Osmotic Excursions Research Articles

Osmotic injury and cytotoxicity for hMSCs in contact with Me2SO: The effect of cell size distribution

The paper discusses the impact of cell size on cytotoxicity and expansion lysis during the osmotic excursions resulting from the contact of hMSCs from UCB with Me2SO. It builds upon the mathematical model recently presented by the authors, which pertains to a population of cells with uniform size. The objective is to enhance the model's relevance by incorporating the more realistic scenario of cell size distribution, utilizing a Population Balance Equations approach. The study compares the capability of the multiple-sized model to the single-sized one to describe system behavior experimentally measured through cytofluorimetry and Coulter counter when, first, suspending hMSCs in hypertonic solutions of Me2SO (at varying osmolality, system temperature, and contact times), and then (at room temperature) pelleting by centrifugation before suspending the cells back to isotonic conditions. Simulations demonstrate that expansion lysis and cytotoxic effect are not affected by cell size for the specific system hMSCs/Me2SO, thus confirming what was found so far by the authors through a single-size model. On the other hand, simulations show that, when varying the adjustable parameters of the model that are expected to change from cell to cell lineages, expansion lysis is sensitive to cell size, while cytotoxicity is not, being mainly influenced by external CPA concentration and contact duration. More specifically, it is found that smaller cells suffer expansion lysis more than larger ones. The findings suggest that different cells from hMSCs may require a multiple-sized model to assess cell damage during osmotic excursions in cryopreservation.

Modelling the osmotic behaviour of human mesenchymal stem cells

In this work, a novel mathematical model for the description of the osmotic behavior during the cryopreservation of human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB) is proposed. In cryopreservation, the two-parameter formalism of perfect osmometer behavior is typically adopted and preferred due to its simplicity: cell volume osmotic excursions are described as due only to the passive trans-membrane transport of water and permeant solutes such as cryoprotectant agents (CPAs); intracellular solutes, responsible of the isotonic osmolality, are assumed to be impermeant. The application of the two-parameter model fails to capture the osmotic response of hMSCs whenever a swelling phase is involved, as demonstrated by the authors. To overcome this limitation, the imperfect osmometer behavior of hMSCs is successfully modelled herein by improving the two-parameter formalism through the coupling of osmosis with cell mechanics and cell membrane Surface Area Regulation (SAR): now the transmembrane permeation of solutes (ions/salt) during the swelling phase through the temporary opening of mechanosensitive channels is allowed. This way cells can reach an equilibrium volume different from the initial isotonic one, when isotonic conditions are re-established after contact with impermeant or permeant solutes, such as sucrose or dimethyl-sulfoxide (DMSO), respectively. The sequential best-fit procedure adopted to adjust model parameters is reported herein along with model validation through full predictions.

A06 Plenary Lecture 2 : Cryosurgery as the anti-christ of cryopreservation

This presentation will attempt to relate some fundamental aspects of cryobiology to the problems of cryosurgery. Much understanding of the fundamentals has come from studies on cryopreservation, but cryopreservation and cryosurgery differ in important ways aside from their ultimate aims. The following are the topics to be discussed: A. Extrinsic factors or variables: • Extracellular medium • Cooling rate • Can be constant in cryopreservation • * Can not be constant in cryosurgical freezing • Temperature • Minimum temperature • Usually uniform in cryopreservation • Can not be uniform in cryosurgical freezing • Time at subzero temperature • Warming rate • Number of repeat freezing thawing cycles B. Physical events: • Extra-cellular and inter-cellular ice formation • Osmotic dehydration • Permeation of permeating solutes • Cell supercoiling • Is cell position perturbed by ice front? • Intracellular ice formation • Vitrification • Recrystallization of ice • Post-thaw osmotic excursions C. Intrinsic cellular factors • Cell size • Permeability to water • Permeability to solutes. • Possession of aquaporins or not • Chill sensitivity • Sensitivity to osmotic excursions D. Major differences between: Cryopreservation freezing Cryosurgical freezing Cooling is from outside-In Cooling is from inside-out All parts of the system reach equilibrium with temperature of −196 °C throughout System reaches steady state with temps.varying from −160 °C near probe to −0.5 °C at ice-ball surface Cryoprotective agents are always added Cryoprotective agents are never added. But.. Survivals of ⩾50% are usually acceptable Survivals of <1% are mandatory

Androcoll-E large selects a subset of live stallion spermatozoa capable of producing ROS

