Effect of Cell-to-Surface Interaction on Freeze Tolerance and Osmotic Response of Cells

2008 ◽  
Author(s):  
Takashi Yoshimori ◽  
Masaki Fukagawa ◽  
Hiroshi Takamatsu
Keyword(s):  
Cell Suspension ◽  
Cell Injury ◽  
Freeze Tolerance ◽  
Surface Interaction ◽  
Artificial Organs ◽  
Freezing Injury ◽  
Elevated Concentration ◽  
Freeze Thaw ◽  
Osmotic Response ◽  
The Difference

Cryopreservation of tissues and organs, including artificial organs, could be one of the important steps in the medical service that brings the progress in the tissue engineering to realization. In this case, high viability of cryopreserved cells is critical to recovery after transplantation. In contrast, in the cryosurgery, which is expected to expand its application as a minimally invasive treatment of cancer, malignant cells should be destructed completely to prevent from recurrence. The appropriate freeze-thaw protocol is therefore needed to be established for cryopreservation or cryosurgery depending on specific type of tissues and organs. Although it is determined empirically, the underlying mechanism of cell injury by freezing has been explored for a long time to give a scientific basis of the process. The experiments with a cell suspension showed that the cell injury during slow freezing to a relatively higher sub-zero temperature was attributed to the mechanical stress from the extracellular ice, while the effect of elevated concentration of solutes became more crucial to cell damage at lower temperatures [1]. However, there are some studies that indicates the difference in the freeze tolerance between cell suspensions and attached monolayers, some of which indicated higher susceptibility of monolayers to freezing than cell suspension [2] and the other suggested reverse [3,4]. The goal of our study is thus to validate the difference in freezing injury between isolated cells and tissues that are more important in aforementioned applications and clarify the mechanism. We used cells adhered to a surface as a first simple model of cells in tissues. The cells adhered on a surface at low number density were used to highlight the effect of cell-to-surface interaction without cell-to-cell interactions. In the present study we first demonstrate that the survival of cells adhered on a surface is lower than those in the suspension after a freeze-thaw manipulation. Then the osmotic response to concentration increase was examined to clarify if the extent of dehydration is different between these two types of cells. The cells were observed by a laser confocal scanning microscope that allows real-time 3-D observation.

scholarly journals Aquaporin-Mediated Improvement of Freeze Tolerance of Saccharomyces cerevisiae Is Restricted to Rapid Freezing Conditions

2004 ◽  
Vol 70 (6) ◽  
pp. 3377-3382 ◽  
Author(s):  
An Tanghe ◽  
Patrick Van Dijck ◽  
Didier Colavizza ◽  
Johan M. Thevelein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Freeze Tolerance ◽  
Baker’S Yeast ◽  
Yeast Cells ◽  
Freezing Injury ◽  
Crystal Formation ◽  
Rapid Freezing ◽  
Baker's Yeast ◽  
Cooling Rates ◽  
The Difference

ABSTRACT Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.

Osmotic tolerance of human granulocytes

1984 ◽  
Vol 247 (5) ◽  
pp. C373-C381 ◽  
Author(s):  
W. J. Armitage ◽  
P. Mazur
Keyword(s):  
Cell Injury ◽  
Membrane Integrity ◽  
Solute Concentration ◽  
Freezing Injury ◽  
Computer Assisted ◽  
Critical Surface ◽  
Freezing And Thawing ◽  
Osmotic Tolerance ◽  
Sucrose Solutions ◽  
Human Granulocytes

