Development of optimal techniques for cryopreservation of human platelets. I. Platelet activation during cold storage (at 22 and 8 degrees C) and cryopreservation.

Using the current blood bank storage conditions at 22 degrees C, the viability and function of human platelets can be maintained for only 5 days. This does not allow for the necessary and extensive banking of platelets needed to treat patients afflicted with thrombocytopenia, a side effect of many invasive surgeries such as cardiopulmonary bypass or bone marrow transplantation. The development of optimal techniques for long-term cryopreservation and banking of human platelets would provide the ability to greatly extend the viable life of the platelet and would fulfill an increasing and urgent need in many clinical applications. To determine the optimal techniques for platelet preservation, the expression of an activation marker, phosphatidylserine, on the platelet membrane during storage at 22 and 8 degrees C as well as during the different freezing preservation processes was examined using flow cytometry and annexin V binding assay. Human platelets were identified by both CD41 and light scatter in flow cytometry. In cryopreservation experiments, effects of the following factors on platelet activation were evaluated: (a) cryoprotective agents (CPAs) type: dimethyl sulfoxide (Me2SO), ethylene glycol (EG), and propylene glycol (PG), (b) CPA concentration ranging from 0 to 3 M, and (c) ending temperatures of a slow cooling process at -1 degrees C/min. Our results demonstrated that (a) approximately 50% of platelets were activated on days 7 and 16 at 22 and 8 degrees C, respectively; (b) platelets were not significantly activated after 30-min exposure to 1 M Me2SO, EG, and PG at 22 degrees C, respectively, and (c) there was a significant difference in cryoprotective efficacy among these three CPAs in preventing platelets from cryoinjury. After being cooled to -10 degrees C, 74% of the cryopreserved platelets survived (nonactivated) in 1 M Me2SO solution, while in 1 M EG and 1 M PG solutions, 62 and 42% of the platelets survived, respectively. Using the information that Me2SO consistently yields higher percentages of nonactivated platelets and does not seem to be cytotoxic to platelets for 30-min exposure time, this was found to be the optimal cryoprotective agent for platelets. In addition, significant Me2SO toxicity to platelets was not noted until Me2SO concentrations exceeded 2 M. Finally, a concentration of 1 M Me2SO proved to be the most effective at all cryopreservation ending temperatures tested (-10, -30, -60, and -196 degrees C). In conclusion, under the present experimental conditions, a storage temperature of 8 degrees C appeared to be much better than 22 degrees C. Although the potential chemical toxicity of 1 M Me2SO, EG, or PG is negligible, 1 M Me2SO was found to be optimum for cryopreservation of human platelets. PG has the least cryoprotective function for low-temperature platelet survival.     

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.     

Cryopreservation of seabream (Sparus aurata) spermatozoa

The aim of this research was to optimize protocols for freezing spermatozoa of seabream (Sparus aurata). All the phases of the cryopreservation procedure (sampling, choosing the cryoprotective extender, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa under examination, so as to be able to restore on thawing the morphological and physiological characteristics of fresh semen. Seabream spermatozoa were collected by stripping and transported to the laboratory chilled (0-2 degrees C). Five cryoprotectants, dimethyl sulfoxide (Me(2)SO), ethylene glycol (EG), 1,2-propylene glycol (PG), glycerol, and methanol, were tested at concentrations between 5 and 15% by volume to evaluate their effect on the motility of semen exposed for up to 30 min at 26 degrees C. The less toxic cryoprotectants, 10% EG, 10% PG, and 5% Me(2)SO, respectively, were added to 1% NaCl to formulate the extenders for freezing. The semen was diluted 1:6 with the extender, inserted into 0.25-ml plastic straws by Pasteur pipette, and frozen using a cooling rate of either 10 or 15 degrees C/min to -150 degrees C followed by transfer and storage in liquid nitrogen (-196 degrees C). The straws were thawed at 15 degrees C/s. On thawing, the best motility was obtained with 5% Me(2)SO, although both 10% PG and EG showed good results; no differences were found between the two freezing gradients, although semen frozen with the 10 degrees C/min gradient showed a slightly higher and more prolonged motility.     

