Biomedical subjects

L E McGann

Publications and source records attributed to L E McGann.

At least 19 recordsLinked to original sources

PubMed2006

Storage of porcine articular cartilage at high subzero temperatures.

OBJECTIVE: Transplantation of osteochondral allograft tissue can treat large joint defects but is limited by tissue availability, surgical timing, and infectious disease transmission. Fresh allografts perform the best but requirements for infectious disease testing delay the procedure with subsequent decrease in cell viability and function. Hypothermic storage at lower temperatures can extend tissue banking time without loss of cell viability and, therefore, increase the supply of allograft tissue. This study investigated the effects of different cryoprotectant solutions on intact AC at various subzero temperatures. DESIGN: 10 mm porcine osteochondral dowels were immersed for 30 minutes in various combinations of solutions [(XVIVO, propylene glycol (51% w/w), sucrose (46% w/w)] cooled to various subzero temperatures (-10, -15, and -20 degrees C), and held for 30 min. After warming, 70 mum slices were stained with membrane integrity dyes, viewed under fluorescence microscopy and cell recovery calculated relative to fresh controls. RESULTS: Results demonstrated excellent cell recovery (>75%) at -10 degrees C provided ice did not form. Excellent cell recovery (>70%) occurred at -15 degrees C in solutions containing 51% propylene glycol but formation of extra-matrix ice in other solutions resulted in significant cell loss. All groups had <6% cell recovery at -20 degrees C and propylene glycol did not provide a protective effect even though extra-matrix ice did not form CONCLUSIONS: These results suggest that extra-matrix ice plays an important role in cell damage during cryopreservation. Excellent cell recovery can be obtained after storage at subzero temperatures if ice does not form. Hypothermic preservation at high subzero temperatures may extend AC storage time in tissue banks compared to current techniques.

Animals↗

PubMed2005

Cryoprotectant equilibration in tissues.

The first step in the cryopreservation of cells or tissues is often the movement of a permeating cryoprotectant into the cells or tissues from the solution into which they have been placed. The cryoprotectant enters the cells or tissues by thermodynamic equilibration with the surroundings. In the reverse case, thermodynamic equilibration also drives the removal of permeating cryoprotectants by a dilution solution at the end of the preservation process when the cells or tissues are being readied for use. There have been reports of tissues having equilibrium cryoprotectant concentrations lower than that of the surrounding carrier solution. For various tissues, the equilibrium concentration of cryoprotectant inside the tissue is either equal to, or lower than the cryoprotectant concentration of the surrounding solution. A simple thermodynamic treatment of the solution-tissue equilibrium shows that an equilibrium concentration difference can exist between a tissue and the surrounding solution if a pressure difference can be maintained.

Animals↗

PubMed2005

Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood.

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.

Antigens, CD34↗

PubMed2005

Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment.

In all, 78 peripheral hematopoietic progenitor cell collections from 52 patients were evaluated using our previously published validated post-thaw assays at the time of collection and following transplantation by assessment of viable CD34(+) cells, and granulocyte-macrophage colony-forming units (CFU-GM) cryopreserved in quality control vials. The median (range) post-thaw recovery of viable CD34(+) cells and CFU-GM was 66.4% (36.1-93.6%) and 63.0% (28.6-85.7%), respectively, which did not show significant correlation with the engraftment of either neutrophils (P=0.136 and 0.417, respectively) or platelets (P=0.88 and 0.126, respectively). However, the reinfused viable CD34(+) cells/kg of patient weight pre- or post-cryopreservation showed significant correlation to engraftment of neutrophils (P=0.0001 and 0.001, respectively) and platelets (P=0.023 and 0.010, respectively), whereas CFU-GM pre- or post-cryopreservation was significantly correlated to neutrophils (P=0.011 and 0.007, respectively) but not to platelets (P=0.112 and 0.100, respectively). The results show that post-cryopreservation assessment of viable CD34(+) cells or CFU-GM is as reliable a predictor of rapid engraftment as that of pre-cryopreservation measures. Therefore, the post-cryopreservation number of viable CD34(+) cells or CFU-GM should be used to eliminate the risks of unforeseen cell loss that could occur during cryopreservation or long-term storage.

Adult↗

PubMed2004

Cryoprotectant permeability parameters for cells used in a bioengineered human corneal equivalent and applications for cryopreservation.