Human granulocytes are injured when returned to isotonic conditions after exposure at 0 degree C to hyperosmotic solutions of NaCl or sucrose with osmolalities above 0.6 osmolal. The damage was expressed as a loss of membrane integrity [fluorescein diacetate (FDA) assay] only after 60-90 min incubation at 37 degrees C. Survival after exposure to a 1.4-osmolal solution at 0 degree C was dependent on the extent of subsequent dilution. Dilution to below 0.6 osmolal was damaging, but cells could be returned to near-osmotic conditions provided that the solute concentration was increased again to 0.64 osmolal before the cells were incubated at 37 degrees C. Granulocyte cell volumes were measured under various osmotic conditions by computer-assisted micrometry. The cells did not display a minimum volume but behaved as osmometers over the observed range of 0.2-1.4 osmolal. Granulocyte volume at a given osmolality was independent of whether the cells had first been exposed to a strongly hyperosmotic medium, indicating that no solute loading occurred in hyperosmotic sucrose solutions. Even though the cells did not survive sequential exposure to greater than 0.6 osmolal solutions, subsequent return to isotonicity, and incubation at 37 degrees C, neither cell lysis nor loss in FDA-positive cells occurred after the first two steps. This finding is not consistent with the critical-surface area-increment theory of freezing injury. The mechanism of cell injury in hyperosmotic solutions is thus not known. However, the results show that osmotic stress is potentially a major damaging factor both in the equilibration of cells with protective additives and during freezing and thawing.

Effect of Saturation Degree on Concrete Deterioration due to Freeze-Thaw Action

2011 ◽  
Vol 477 ◽  
pp. 404-408 ◽  
Author(s):  
Wen Cui Yang ◽  
Yong Ge ◽  
Bao Sheng Zhang ◽  
Jie Yuan
Keyword(s):  
Frost Resistance ◽  
Water Pressure ◽  
High Water ◽  
Frost Damage ◽  
Critical Value ◽  
Cold Weather ◽  
Saturation Degree ◽  
Freeze Thaw ◽  
Air Content ◽  
The Difference

Freezing-thawing durability of cement concrete is extremely important in cold weather, to better understand mechanism of frost damage and air-entraining，saturation degree of pores in concrete and its relation with frost resistance were studied in this paper. Concrete specimens with different saturation degree from 0 to 100% were prepared used a sealed tin with a high water pressure pump. Then these specimens were subjected to six freezing-thawing cycles and the relative dynamic modulus of elasticity was examined. The results showed that critical saturation degree of concrete with water- binder ratio of 0.30 and 0.47, air content of 1%, 4% and 6% were from 0.60 to 0.80. When its saturation degree exceeded the critical value, concrete was deteriorated significantly after only six freeze-thaw cycles. The critical saturation degree was mainly related to the air content of concrete mixture, and it decreased with the increasing of air content. The difference between the saturation degree and the critical value can be used to evaluate potential frost resistance of concrete, and its result was consistent with the result of frost tests very well.

Freeze-thaw injury in erythrocytes of the freeze-tolerant wood frog, Rana sylvatica

1991 ◽  
Vol 261 (6) ◽  
pp. R1346-R1350 ◽  
Author(s):  
J. P. Costanzo ◽  
R. E. Lee
Keyword(s):  
Cell Injury ◽  
Osmotic Fragility ◽  
Rana Sylvatica ◽  
Freeze Tolerance ◽  
Wood Frog ◽  
In Vitro Tests ◽  
Freezing And Thawing ◽  
Freeze Tolerant ◽  
High Concentrations

Erythrocytes from the freeze-tolerant wood frog (Rana sylvatica) were subjected to in vitro tests of freeze tolerance, cryoprotection, and osmotic fragility. The responses of cells from frogs acclimated to 4 or 15 degrees C were similar. Erythrocytes that were frozen in saline hemolyzed at -4 degrees C or lower. The addition of high concentrations (150 and 1,500 mM) of glucose or glycerol, cryoprotectants produced naturally by freeze-tolerant frogs, significantly reduced cell injury at -8 degrees C, but concentrations of 1.5 or 15 mM were ineffective. Hemolysis was reduced by 94% with 1,500 mM glycerol and by 84% with 1,500 mM glucose; thus glycerol was the more effective cryoprotectant. Mean fragility values for frog erythrocytes incubated in hypertonic and hypotonic saline were 1,938 and 49 mosM, respectively. Survival in freeze tolerance and cryoprotection experiments was comparable for erythrocytes from frogs and humans, suggesting that these cells may respond similarly to freezing-related stresses. However, the breadth of osmotic tolerance, standardized for differences in isotonicity, was greater for frog erythrocytes than for human erythrocytes. Our data suggest that erythrocytes from R. sylvatica are adequately protected by glucose under natural conditions of freezing and thawing.

