Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution

Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me₂SO) and stored for extended periods at −80 °C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me₂SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at −80 °C. The cryopreserved platelet units (n = 9) were rapidly thawed at 37 °C, reconstituted in 50% SSP+/plasma and stored at 22 °C. Platelet recovery and quality were examined 1 and 24 h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24 h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me₂SO and additive solution produces acceptable in vitro platelet quality.     

Platelet cryopreservation with glycerol, dextran, and mannitol: recovery of 5-hydroxytryptamine uptake and hypotonic stress response

Human platelets were frozen in 0.5 M glycerol, 0.5 M glycerol + 3% Dextran T40, or 0.5 M glycerol + 5% mannitol. The recovery of active transport of 5-hydroxytryptamine (5-HT) and the hypotonic stress response after freezing were dependent on the rate of cooling: the optimum range of rates was between 12 and 23 degrees C/min. The numerical recovery of cells was independent of cooling rate, but freezing altered the cell-size distribution. The combination of dextran and glycerol was no better than glycerol alone at protecting platelets against freezing damage. Mannitol, however, adversely affected platelet 5-HT uptake, and this was reflected in a low recovery of that activity after freezing platelets in glycerol supplemented with mannitol.     

Cooling and freezing damage platelet membrane integrity.

Cytoskeletal rearrangements and a membrane lipid phase transition (liquid crystalline to gel) occur in platelets on cooling from 23 to 4 degrees C. A consequence of these structural alterations is irreversible cellular damage. We investigated whether platelet membrane integrity could be preserved by (a) previously studied combinations of a calcium chelator (EGTA) and microfilament stabilizer (cytochalasin B) with apparent benefit in protecting platelets from cooling injury or (b) agents of known benefit in protecting membranes and proteins from freezing injury. Platelet function and activation before and after freezing or cooling were measured by agglutination with ristocetin, aggregation with thrombin or ADP, platelet-induced clot retraction (PICR), and expression of P-selectin. Platelets were loaded with 10 nM fluorescein diacetate. After freezing or cooling, the preparations were centrifuged and the supernatant was measured for fluorescein. For cooling experiments, fresh platelets were chilled at 4 degrees C for 1 to 21 days with or without the combination of 80 microM EGTA/AM and 2 microM cytochalasin B (EGTA/AM-CytoB) and then warmed rapidly at 37 degrees C. For freezing experiments, 5% dimethyl sulfoxide (Me2SO) or 5 mM glycerol were added to fresh platelets. The preparations were then frozen at -1 degrees C/min to -70 degrees C and then thawed rapidly at 37 degrees C. Platelet membrane integrity, as measured by supernatant levels of fluorescein, correlated inversely with platelet function. Chilling platelets at 4 degrees C with EGTA/AM-CytoB showed a gradual loss of membrane integrity, with maximum loss reached on day 7. The loss of membrane integrity preceded complete loss of function as demonstrated by PICR. In contrast, platelets chilled without these agents had complete loss of membrane integrity and function after 1 day of storage. Freezing platelets in Me2SO resulted in far less release of fluorescein than did freezing with or without other cryoprotectants (P < 0.001). This result correlated with enhanced function as demonstrated by PICR and supports earlier observations that Me2SO protects platelet membranes from freezing injury. Release of fluorescein into the surrounding medium reflected loss of membrane integrity and function in both cooled and frozen platelets. Membrane cytoskeletal rearrangements are linked to membrane changes during storage. These results may be generally applicable to the study of platelet storage.     

Platelet cryopreservation. 1. In vitro aggregation studies

Platelets were harvested by a Hemonetics Model-30 discontinuous cell separator from 20 normal volunteers and were cryopreserved in the presence of 5% DMSO at a controlled rate of freezing of -1 degrees C/min and stored in liquid nitrogen for up to 3 months. A significant loss of platelets occurred at the platelet concentration step through adhesion of platelets to the bag walls. A small reduction in aggregation associated with this was also seen and may reflect some damage to the platelets during the pheresis procedure. A small, but significant loss of platelet aggregation was seen with all agents following cryopreservation. Mean percentage aggregation post-thaw for all the agents was 75.4% (range 74-78%) and platelet recovery was approximately 90%. No significant changes in aggregation or recovery were seen over the 3 months' storage period. The cryoprotectant DMSO was shown to have no deleterious effect on platelet function in vitro.     