A human corneal equivalent is being developed with applications in pharmaceutical testing and biomedical research, but the distribution of this engineered tissue, depends on successful cryopreservation. Cryopreservation of tissues depends on the presence of cryoprotectants, their addition and removal, and exposure to conditions during freezing and thawing, all of which depend on cellular membrane permeabilities to water and cryoprotectant. This study defines the permeability properties that define the rate of water and cryoprotectant movement across the plasma membrane of isolated human corneal endothelial, keratocyte, and epithelial cells. Cells were transferred from isotonic conditions (300 mosm/kg) to 0.5, 1, or 2 M dimethyl sulfoxide and propylene glycol solutions at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after cryoprotectant exposure allowed calculation of hydraulic conductivity (Lp), cryoprotectant permeability (Ps), and the reflection coefficient (sigma). Experimental values for Lp and Ps at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (Ea). Defining the permeability parameters and temperature dependencies allows simulation of responses of human corneal cells to addition and removal of cryoprotectants and to freezing conditions, allowing amount of supercooling, intracellular electrolyte concentration, and intracellular cryoprotectant concentration to be calculated. Simulations also show that the constituent cells in the bioengineered cornea respond differently to addition and removal of cryoprotectants and to freezing. This study has defined the requirements during cryopreservation for the corneal cells; future work will define the matrix requirements which will allow the development of a cryopreservation protocol.

Cell Membrane Permeability↗

PubMed2004

Intramatrix events during cryopreservation of porcine articular cartilage using rapid cooling.

Cryopreservation of articular cartilage may improve long-term transplantation results if cell and matrix integrity can be maintained. This study examined intramatrix events in intact porcine articular cartilage that occurred during a rapid-cooling technique with various concentrations of dimethyl sulfoxide (DMSO) (1, 3, 5, 6 and 7 M). Thermocouples were inserted into the solution and in the cartilage matrix to record the temperature during rapid cooling. In addition, scanning electron microscopy of freeze-substituted samples was performed and quantitatively evaluated for the areas representing ice in the matrix. The results of this study showed that low concentrations of DMSO resulted in the largest temperature gradient between the matrix and the surrounding solution, which occurred near the freezing point of the cryoprotectant solution. At higher concentrations of DMSO, the peak temperature gradient occurred near the glass transition temperature. The temperature measurements suggested that a significant amount of ice formed within the matrix at lower DMSO concentrations. At higher DMSO concentrations that resulted in vitrification of the external solution, there was evidence of some ice in the matrix. The scanning electron micrographs demonstrated significantly more matrix disruption (likely due to ice formation) (P<0.02) in the lower DMSO concentrations (1 and 5 M) while the 6 M DMSO concentration demonstrated minimal matrix disruption. Cryopreservation of articular cartilage with a rapid-cooling technique and high concentrations of DMSO resulted in partial vitrification of the matrix and significantly less matrix disruption. It appears that successful cryopreservation of viability and function in articular cartilage will require high concentrations of cryoprotectants and rapid cooling.

Animals↗

PubMed2003

Validation and reproducibility of computerised cell-viability analysis of tissue slices.

BACKGROUND: The identification of live cells using membrane integrity dyes has become a frequently used technique, especially with articular cartilage and chondrocytes in situ where tissue slices are used to assess cell recovery as a function of location. The development of a reproducible computerised method of cell evaluation would eliminate many variables associated with manual counting and significantly reduce the amount of time required to evaluate experimental results. METHODS: To validate a custom computerised counting program, intra-person and inter-person cell counts of nine human evaluators (three groups - unskilled, novice, and experienced) were compared with repeated pixel counts of the custom program on 15 digitised images (in triplicate) of chondrocytes in situ stained with fluorescent dyes. RESULTS: Results indicated increased reproducibility with increased experience within evaluators [Intraclass Correlation Coefficient (ICC) range = 0.67 (unskilled) to 0.99 (experienced)] and between evaluators [ICC = 0.47 (unskilled), 0.85 (novice), 0.93 (experienced)]. The computer program had perfect reproducibility (ICC = 1.0). There was a significant relationship between the average of the experienced evaluators results and the custom program results (ICC = 0.77). CONCLUSIONS: This study demonstrated that increased experience in cell counting resulted in increased reproducibility both within and between human evaluators but confirmed that the computer program was the most reproducible. There was a good correlation between the intact cell recovery determined by the computer program and the experienced human evaluators. The results of this study showed that the computer counting program was a reproducible tool to evaluate intact cell recovery after use of membrane integrity dyes on chondrocytes in situ. This and the significant decrease in the time used to count the cells by the computer program advocate its use in future studies because it has significant advantages.