Low Frequency of Adenylate Kinase Release into Cerebrospinal Fluid during Balanced, Normotensive Anaesthesia and a Non-Orthognathic Surgical Procedure

1997 ◽  
Vol 25 (2) ◽  
pp. 92-97 ◽  
Author(s):  
M Enlund ◽  
O Mentell ◽  
L Edmark ◽  
G Ronquist
Keyword(s):  
Cerebrospinal Fluid ◽  
Adenylate Kinase ◽  
Orthognathic Surgery ◽  
Cell Injury ◽  
Low Frequency ◽  
Brain Cell ◽  
Contributing Factors ◽  
Oscillating Saw ◽  
The Difference ◽  
Increased Activity

Activity of strictly intracellular enzymes in the cerebrospinal fluid (CSF) may indicate leakage from dysfunctional brain cells. Increased activity of adenylate kinase (AK) in the CSF is indicative of brain cell injury arising from several sources, among them orthognathic surgery. The mechanism in the latter case is obscure, but the use of an oscillating saw which generates vibrations, and the site of surgery close to the brain may be contributing factors. Anaesthesia may also play a role. In the present study, CSF-AK activity was measured after hysterectomy and was compared with activity after orthognathic surgery in two other studies. Four of 19 patients (21%) in the present study expressed pathological activity, compared with 34 of 47 (72%) orthognathic patients in the two other studies. No firm conclusion may be drawn from historical comparisons, and the difference in activity seen between the two types of surgery might not necessarily be the result of surgical factors. Until this is investigated further, however, we conclude that there may be a difference in postoperative CSF-AK activity between orthognathic and lower abdominal surgery.

Freezing of Cells: Role of Ice and Solutes in Cell Damage

2007 ◽  
Author(s):  
Hiroshi Takamatsu
Keyword(s):  
Mechanical Stress ◽  
Cell Damage ◽  
Cellular Damage ◽  
Elevated Concentration ◽  
Cellular Injury ◽  
Extracellular Solution ◽  
Different Types ◽  
A Cell ◽  
The Difference

The mechanism of cellular damage associated with freezing of biological cells is discussed by summarizing the author’s recent studies that consists of four different types of experiments. The “solution effects” that designate the influence of elevated concentration of electrolytes during freezing is examined first by a nonfreezing experiment that exposes cells to hypertonic solutions using a perfusion microscope. The cell damage due to the solution effect is evaluated directly from a pseudo-freezing experiment, where cells were subjected to the milieu that simulated a freeze-thaw process in the absence of ice. Contribution of ice formed in the extracellular solution is then estimated from the difference in cell survival between the pseudo-freezing experiment and a corresponding freezing experiment. The cellular injury by the mechanical stress is also examined independently by a cell deformation experiment, which mimicked the situation that cells are compressed and deformed between ice crystals. This experiment was designed to examine a complex effect of mechanical stress from ice and elevated concentration of electrolytes. Based on all these experiments, the role of concentrated solutes and ice is revealed as a function of freezing conditions.

scholarly journals Freezing of living cells: mechanisms and implications

1984 ◽  
Vol 247 (3) ◽  
pp. C125-C142 ◽  
Author(s):  
P. Mazur
Keyword(s):  
Solute Concentration ◽  
Living Cells ◽  
Slow Freezing ◽  
Freezing Injury ◽  
Cell Water ◽  
Cell Shrinkage ◽  
Physiological Conditions ◽  
Osmotic Response ◽  
Kinetics Of ◽  
Do So