Effect of cyclic AMP on human blood platelets: induction of pseudopod formation and protein phosphorylation

A variety of techniques, including immunofluorescence, electron microscopy and biochemical analysis, were used to examine shape changes and cytoskeletal reorganization of human blood platelets during treatment with N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dbcAMP), and agent known to elevate the intracellular level of cyclic AMP (cAMP). Cytochemical analysis shows that the unstimulated platelets have a discoid shape with no obvious membrane projections. Platelets treated with dbcAMP produce pseudopod-like structures containing cytoskeletal proteins such as actin and microtubules. Biochemical analysis reveals that a 125,000 dalton phosphoprotein (P-125) is preferentially recruited into cytoskeletal fractions of platelets treated with dbcAMP. This protein, which is one of the substrates for cAMP-dependent kinase(s) and/or is closely associated with the cytoskeleton, may play an important role in regulating the shape changes and cytoskeletal reorganization that occur during the early stages of platelet activation.     

The effects of osmotic stress on human platelets

The effect of osmotic stress on human platelets was investigated at 0, 25, and 37 degrees C. The osmolality of the suspending plasma was decreased by adding water or increased by adding sodium chloride or sucrose. After 5 min, isotonicity was restored by dilution with an excess of isotonic phosphate-buffered saline. After centrifugation, the platelets were resuspended in autologous plasma and then incubated for 1 hr at 37 degrees C before assaying the active transport of 5-hydroxytryptamine (5-HT) and the hypotonic stress response. Anisosmotic conditions had a greater effect on the extent of volume reversal in the hypotonic stress test than on 5-HT uptake. At 25 degrees C, only moderate degrees of hypotonicity (0.25 osmol/kg) or hypertonicity (0.59 osmol/kg) were sufficient to depress the hypotonic stress response. In general, platelets tolerated departures from isotonic conditions better at 0 degree C than at the higher temperatures. Furthermore, at 0 and 25 degrees C approximately equiosmolal concentrations of sucrose and sodium chloride depressed the hypotonic stress response to similar extents, but at 37 degrees C high osmolalities (greater than 2 osmol/kg) were tolerated better when the additive was sucrose than when it was sodium chloride. Platelets shrank when subjected to hyperosmotic conditions, but their discoid shape and the peripheral band of microtubules were maintained.     

Cryopreservation of platelets using trehalose: The role of membrane phase behavior during freezing

In blood banks, platelets are stored at 20–24°C, which limits the maximum time they can be stored. Platelets are chilling sensitive, and they activate when stored at temperatures below 20°C. Cryopreservation could serve as an alternative method for long term storage of platelet concentrates. Recovery rates using dimethyl sulfoxide (DMSO) as cryoprotective agent, however, are low, and removal of DMSO is required before transfusion. In this study, we have explored the use of trehalose for cryopreservation of human platelets while using different cooling rates. Recovery of membrane intact cells and the percentage of nonactivated platelets were used as a measure for survival. In all cases, survival was optimal at intermediate cooling rates of 20°C min^?1. Cryopreservation using DMSO resulted in high percentages of activated platelets; namely 54% of the recovered 94%. When using trehalose, 98% of the platelets had intact membranes after freezing and thawing, whereas 76% were not activated. Using Fourier transform infrared spectroscopy, subzero membrane phase behavior of platelets has been studied in the presence of trehalose and DMSO. Furthermore, membrane hydraulic permeability parameters were derived from these data to predict the cell volume response during cooling. Both trehalose and DMSO decrease the activation energy for subzero water transport across cellular membranes. Platelets display a distinct lyotropic membrane phase transition during freezing, irrespective of the presence of cryoprotective agents. We suggest that concomitant uptake of trehalose during freezing could explain the increased survival of platelets cryopreserved with trehalose. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Actin filament content and organization in unstimulated platelets