The aim of this study was to elucidate if SLC after 24h storage selects the subpopulation of spermatozoa that better withstands osmotic shock. To test this hypothesis, viability, mitochondrial membrane potential (MMP) and superoxide anion (O2−) production of uncentrifuged (UC) and single layer centrifugation (SLC) – selected spermatozoa were analyzed following SLC after storage of the semen. An aliquot of the extended ejaculate (100×106spermatozoa/mL) was centrifuged through a single layer of a silane-coated silica based colloid formulation optimized for equine spermatozoa (Androcoll-E large, SLU, Sweden) and the rest was used as control. UC and SLC-sperm samples were subjected to osmotic challenges (75 and 900mOsm) with a subsequent return to isosmolarity (300mOsm) using Biggers–Whitten–Whittingham (BWW) medium. Viability and MMP decreased after the different osmotic stress in UC and SLC spermatozoa, and return to isosmolarity did not reverse these effects. O2− production was enhanced after SLC in all osmolarities tested. Interestingly, the percentage of living spermatozoa showing O2− production was increased after 900mOsm stress in UC spermatozoa, this increase being more evident in SLC spermatozoa. Returning spermatozoa to 300mOsm enhanced this percentage in UC viable cells but not in SLC spermatozoa. The scenario observed for UC spermatozoa shows that O2− is produced in response to isolated hyperosmolarities and subsequent osmotic excursions. As the viability, MMP and cell volume remained the same between SLC and UC spermatozoa, we conclude that Androcoll-E large is likely selecting a higher percentage of physiologically O2− producing spermatozoa.

The Mitochondria of Stallion Spermatozoa Are More Sensitive Than the Plasmalemma to Osmotic‐Induced Stress: Role of c‐Jun N‐terminal Kinase (JNK) Pathway

Cryopreservation introduces extreme temperature and osmolality changes that impart lethal and sublethal effects on spermatozoa. Additionally, there is evidence that the osmotic stress induced by cryopreservation causes oxidative stress to spermatozoa. The main sources of reactive oxygen species in mammalian sperm are the mitochondria. In view of this, the aim of our study was to test whether or not osmotic stress was able to induce mitochondrial damage and to explore the osmotic tolerance of the mitochondria of stallion spermatozoa. Ejaculates from 7 stallions were subjected to osmolalities ranging from 75 to 1500 mOsm/kg, and the effect on sperm membrane integrity and mitochondrial membrane potential was studied. Additionally, the effects of changes in osmolality from hyposmotic to isosmotic and from hyperosmotic to isosmotic solutions were studied (osmotic excursions). The cellular volume of stallion spermatozoa under isosmotic conditions was 20.4 ± 0.33 μm(3). When exposed to low osmolality, the stallion spermatozoa behaved like a linear osmometer, whereas exposure to high osmolalities up to 900 mOsm/kg resulted in decreased sperm volume. Although sperm membranes were relatively resistant to changes in osmolality, mitochondrial membrane potential decreased when osmolalities were low or very high (10.7 ± 1.74 and 16.5 ± 1.70 at 75 and 150 mOsm/kg, respectively, and 13.1 ± 1.83 at 1500 mOsm/kg), whereas in isosmolar controls the percentage of stallion sperm mitochondria with a high membrane potential was 41.1 ± 1.69 (P < .01). Osmotic excursions induced greater damage than exposure of spermatozoa to a given nonphysiologic osmolality, and again the mitochondria were more prone to damage induced by osmotic excursions than was the sperm plasma membrane. In search of intracellular components that could mediate these changes, we have detected for the first time the c-Jun N-terminal kinase 1/2 in stallion spermatozoa, which are apparently involved in the regulation of the viability of these cells.

Mathematical modeling of cryoprotectant addition and removal for the cryopreservation of engineered or natural tissues

Long-term storage of natural tissues or tissue-engineered constructs is critical to allow off-the-shelf availability. Vitrification is a method of cryopreservation that eliminates ice formation, as ice may be detrimental to the function of natural or bioartificial tissues. In order to achieve the vitreous state, high concentrations of CPAs must be added and later removed. The high concentrations may be deleterious to cells as the CPAs are cytotoxic and single-step addition or removal will result in excessive osmotic excursions and cell death. A previously described mathematical model accounting for the mass transfer of CPAs through the sample matrix and cell membrane was expanded to incorporate heat transfer and CPA cytotoxicity. Simulations were performed for two systems, an encapsulated system of insulin-secreting cells and articular cartilage, each with different transport properties, geometry and size. Cytotoxicity and mass transfer are dependent on temperature, with a higher temperature allowing more rapid mass transfer but also causing increased cytotoxicity. The effects of temperature are exacerbated for articular cartilage, which has larger dimensions and slower mass transport through the matrix. Simulations indicate that addition and removal at 4 °C is preferable to 25 °C, as cell death is higher at 25 °C due to increased cytotoxicity in spite of the faster mass transport. Additionally, the model indicates that less cytotoxic CPAs, especially at high temperature, would significantly improve the cryopreservation outcome. Overall, the mathematical model allows the design of addition and removal protocols that insure CPA equilibration throughout the sample while still minimizing CPA exposure and maximizing cell survival.