Animals↗

PubMed2003

Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation.

Three widely used viability assessments were compared: (1) membrane integrity of nucleated cells using trypan blue (TB) exclusion and a fluorometric membrane integrity assay (SYTO 13 and propidium iodide), (2) enumeration of viable CD34+ cells, and (3) clonogenic assay (granulocyte-macrophage colony-forming units, CFU-GM). Post thaw peripheral hematopoietic progenitor cells (HPC) were incubated at 0, 22, and 37 degrees C for 20-min intervals before assessment. The recovery of viable nucleated cells assessed by TB and SYTO/PI decreased significantly with time at incubation temperatures of 22 and 37 degrees C (P<0.05), and correlated with the concentration of mononuclear cells (MNC) (r=0.936, P<0.05). The decrease in recovery of viable nucleated cells was slower when thawed cells were incubated at 0 degrees C compared with 22 degrees C or 37 degrees C. The recovery, measured by absolute viable CD34+ or CFU-GM, was not affected by 2 h post thaw incubation (P>0.05) at 0, 22, and 37 degrees C (P>0.05). There were no significant differences in the measured recovery of viable CD34+ cells and CFU-GM at all incubation times (P>0.05) and temperatures (P>0.05). Both CFU-GM and absolute CD34+ cells can be used as post thaw viability assays for HPC cryopreserved for transplantation.

Antigens, CD34↗

PubMed2002

The effect of temperature on membrane hydraulic conductivity.

The objective of this study was to use the temperature dependence of water permeability to suggest the physical mechanisms of water transport across membranes of osmotically slowly responding cells and to demonstrate that insight into water transport mechanisms in these cells may be gained from easily performed experiments using an electronic particle counter. Osmotic responses of V-79W Chinese hamster fibroblast cells were measured in hypertonic solutions at various temperatures and the membrane hydraulic conductivity was determined. The results were fit with the general Arrhenius equation with two free parameters, and also fit with two specific membrane models each having only one free parameter. Data from the literature including that for human bone marrow stem cells, hamster pancreatic islets, and bovine articular cartilage chondrocytes were also examined. The results indicated that the membrane models could be used in conjunction with measured permeability data at different temperatures to investigate the method of water movement across various cell membranes. This approach for slower responding cells challenges the current concept that the presence of aqueous pores is always accompanied by an osmotic water permeability value, P(f)>0.01 cm/s. The possibility of water transport through aqueous pores in lower-permeability cells is proposed.

Animals↗

PubMed2002

Osmotic parameters of cells from a bioengineered human corneal equivalent and consequences for cryopreservation.

A human corneal equivalent is under development with potential applications in pharmaceutical testing, biomedical research, and transplantation, but the ability to distribute this engineered tissue, depends on successful cryopreservation. Tissue recovery after exposure to conditions during cryopreservation depends on the response of its constituent cells to the changing environment as ice forms and solutes concentrate. This study defines the osmotic properties that define the rate of water movement across the plasma membrane of isolated human corneal endothelial, stroma, and epithelial cells. Cells were transferred from an isotonic (300 mosm/kg) to an anisotonic (150-1500 mosm/kg) solution at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after anisosmotic exposure allowed calculation of hydraulic conductivity (L(p)) and osmotically inactive volume fraction (V(b)). Experimental values for L(p) at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (E(a)). The L(p) for endothelial, stroma, and epithelial cells at 37 degrees C was 1.98+/-0.32,1.50+/-0.30, and 1.19+/-0.14 microm/min/atm, and the V(b) was 0.28, 0.27, and 0.41, respectively. The E(a) for endothelial, stroma, and epithelial cells was 14.8, 12.0, and 14.1 kcal/mol, respectively, suggesting the absence of aqueous pores. These osmotic parameters and temperature dependencies allow simulation of osmotic responses of human corneal cells to cryopreservation conditions, allowing amount of supercooling to be calculated to indicate the likelihood of intracellular freezing. Simulations show that differences in the osmotic parameters for the constituent cells in the bioengineered cornea result in significant implications for cryopreservation of the engineered corneal equivalent.