The extent of actin polymerization in unstimulated, discoid platelets was measured by DNase I inhibition assay in Triton X-100 lysates of platelets washed at 37 degrees C by gel filtration, or in Triton X-100 lysates of platelets washed at ambient temperatures by centrifugation in the presence of prostacyclin. About 40% of the actin in the discoid platelets obtained by either method existed as filaments. These filaments could be visualized by electron microscopy of thin sections. Similar results were obtained when the actin filament content of discoid platelets was measured by sedimentation of filaments from Triton X-100 lysates at high g forces (145,000 g for 45 min). However, few of these filaments sedimented at the lower g forces often used to isolate networks of actin filaments from cell extracts. These results indicate that actin filaments in discoid cells are not highly crosslinked. Platelets isolated by centrifugation in the absence of prostacyclin were not discoid, but were instead irregular with one or more pseudopodia. These platelets also contained approximately 40-50% of their actin in a filamentous form; many of these filaments sedimented at low g forces, however, indicating that they were organized into networks. The discoid shape of these centrifuged platelets could be restored by incubating them for 1-3 h at 37 degrees C, which resulted in the reversal of filament organization. High g forces were then required for the sedimentation of the actin. Approximately 80-90% of the actin in platelets washed at 4 degrees C was filamentous; this high actin filament content could be attributed to actin polymerization during the preparation of the platelets at low temperatures. These studies show that platelet activation involves mechanisms for the structural reorganization of existing filaments, in addition to those previously described for mediating actin polymerization.     

In vitro study of platelet concentrate preparations from whole blood using N-(2-mercaptopropionyl)-glycine

The sulfhydryl group containing drug N-(2-mercaptopropionyl)-glycine (MPG) which inhibits platelet aggregation in a reversible manner permits to prepare platelet concentrates in non-siliconized glass containers at pH 7.4. Resuspension of platelets is possible immediately after centrifugation. In vitro platelets tests were carried out after washing out the MPG in MPG-free plasma. Thereafter, no inhibitory effects on platelet functions were found. Platelets concentrated in presence of MPG were significantly better with respect to yield, maintenance of discoid shape, aggregability, and hypotonic shock response compared with control platelets concentrated in absence of MPG.     

Platelet cryopreservation using a combination of epinephrine and dimethyl sulfoxide as cryoprotectants

Current methods of platelet storage are unsatisfactory because of the short shelf life of platelets and the rapid loss of platelet viability. We have developed a cryopreservation method that results in less damage from freezing and higher recovered function of platelets. Platelets were cryopreserved using a combination of epinephrine (EPN) and dimethyl sulfoxide (Me(2)SO) as cryoprotectants. The response of platelets to agonists was studied by flow cytometry and aggregation tests. Cryopreserving platelets with Me(2)SO decreased platelet annexin V binding due to freezing. The combination of EPN with Me(2)SO enhanced Me(2)SO cryoprotection and decreased platelet microparticle generation, suggesting that cryopreserving platelets using this combination is associated with increased platelet integrity. Platelet cryopreservation with an Me(2)SO/EPN combination also increased platelet aggregability, which was demonstrated by decreasing the lag phase and increasing the aggregation density to 66.39% +/- 6.6 that of fresh platelet-rich plasmas. We conclude that adding EPN as a combined cryoprotectant improves the quality of Me(2)SO-frozen platelets. As a method of aggregation of cryopreserved platelets, this method is comparable to that of normal fresh platelets and may improve the conditions for platelet transfusion.     

Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.     

Seleno-DL-methionine and the protection of human platelets against freezing injury

The effect of seleno-DL-methionine, which has antioxidative properties, on the recovery of human platelets after freezing with 0.5 mol/liter glycerol or 0.7 mol/liter (5% v/v) dimethyl sulfoxide was investigated. Incubation of platelets with 2 mumol/liter seleno-DL-methionine for 30 min at room temperature before equilibration with the protective additives improved the post-thaw uptake of 5-hydroxytryptamine and the percentage reversal in the hypotonic stress test. The effect was small, but in view of the ability of seleno-DL-methionine to inhibit lipid peroxidation in membranes, the results suggest that oxidative damage is implicated in freezing injury. The dimethyl sulfoxide protocol apparently afforded greater protection to the platelets than the glycerol protocol. But, if the platelets were incubated for 24 hr at 37 degrees C after thawing, there was a marked improvement in the response of cells in the hypotonic stress test, particularly in the samples frozen with glycerol, and there was no longer any difference between the two additives. There was, however, a concomitant loss of almost half the number of cells in the thawed suspensions during the prolonged incubation at 37 degrees C.     

Freeze preservation of platelets using hydroxyethyl starch (HES); a preliminary report.