Osmotic water permeability of plasma and vacuolar membranes in protoplasts II. Theoretical basis

Water permeability of the plasma membrane (PM) and the vacuolar membrane (VM) is important for intracellular and transcellular water movement in plants, because mature plant cells have large central vacuoles. We have developed a new method for measuring the osmotic water permeability of the PM and VM (P ( f1) and P ( f2), respectively) in individual plant cells. Here, the theoretical basis and procedure of the method are discussed. Protoplasts isolated from higher plant tissues are used to measure P ( f1) and P ( f2). Because of the semi-permeability (selective permeability) of cellular membranes, protoplasts swell or shrink under hypotonic or hypertonic conditions. A theoretical three-compartment model is presented for simulating time-dependent volume changes in the vacuolar and cytoplasmic spaces in a protoplast during osmotic excursions. The model describes the theoretical relationships between P ( f1), P ( f2) and the bulk osmotic water permeability of protoplasts (P ( f(bulk))). The procedure for measuring the osmotic water permeability is: (1) P ( f(bulk)) is calculated from the time when half of the total change in protoplast volume is completed, by assuming that the protoplast has a single barrier to water movement across it (two-compartment model); (2) P ( f2) of vacuoles isolated from protoplasts is obtained in the same manner; and (3) P ( f1) is determined from P ( f(bulk)) and P ( f2) according to the three-compartment model. The theoretical relationship between P ( fl ) (m s(-1)) and L ( Pl ) (hydraulic conductivity, l=1, 2) (m s(-1) Pa(-1)) is also discussed.

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.

Frozen/Thawed Platelets: Importance of Osmotic Tolerance and Platelet Subpopulations

Me2SO cryopreserved platelets circulatein vivo,reduce bleeding time, and have hemostatic properties but their functional recovery is only half that of the fresh material. Poor osmotic response is often reported as the cause of the freezing injury. Osmotic excursions on 1- and 5-day-old platelets have been studied. Platelets stored for 5 days have a lesser capability to regulate their volume particularly after an initial swelling. This is attributed to the reduction of discoid cell number, 80% vs 62% for 1-day-old and 5-day-old platelets, respectively. After freezing, hypotonic stress response is reduced from 86 to 39% for 1-day-old and 73 to 31% for 5-day-old platelets. This reduction in function is supported by a similar reduction of discoid cells from 80 to 40% for 1-day-old and 62 to 32% for 5-day-old platelets. The integrity of the cytoskeleton is critical for the osmotic response. Freezing recovery is significantly lowered in the presence of propylene glycol, which alters actin. This contrasts with the recovery of platelets treated with anti-aggregating agents. Platelets show a greater viability after freezing and thawing when PGI2is added. It is postulated that freshly collected platelets, which are heterogeneous, contain populations of cells that are more sensitive to freezing than others. More tolerant cells remain discoid after freezing and are also less susceptible to storage lesions. Therefore, the maintenance of the integrity of the membrane and the cytoskeleton should be considered for the development of preservation methodologies.

Cryopreservation of Rye Protoplasts by Vitrification

A procedure has been developed for the vitrification of mesophyll protoplasts isolated from leaves of nonacclimated (NA) and cold-acclimated (ACC) winter rye seedlings (Secale cereale L. cv Puma). The procedure involves (a) equilibration (loading) of the protoplasts with an intermediate concentration (1.5, 1.75, or 2.0 molar) of ethylene glycol (EG) at 20 degrees C; (b) dehydration of the protoplasts in a concentrated vitrification solution made of 7 molar EG + 0.88 molar sorbitol + 6% (w/v) bovine serum albumin (BSA) at 0 degrees C; (c) placing the protoplasts into polypropylene straws and quenching in liquid nitrogen (LN(2)); and (d) recovery of the protoplasts from LN(2) and removal (unloading) of the vitrification solution. For NA protoplasts, 47 + 1% survival was obtained following recovery from LN(2) if the protoplasts were first loaded with 1.75 molar EG prior to the dehydration step. However, to achieve this level of survival, NA protoplasts had to be unloaded in a hypertonic (2.0 osmolal [osm]) sorbitol solution. If they were unloaded in an isotonic solution (0.53 osm), survival was 3+/-2%. In contrast, survival of ACC protoplasts following recovery from LN(2) was 34 +/- 10% when the protoplasts were loaded in a 2.0 molar EG solution and unloaded in an isotonic sorbitol solution (1.03 osm). If ACC protoplasts were unloaded in an hypertonic sorbitol solution (1.5 osm), survival was 51 +/- 9%. These results indicate that the osmotic excursions incurred during the procedure are a major factor affecting survival.

