can we be convinced that these two factors comprise the bulk of the story?…”

Section: Discussionmentioning

confidence: 99%

“…Despite the opposite objectives of these applications, both require an understand of cell-injury mechanisms during freezing to achieve their desired outcomes-maximum post-thaw survival and preservation of biological functions for cryopreservation, or complete cell/tissue destruction for cryosurgery (Han and Bischof, 2004). During the process of cryopreservation, cellular membranes must withstand a variety of stresses, including: (1) volumetric changes and associated membrane shrinkage and stretching in response to hyperosmotic cryoprotectant solutions (Mazur and Cole, 1989), (2) cryoprotectant toxicity effects (Fiser and Fairfull, 1986), (3) chilling from body temperature and consequent chilling injury (Saragusty et al, 2005), (4) thermotropic phase transition of membrane phospholipids in response to decreasing temperature and changes in membrane lipid composition (Darin-Bennett et al, 1973;Drobnis et al, 1993;Quinn, 1985), (5) freezeinduced dehydration (Devireddy et al, 2000), (6) mechanical stresses induced by extracellular ice formation as well as cell-cell and cell-container interactions (Hubel et al, 2007;Mazur and Cole, 1985;Nei, 1967Nei, , 1968Takamatsu and Rubinsky, 1999), (7) ionic and electrical effects resulting from preferential incorporation of some ionic species into the ice (Zimmermann, 1982), (8) the effects of elevated solute concentration and intracellular ice formation (IIF), which are cooling-rate dependent (Muldrew and McGann, 1988), (9) cryopreservation-induced lipid peroxidation and loss of superoxide dismutase activity (Alvarez and Storey, 1992), and (10) recrystallization, which is warming-rate dependent (Arav et al, 1994;Mazur, 1977). To be fully functional, spermatozoa must be able to capacitate, reach the oocyte, undergo the acrosome reaction, penetrate the zona pellucida, fuse with the oocyte's plasma membrane, and deliver intact genetic material to form a viable zygote (Willoughby et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

“…A third important mechanism, the mechanical effect, has traditionally been largely ignored since the mid 1950s, when two articles claimed it to be a redundant factor (Harrison and Cerroni, 1956;Sherman, 1957). Ten years later, Nei (1967Nei ( , 1968 showed that ice crystals do damage cells via mechanical interaction between the ice crystals and the cells trapped between them. Mazur et al (1981) and Mazur and Rigopoulos (1983) referred to this as the ''unfrozen fraction hypothesis.''…”

Section: Introductionmentioning

confidence: 99%

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Do physical forces contribute to cryodamage?

Saragusty

Gacitua

Rozenboim

et al. 2009

Biotech & Bioengineering

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To achieve the ultimate goal of both cryosurgery and cryopreservation, a thorough understanding of the processes responsible for cell and tissue damage is desired. The general belief is that cells are damaged primarily due to osmotic effects at slow cooling rates and intracellular ice formation at high cooling rates, together termed the "two factor theory." The present study deals with a third, largely ignored component--mechanical damage. Using pooled bull sperm cells as a model and directional freezing in large volumes, samples were frozen in the presence or absence of glass balls of three different diameters: 70-110, 250-500, and 1,000-1,250 microm, as a means of altering the surface area with which the cells come in contact. Post-thaw evaluation included motility at 0 h and after 3 h at 37 degrees C, viability, acrosome integrity, and hypoosmotic swelling test. Interactions among glass balls, sperm cells, and ice crystals were observed by directional freezing cryomicroscopy. Intra-container pressure in relation to volume was also evaluated. The series of studies presented here indicate that the higher the surface area with which the cells come in contact, the greater the damage, possibly because the cells are squeezed between the ice crystals and the surface. We further demonstrate that with a decrease in volume, and thus increase in surface area-to-volume ratio, the intra-container pressure during freezing increases. It is suggested that large volume freezing, given that heat dissipation is solved, will inflict less cryodamage to the cells than the current practice of small volume freezing.

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Mechanism of hemolysis of erythrocytes by freezing at near-zero temperatures

Cited by 28 publications

References 3 publications

Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes

Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes

Intracellular Ice Formation during the Freezing of Hepatocytes Cultured in a Double Collagen Gel

Do physical forces contribute to cryodamage?

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