A comparative study has been made of platelets stored by freeze preservation following treatment with dimethyl sulfoxide (DMSO) or hydroxyethyl starch (HES) with fresh platelets and platelets stored at 4 °C for 48 hr. The indices studied were platelet recovery, pH, light microscope morphology, platelet Factor 3 (PF₃) availability and the hypotonic stress response. The DMSO preserved platelets gave a better response to hypotonic stress and incurred lesser degrees of membrane damage as demonstrated by PF₃ availability. There was however a significantly higher recovery of platelets treated with HES; with DMSO the osmotic damage inflicted during removal caused considerable lysis. Platelets frozen by DMSO or HES gave consistently better in vitro results than platelets stored at 4 °C for 48 hr. A preliminary clinical trial of HES preserved platelets has confirmed haemostatic effectiveness in vivo. HES being relatively nontoxic, platelets can be infused immediately after thawing and with minimal post thaw manipulation, thus maintaining a relatively closed system. It is concluded that cryopreservation with HES is a practical and effective means for long term platelet storage.     

The MEK kinase Ssk2p promotes actin cytoskeleton recovery after osmotic stress

Saccharomyces cerevisiae adapts to osmotic stress through the activation of a conserved high-osmolarity growth (HOG) mitogen-activated protein (MAP) kinase pathway. Transmission through the HOG pathway is very well understood, yet other aspects of the cellular response to osmotic stress remain poorly understood, most notably regulation of actin organization. The actin cytoskeleton rapidly disassembles in response to osmotic insult and is induced to reassemble only after osmotic balance with the environment is reestablished. Here, we show that one of three MEK kinases of the HOG pathway, Ssk2p, is specialized to facilitate actin cytoskeleton reassembly after osmotic stress. Within minutes of cells' experiencing osmotic stress or catastrophic disassembly of the actin cytoskeleton through latrunculin A treatment, Ssk2p concentrates in the neck of budding yeast cells and concurrently forms a 1:1 complex with actin. These observations suggest that Ssk2p has a novel, previously undescribed function in sensing damage to the actin cytoskeleton. We also describe a second function for Ssk2p in facilitating reassembly of a polarized actin cytoskeleton at the end of the cell cycle, a prerequisite for efficient cell cycle completion. Loss of Ssk2p, its kinase activity, or its ability to localize and interact with actin led to delays in actin recovery and a resulting delay in cell cycle completion. These unique capabilities of Ssk2p are activated by a novel mechanism that does not involve known components of the HOG pathway.     

Platelets Recognize Brain-Specific Glycolipid Structures,Respond to Neurovascular Damage and Promote Neuroinflammation

Platelets respond to vascular damage and contribute to inflammation, but their role in the neurodegenerative diseases is unknown. We found that the systemic administration of brain lipid rafts induced a massive platelet activation and degranulation resulting in a life-threatening anaphylactic-like response in mice. Platelets were engaged by the sialated glycosphingolipids (gangliosides) integrated in the rigid structures of astroglial and neuronal lipid rafts. The brain-abundant gangliosides GT1b and GQ1b were specifically recognized by the platelets and this recognition involved multiple receptors with P-selectin (CD62P) playing the central role. During the neuroinflammation, platelets accumulated in the central nervous system parenchyma, acquired an activated phenotype and secreted proinflammatory factors, thereby triggering immune response cascades. This study determines a new role of platelets which directly recognize a neuronal damage and communicate with the cells of the immune system in the pathogenesis of neurodegenerative diseases.     