The influence of cold acclimation on the lipid composition and cryobehaviour of the plasma membrane of isolated rye protoplasts

Destabilization of the plasma membrane, which is a primary cause of freezing injury, is a consequence of freeze-induced osmotic stresses and cell dehydration. However, the mechanism of injury depends on the magnitude of the osmotic stress and the extent of cell dehydration. Over the range of 0 to — 5 °C, destabilization of the plasma membrane in protoplasts isolated from non-acclimated rye leaves is a result of osmotic excursions, because freeze-induced osmotic contraction results in endocytotic vesiculation of the plasma membrane and sufficiently large area reductions are irreversible. At lower temperatures, the protoplasts are subjected to extremely large osmotic pressures ( — 12 MPa at —10 °C), and there are several changes in the ultrastructure of the plasma membrane, including the formation of aparticulate domains and lamellar- to hexagonaln-phase transitions. These changes, which are manifestations of demixing of the membrane components, are predicted by a theory of bilayer interactions at low levels of hydration. During cold acclimation, the cryobehaviour of the plasma membrane is altered; osmotic contraction results in the reversible formation of exocytotic extrusions and the propensity for dehydration-induced demixing and lamellar- to hexagonaln-phase transitions is decreased. In both cases, the differential behaviour is also observed in liposomes prepared from plasma membrane lipids isolated from non-acclimated and cold-acclimated leaves. However, as no lipid species are unique to the plasma membrane of either non-acclimated or cold-acclimated leaves, the differential behaviour is caused by altered lipid-lipid interactions because of different proportions of the lipid species. Hence the behaviour of the plasma membrane can be altered by using a protoplast-liposome fusion procedure to selectively modify the lipid composition of the plasma membrane. These studies provide direct evidence that the increased cryostability of the plasma membrane is a consequence of alterations in its lipid composition.

Cryopreservation of rye protoplasts by vitrification

A procedure has been developed for the vitrification of mesophyll protoplasts isolated from leaves of nonacclimated (NA) and cold-acclimated (ACC) winter rye seedlings (Secale cereale L. cv Puma). The procedure involves (a) equilibration (loading) of the protoplasts with an intermediate concentration (1.5, 1.75, or 2.0 molar) of ethylene glycol (EG) at 20°C; (b) dehydration of the protoplasts in a concentrated vitrification solution made of 7 molar EG + 0.88 molar sorbitol + 6% (w/v) bovine serum albumin (BSA) at 0°C; (c) placing the protoplasts into polypropylene straws and quenching in liquid nitrogen (LN2); and (d) recovery of the protoplasts from LN2 and removal (unloading) of the vitrification solution. For NA protoplasts, 47 + 1% survival was obtained following recovery from LN2 if the protoplasts were first loaded with 1.75 molar EG prior to the dehydration step. However, to achieve this level of survival, NA protoplasts had to be unloaded in a hypertonic (2.0 osmolal [osm]) sorbitol solution. If they were unloaded in an isotonic solution (0.53 osm), survival was 3±2%. In contrast, survival of ACC protoplasts following recovery from LN2 was 34 ± 10% when the protoplasts were loaded in a 2.0 molar EG solution and unloaded in an isotonic sorbitol solution (1.03 osm). If ACC protoplasts were unloaded in an hypertonic sorbitol solution (1.5 osm), survival was 51 ± 9%. These results indicate that the osmotic excursions incurred during the procedure are a major factor affecting survival.

Freeze/thaw-induced destabilization of the plasma membrane and the effects of cold acclimation.

Disruption of the plasma membrane is a primary cause of freezing injury. In this review, the mechanisms of injury resulting from freeze-induced cell dehydration are presented, including destabilization of the plasma membrane resulting from (a) freeze/thaw-induced osmotic excursions and (b) lyotropic phase transitions in the plasma membrane lipids. Cold acclimation dramatically alters the behavior of the plasma membrane during a freeze/thaw cycle--increasing the tolerance to osmotic excursions and decreasing the propensity for dehydration-induced lamellar to hexagonal-II phase transitions. Evidence for a casual relationship between the increased cryostability of the plasma membrane and alterations in the lipid composition is reviewed.