Cells can endure storage at low temperatures such as--196 degrees C for centuries. The challenge is to determine how they can survive both the cooling to such temperatures and the subsequent return to physiological conditions. A major factor is whether they freeze intracellularly. They do so if cooling is too rapid, because with rapid cooling insufficient cell water is removed osmotically to eliminate supercooling. Equations have been developed that describe the kinetics of this water loss and permit one to predict the likelihood of intracellular freezing as a function of cooling rate. Such predictions agree well with observations. Although the avoidance of intracellular freezing is usually necessary for survival, it is not sufficient. Slow freezing itself can be injurious. As ice forms outside the cell, the residual unfrozen medium forms channels of decreasing size and increasing solute concentration. The cells lie in the channels and shrink in osmotic response to the rising solute concentration. Prior theories have ascribed slow freezing injury to the concentration of solutes or the cell shrinkage. Recent experiments, however, indicate that the damage is due more to the decrease in the size of the unfrozen channels. This new view of the mechanism of slow freezing injury ought to facilitate the development of procedures for the preservation of complex assemblages of cells of biological, medical, and agricultural significance.

scholarly journals Supercooling in Dormant Flower Buds of Forsythia, and the Correlation between Pistil Size and Bud Hardiness

1999 ◽  
Vol 17 (2) ◽  
pp. 57-62 ◽  
Author(s):  
Cindy L. Flinn ◽  
Edward N. Ashworth
Keyword(s):  
Low Temperature ◽  
Supercooled Water ◽  
Freeze Tolerance ◽  
Freezing Injury ◽  
Laboratory Studies ◽  
Flower Buds ◽  
Deep Supercooling ◽  
Freeze Stress ◽  
Stem Segments ◽  
Accurate Indicator

Abstract Experiments were conducted to determine if dormant buds of Forsythia taxa exhibit the deep supercooling characteristic. Specimens were collected from thirteen Forsythia taxa including: F. suspensa (Thunb.) Vahl, F. x intermedia cv. Spectabilis (Koehne), F. x intermedia cv. Lynwood (G.E. Peterson), F. europaea (Degen and Baldacci), F. giraldiana (Lingelsh), F. japonica (Makino) var. saxatilis (Nakai), F. mandshurica (Uyeki), F. ovata (Nakai), F. suspensa var. fortunei (Lindl.), F. viridissima (Lindl.), F. x intermedia cv. Arnold Giant (Sax), F. cv. Arnold's Dwarf, and F. cv. Meadowlark (Flint). Buds and attached stem segments, were cooled at 2C (3.6F) per hour, and the temperature at which freezing occurred was determined by thermal analysis. Typically, two distinct freezing events were detected within Forsythia buds. The first freezing event, or high temperature exotherm, occurred just below 0C (32F), while the second freezing event, or low temperature exotherm, occurred between −16C (3.2F) and −28C (−18.4F). The low temperature exotherm corresponded to the freezing of supercooled water within dormant buds, and the detection of low temperature exotherms in buds of all 13 Forsythia taxa indicated that deep supercooling is common among members of this genus. In nine of the 13 Forsythia taxa, the temperature of the low temperature exotherm was an accurate indicator of bud freeze-tolerance (LT50), as determined by a laboratory freeze-stress protocol. The discrepancies noted in the other four taxa were apparently due to the occurrence of field freezing injury prior to conducting these laboratory studies. Evidence indicated a relationship between the extent of supercooling and the size of the pistil in dormant Forsythia buds.

scholarly journals Increased plasma levels of interleukin-6 in sepsis [see comments]