Biomedical Engineering↗

PubMed2002

Cryopreservation of intact human articular cartilage.

Damaged articular cartilage (AC) impairs joint function and many treatment techniques are being investigated to determine their long term results. Successful cryopreservation of AC can provide a reliable source of intact matrix with viable chondrocytes to maintain the cartilage over long periods of time. This study investigated the application of an established cryopreservation protocol to determine the recovery of intact chondrocytes from human AC. Ten millimeter diameter osteochondral dowels were harvested from two human donors. The cryopreservation protocol was performed and the samples were rapidly warmed from varying experimental holding temperatures (-10, -20, -30, -40 degrees C), with and without plunging into liquid nitrogen, using 1 M dimethyl sulfoxide as cryoprotectant. The cartilage was stained with membrane integrity dyes and viewed under fluorescence microscopy. The percent of intact chondrocytes was compared to fresh controls. Low recovery of intact chondrocytes was recorded from all temperature levels with and without cryoprotectant. The results of this experiment demonstrated that the cryopreservation procedure used to achieve moderate success with intact sheep AC was not successful with intact human AC and further investigation is required.

Adolescent↗

PubMed2001

Transplantation of articular cartilage following a step-cooling cryopreservation protocol.

Using a step-cooling cryopreservation protocol that held the tissue 60 min at -4 degrees C, 30 min at -8 degrees C, and 10 min at -40 degrees C before plunging into liquid nitrogen, we were able to get a substantial improvement in the magnitude and pattern of chondrocyte recovery following cryopreservation, achieving postthaw recoveries of 62 +/- 13%. These results are consistent with the hypothesis that ice growth within articular cartilage is planar, but they provide no direct support for that hypothesis. Transplanting (step-cooled) cryopreserved osteochondral allografts into adult Suffolk/Romanoff crossbred sheep for periods of 3 months and 1 year further tested the efficacy of the cryopreservation protocol. Unfortunately, the cryoinjury sustained by the chondrocytes during cryopreservation, although apparently nonlethal immediately after thawing in many cases, was not innocuous in the long term. The presence of large clusters of chondrocytes at 1 year after transplantation illustrates that cryoinjury not detectable with a membrane integrity assay can still have far-reaching effects on transplanted tissue.

Animals↗

PubMed2001

Intercellular ice propagation: experimental evidence for ice growth through membrane pores.

Propagation of intracellular ice between cells significantly increases the prevalence of intracellular ice in confluent monolayers and tissues. It has been proposed that gap junctions facilitate ice propagation between cells. This study develops an equation for capillary freezing-point depression to determine the effect of temperature on the equilibrium radius of an ice crystal sufficiently small to grow through gap junctions. Convection cryomicroscopy and video image analysis were used to examine the incidence and pattern of intracellular ice formation (IIF) in the confluent monolayers of cell lines that do (MDCK) and do not (V-79W) form gap junctions. The effect of gap junctions on intracellular ice propagation was strongly temperature-dependent. For cells with gap junctions, IIF occurred in a directed wave-like pattern in 100% of the cells below -3 degrees C. At temperatures above -3 degrees C, there was a marked drop in the incidence of IIF, with isolated individual cells initially freezing randomly throughout the sample. This random pattern of IIF was also observed in the V-79W monolayers and in MDCK monolayers treated to prevent gap junction formation. The significant change in the low temperature behavior of confluent MDCK monolayers at -3 degrees C is likely the result of the inhibition of gap junction-facilitated ice propagation, and supports the theory that gap junctions facilitate ice nucleation between cells.

Animals↗

PubMed2001

High-efficiency volume reduction of cord blood using pentastarch.