FREEZING STRESS AND OSMOTIC DEHYDRATION IN FUCUS DISTICHUS (PHAEOPHYTA): EVIDENCE FOR PHYSIOLOGICAL SIMILARITY1

The effects of osmotic dehydration and freezing on photosynthesis were studied in the brown alga Fucus distichus L. The data indicated that F. distichus exhibits similar physiological responses to both osmotic dehydration and freezing stress and that these responses resemble those in the literature for the effect of desiccation in air. Both stresses inhibited light-limited (P_subsat) and light-saturated (P_max) photosynthesis measured immediately after plants were reimmersed in seawater. The degree of initial inhibition and subsequent recovery of photosynthesis were proportional to the severity of the dehydration or freezing treatment. P_subsat and P_max recovered completely from osmotic dehydration for 3 h in 200% and 3 hr at – 10°C, but recovery was only partial following 3 h in 300%o or 3 h at – 15°C. In most cases, recovery was complete within 2 h following dehydration, with little further recovery occurring between 2 and 24 h posttreatment. No time-dependent recovery occurred following severe freezing. Observations using the vital stain fluorescein diacetate suggested that the lack of complete recovery might be due to severe damage or death of a proportion of cells in the thallus. There were no clear effects of either osmotic dehydration or freezing on dark respiration (R_d), although R_d was stimulated in all emersed treatments (frozen plants and 5° C controls) immediately following reimmersion. Measurement of chlorophyll fluorescence induction kinetics indicated that both osmotic dehydration and freezing reduced the ratio of variable to maximum florescence (F_v/F_m), indicating a decrease in the quantum efficiency of photosystem I. Based on these data, we suggest that there are common cellular and physiological components involved in the response of fucoid algae to a range of water stresses. This hypothesis was supported by experiments that showed that osmoacclimation in hyperosmotic seawater (51%o)for 2 weeks increased the ability of F. distichus to recover from freezing at – 15° C. During acclimation, mannitol content increased under hyperosmotic conditions and decreased under hypoosmotic conditions. Changes in plasma membrane integrity, determined by fresh weight: dry weight ratio, and amino acid release following freezing indicated an increasing gradient of freezing tolerance from low to high salinity. However, none of these physiological changes fully explained the marked increase in the freezing tolerance of photosynthesis observed in plants acclimated under hyperosmotic conditions.     

Actin cytoskeleton of resting bovine platelets

Actin filaments in resting discoid bovine platelets were examined by fluorescence and electron microscopy. Rhodamine-phalloidin staining patterns showed a characteristic wheel-like structure which consisted of a central small circle connected by several radial spokes to a large peripheral circle. This wheel-like structure was composed of actin filaments forming a characteristic arrowhead structure with heavy meromyosin from muscle. Actin filaments were densely arrayed in parallel with a marginal microtubule band and radiated out from the center to the periphery. Platelets treated with colchicine lost their marginal microtubule band but retained their wheel-like structure and normal discoid form. Cytochalasin B disrupted the wheel-like structure but not the marginal microtubule band or the normal discoid form. After simultaneous treatment with both cytochalasin B and colchicine, platelets lost their discoid shape. These results suggest that actin filaments and microtubules both play important roles in the maintenance of the discoid shape of resting bovine platelets.     

Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential

Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sperm parameters evaluated included cell volume, membrane integrity, lipid peroxidation, caspase 3, 7, and 8 activation, mitochondrial membrane potential, and integrity of the cytoskeleton. Glycerol exerted toxicity at concentrations ≥ 3.5% and the maximal toxicity was observed at 5%. The actin cytoskeleton was especially sensitive to glycerol presence, inducing rapid F actin depolymerization at concentrations over 1.5%. The sperm membrane and the mitochondria were other structures affected. The toxicity of glycerol is apparently related to osmotic and nonosmotic effects. In view of our results the concentration of glycerol in the freezing media for stallion spermatozoa should not surpass 2.5%.     

Investigating cryoinjury using simulations and experiments. 1: TF-1 cells during two-step freezing (rapid cooling interrupted with a hold time)

There is significant interest in designing a cryopreservation protocol for hematopoietic stem cells (HSC) which does not rely on dimethyl sulfoxide (Me₂SO) as a cryoprotectant. Computer simulations that describe cellular osmotic responses during cooling and warming can be used to optimize the viability of cryopreserved HSC; however, a better understanding of cellular osmotic parameters is required for these simulations. As a model for HSC, the erythroleukemic human cell line TF-1 was used in this study. Simulations, based on the osmotic properties of TF-1 cells and on the solution properties of the intra- and extracellular compartments, were used to interpret cryoinjury associated with a two-step cryopreservation protocol. Calculated intracellular supercooling was used as an indicator of cryoinjury related to intracellular ice formation. Simulations were applied to the two-step cooling protocol (rapid cooling interrupted with a hold time) for TF-1 cells in the absence of Me₂SO or other cryoprotectants and optimized by minimizing the indicator of cryoinjury. A comparison of simulations and experimental measurements of membrane integrity supports the concept that, for two-step cooling, increasing intracellular supercooling is the primary contributor to potential freezing injury due to the increase in the likelihood of intracellular ice formation. By calculating intracellular supercooling for each step separately and comparing these calculations with cell recovery data, it was demonstrated that it is not optimal simply to limit overall supercooling during two-step freezing procedures. More aptly, appropriate limitations of supercooling differ from the first step to the second step. This study also demonstrates why high cell recovery after cryopreservation could be achieved in the absence of traditional cryoprotectants.