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1704-1710 ◽  
Author(s):  
CE Hack ◽  
ER De Groot ◽  
RJ Felt-Bersma ◽  
JH Nuijens ◽  
RJ Strack Van Schijndel ◽  
...  
Keyword(s):  
Septic Shock ◽  
Interleukin 6 ◽  
Cell Injury ◽  
Prognostic Significance ◽  
Inverse Correlation ◽  
P Value ◽  
Classical Pathway ◽  
Significance Levels ◽  
Lethal Complications ◽  
The Difference

Interleukin-6 (IL-6) is likely to be an important mediator of the inflammatory response. We measured levels of this cytokine in plasma samples from 37 patients with sepsis or septic shock obtained at the time of admission to the intensive care unit and related these levels to hemodynamic and biochemical parameters as well as to clinical outcome. In 32 of the 37 patients, increased levels of IL-6 were found, occasionally up to 7,500 times the normal level. The highest IL-6 levels were encountered in patients who suffered from septic shock (P value of the difference between patients with and without shock less than .0001). In addition, IL-6 significantly correlated with plasma lactate (P less than .0001), heart rate (P = .05) and, inversely, with mean arterial pressure (P = .01) and platelet counts (P = .0002). Significant correlations of IL-6 with the anaphylatoxins C3a (P = .0001) and C4a (P = .0002) and with the main inhibitor of the classical pathway of complement, C1-inhibitor (inverse correlation, P = .05), were also observed. IL-6 on admission appeared to be of prognostic significance: levels were higher in septic patients who subsequently died than in those who survived (P = .0003), in particular when only patients with septic shock were considered (P less than .0001). All nine septic patients with levels of less than 40 U/mL on admission survived, whereas 89% of the nine patients with levels exceeding 7,500 U/mL died. These data provide evidence for a role of IL-6 in the pathophysiology of septic shock. Further studies are needed to reveal whether IL-6 in sepsis is directly involved in mediating lethal complications or whether it is to be considered as an “alarm hormone” that reflects endothelial cell injury probably mediated by the anaphylatoxines.

Long-Term Functional Recovery of Hepatocytes after Cryopreservation in a Three-Dimensional Culture Configuration

1992 ◽  
Vol 1 (4) ◽  
pp. 281-292 ◽  
Author(s):  
Inne H.M. Borel Rinkes ◽  
Mehmet Toner ◽  
Sean J. Sheehan ◽  
Ronald G. Tompkins ◽  
Martin L. Yarmush
Keyword(s):  
Protein Secretion ◽  
Freezing Temperature ◽  
Freezing Injury ◽  
Albumin Secretion ◽  
Liver Support ◽  
Freeze Thaw ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle ◽  
Optimized Conditions

Hepatocyte cryopreservation is essential to ensure a ready supply of cells for use in transplantation or as part of an extracorporeal liver assist device to provide on-demand liver support. To date, most of the work on hepatocyte cryopreservation has been performed on isolated hepatocytes, and has generally yielded cells which display low viability and greatly reduced short-term function. This report presents the development of a freezing procedure for hepatocytes cultured in a sandwich configuration. A specially designed freezing unit was used to provide controlled temperatures throughout the freeze-thaw cycle. Cooling rate, warming rate, and final freezing temperature were evaluated as to their effect on hepatocyte function as judged by albumin secretion. Under optimized conditions (cooling at 5°C/min and warming at ≥400°C/min), freezing to −40°C resulted in full recovery of albumin secretion within 2-3 days post-freezing, whereafter albumin secretion levels remained normal for the duration of the experiments (2 wks). Freezing to −80°C lead to an approximate 70% recovery of long-term protein secretion when compared to control cultures. In addition, the overall hepatocyte morphology as judged by light microscopy, closely followed the functional results. The sandwich culture configuration, thus, enables hepatocytes to maintain a satisfactory level of long-term protein secretion after a freeze-thaw cycle under optimized conditions, and offers an attractive tool for further studies into the mechanisms of freezing injury and subsequent hepatocellular recovery. These results are a promising step in the development of satisfactory storage procedures for hepatocytes.

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