Human umbilical cord blood (UCB) has been used successfully to treat a variety of genetic, hematological, and oncologic disorders. However, the low number of hematopoietic progenitor cells available in donated cord blood samples limits transplantation of cord blood to children and small adults. Reduction of the volume of umbilical cord blood is widely used in cord blood banking to reduce the storage requirements in large-scale UCB banks. Unfortunately, during the volume reduction process, up to 40% or more of the progenitor cells are lost using current reduction methods. This study describes a highly reproducible, double collection technique using Pentaspan to reduce UCB volume by red cell depletion. This results in the preservation of critical hematopoietic progenitor cells. The final volume of the leukocyte concentrates (LC) was 19.8 +/- 0.4 ml with 95% red cell depletion. The recovery of nucleated cells (NC), mononuclear cells (MNC), CD34(+) cells and colony-forming units (CFU) following double collection was 89%, 94%, 96%, and 106%, respectively. This is significantly higher than the recovery from single collections, where recovery was 74%, 77%, 84%, and 91% for NC, MNC, CD34(+) and CFU, respectively. The double collection technique provides an efficient and highly reproducible method for the preparation of UCB for long-term storage and transplantation.

Blood Banks↗

PubMed2001

Evidence of chondrocyte repopulation in adult ovine articular cartilage following cryoinjury and long-term transplantation.

OBJECTIVE: To characterize the response of articular chondrocytes to a specific cryoinjury that leads to cluster formation following long-term transplantation. DESIGN: Osteochondral dowels from 20 adult sheep were cryopreserved to optimize the recovery of chondrocytes immediately after thawing. The dowels were transplanted as allografts and observed at 3 and 12 months. Chondrocyte distribution and viability was assessed using paravital dyes after transplantation. Chondrocyte phenotype was assessed by in situ hybridization and immunohistochemistry to detect type II collagen. An anticentrosome antibody was used to identify cells undergoing cell cycle progression towards mitosis. RESULTS: All cryopreserved grafts showed the presence of spheroidal clusters of chondrocytes 1 year after transplantation while the host cartilage adjacent to the graft appeared morphologically normal. The average size of the clusters increased from four cells at 3 months to 12 cells at 1 year. The chondrocytes in the clusters displayed newly formed type II collagen protein and mRNA. Some cells within clusters were observed with two centrosomes, indicative of cells progressing through the S phase of the cell cycle. CONCLUSION: Adult articular chondrocytes retain the ability to repopulate the matrix, an ability which is demonstrated with this specific cryoinjury. This may be an initial stage of cartilage regeneration.

Animals↗

PubMed2001

Damage and protection of UC blood cells during cryopreservation.

BACKGROUND: Current procedures for the cryopreservation of umbilical cord blood (UCB) progenitor cells, which are based on techniques used for BM, have had varying degrees of success (survival 9-118%). Improving the effectiveness of UCB cell therapies demands a more comprehensive understanding of freezing injury during cryopreservation. METHODS: Leukocyte concentrates from UCB, with or without 10% DMSO were cooled at 1 degrees C/min to different subzero temperatures (-5 to -50 degrees C), then either thawed directly (thaw) or plunged into liquid nitrogen before thawing (plunge). Single-platform flow cytometry with 7-amino-actinomycin D was used to directly quantify survival of CD34(+) cells. Fluorescent microscopy was used to examine plasma membrane integrity of nucleated cells. RESULTS: Without DMSO, recovery of nucleated cells was approximately 80% for both thaw and plunge. Survival was 9%, indicating damage to the plasma membrane. With 10% DMSO, nucleated cell recovery was also approximately 80%, indicating that DMSO does not improve recovery of nucleated cells. Survival, however, was much higher with DMSO, > 60% for nucleated cells thawed directly, and 30-55% for cells thawed from plunge, demonstrating cryoprotection conferred by DMSO. With DMSO, survival of CD34(+) cells was higher than that of nucleated cells, indicating that CD34(+) cells with 10% DMSO are more tolerant to cryopreservation than the total nucleated cell population. DISCUSSION: This study provides the necessary data on the low temperature response of UCB progenitor cells that are critical for the development of standards for the cryopreservation of UCB.

Antigens, CD34↗

PubMed2001

Osmotic and cryoprotectant permeation characteristics of islet cells isolated from the newborn pig pancreas.

The development of effective protocols for the low-temperature banking of pancreatic islets is an important step in islet transplantation for the treatment of type I diabetes mellitus. We have been exploring the use of islets from the newborn pig as an alternative source of tissue for transplantation. Current cryopreservation protocols are empirically derived, but may be optimized by modeling osmotic responses during the cryopreservation process. This study determined the osmotic and cryoprotectant permeability parameters of cells isolated from the pancreas of newborn pigs. Key parameters are: the osmotically inactive fraction of cell volume, hydraulic conductivity, the permeability coefficients of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) at varying temperatures, and the activation energies of these transport processes. Newborn pig islets were dispersed into single cells and kinetic and equilibrium cell volumes were recorded during osmotic excursions using an electronic particle counter interfaced to a computer. Data were fitted to theoretical descriptions of the osmotic responses of cells, based on the Kedem-Katchalsky approach. The hydraulic conductivity (Lp) in the absence of cryoprotectant was calculated as 0.050 +/- 0.005, 0.071 +/- 0.006, and 0.300 +/- 0.016 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees C, respectively (mean +/- SEM, n = 7, 6, or 9). These values give an activation energy value of 16.69 kcal/mol when put into an Arrhenius plot. The solute permeability (Ps) values for 1 M DMSO were 0.89 +/- 0.12, 1.86 +/- 0.28, and 5.33 +/- 0.26 microm/min at 4 degrees C, 10 degrees C, and 22 degrees C, respectively (n = 11, 8, or 10) giving an activation energy of 15.98 kcal/mol. The Lp values for cells exposed to 1 M DMSO were 0.071 +/- 0.006, 0.084 +/- 0.008, and 0.185 +/- 0.014 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees C, respectively. The activation energy for these values was 8.95 kcal/mol. The Ps values for 2 M DMSO were 1.11 +/- 0.13, 1.74 +/- 0.19, and 7.68 +/- 0.12 microm/min for the same temperatures, with a calculated activation energy of 17.89 kcal/mol. The Lp values in the presence of 2 M DMSO were 0.070 +/- 0.006, 0.085 +/- 0.008, and 0.192 +/- 0.009 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees C, respectively, with an activation energy of 9.40 kcal/mol. Solutions of 1 M EG gave Ps values of 1.01 +/- 0.13, 1.45 +/- 0.25, and 4.90 +/- 0.48 microm/min at the three test temperatures. The resulting activation energy was 14.60 kcal/mol. The corresponding Lp values were 0.071 +/- 0.007, 0.068 +/- 0.006, and 0.219 +/- 0.012 microm/min/atm with an activation energy of 10.96 kcal/mol. The solute permeabilities in the presence of 2 M EG for newborn pig islet cells were 1.03 +/- 0.15, 1.42 +/- 0.23, and 5.56 +/- 0.22 microm/min; the activation energy was 15.70. The Lp values for cells in the presence of 2 M EG were 0.068 +/- 0.008, 0.071 +/- 0.006, and 0.225 +/- 0.010 microm/min/atm; the activation energy for these values was 11.49 kcal/mol. These key cryobiological parameters permit the mathematical modeling of osmotic responses of intact islets during the cryopreservation process, which may lead to further improvements in the low temperature storage of islets from newborn pigs.

Animals↗

PubMed2000

Cell-cell contact affects membrane integrity after intracellular freezing.

The response of cells to freezing depends critically on the presence of an intact cell membrane. During rapid cooling, the cell plasma membrane may no longer be an effective barrier to ice propagation and can be breached by extracellular ice resulting in the nucleation of the supercooled cytoplasm. In tissues, the formation of intracellular ice is compounded by the presence of cell-cell and cell-surface interactions. Three different hamster fibroblast model systems were used to simulate structures found in organized tissues. Samples were supercooled to an experimental temperature on a cryostage and ice nucleated at the constant temperature. A dual fluorescent staining technique was used for the quantitative assessment of the integrity of the cell plasma membrane. A novel technique using the fluorescent stain SYTO was used for the detection of intracellular ice formation (IIF) in cell monolayers. The cumulative incidence of cells with a loss of membrane integrity and the cumulative incidence of IIF were determined as a function of temperature. Cells in suspension and individual attached cells showed no significant difference in the number of cells that formed intracellular ice and those that lost membrane integrity. For cells in a monolayer, with cell-cell contact, intracellular ice formation did not result in the immediate disruption of the plasma membrane in the majority of cells. This introduces the potential for minimizing damage due to IIF and for developing strategies for the cryoprotection of tissues during rapid cooling.

Animals↗