Cryopreservation of umbilical cord blood: 2. Tolerance of CD34(+) cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing

Cryopreservation protocols for umbilical cord blood have been based on methods established for bone marrow (BM) and peripheral blood stem cells (PBSC). The a priori assumption that these methods are optimal for progenitor cells from UCB has not been investigated systematically. Optimal cryopreservation protocols utilising penetrating cryoprotectants require that a number of major factors are controlled: osmotic damage during the addition and removal of the cryoprotectant; chemical toxicity of the cryoprotectant to the target cell and the interrelationship between cryoprotectant concentration and cooling rate. We have established addition and elution protocols that prevent osmotic damage and have used these to investigate the effect of multimolar concentrations of Me(2)SO on membrane integrity and functional recovery. We have investigated the effect of freezing and thawing over a range of cooling rates and cryoprotectant concentrations. CD34(+) cells tolerate up to 60 min exposure to 25% w/w (3.2M) Me(2)SO at +2 degrees C with no significant loss in clonogenic capacity. Exposure at +20 degrees C for a similar period of time induced significant damage. CD34(+) cells showed an optimal cooling range between 1 degrees C and 2.5 degrees C/min. At or above 1 degrees C/min, increasing the Me(2)SO concentration above 10% w/w provided little extra protection. At the lowest cooling rate tested (0.1 degrees C/min), increasing the Me(2)SO concentration had a statistically significant beneficial effect on functional recovery of progenitor cells. Our findings support the conclusion that optimal recovery of CD34(+) cells requires serial addition of Me(2)SO, slow cooling at rates between 1 degrees C and 2.5 degrees C/min and serial elution of the cryoprotectant after thawing. A concentration of 10% w/w Me(2)SO is optimal. At this concentration, equilibration temperature is unlikely to be of practical importance with regard to chemical toxicity.     

The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing

Dendritic cells (DCs) are now regarded as specialized leucocytes with distinctive morphological and functional characteristics as accessory or stimulator cells for many lymphocyte responses. While knowledge of the response of other leucocytes (e.g., lymphocytes, macrophages, and granulocytes) to freezing and thawing has been established for some years, an understanding of the cryobiological properties of DCs has not, hitherto, been determined specifically. Such information is important both for establishing procedures for the long-term storage of these cells for use in immunological procedures and for defining freezing conditions that might selectively kill DCs in attempts to modulate the immunogenicity of transplantable tissues during cryopreservation. Preparations of rat and human spleen cells enriched for DCs were frozen to -60 degrees C at one of six cooling rates (0.3, 1.5, 10, 20, 70, or 150 degrees C/min) using a procedure that was established for pancreatic islets with 2 M dimethyl sulfoxide (Me2SO) as the cryoprotectant. Following storage at -196 degrees C the survival of thawed cells was assessed by evaluating both the numbers of cells recovered after the complete process and the membrane integrity of the recovered cells using a supravital fluorescent probe assay. Survival profiles for DCs showed a dependence upon cooling rate similar to other lymphoid cells but DCs were more sensitive to freezing injury than either lymphocytes or macrophages: Optimum survival (75% recovery of numbers and 57% membrane integrity) of rat DCs was achieved by slow cooling (0.3 degrees C/min). Optimal recovery of human DCs was significantly higher (83% recovery of numbers and 72% membrane integrity) after cooling at either 0.3 or 1.5 degrees C/min. The viable yield of DCs from both species declined abruptly as cooling rate was increased, with less than 10% survival after cooling at 20 degrees C/min and negligible survival after cooling at 70 degrees C/min or greater. Analysis of variance of the survival data showed that the response of DCs to freezing and thawing was significantly different (P less than 0.005) from that of either lymphocytes or macrophages, thus providing additional evidence that DCs are distinct from other leucocytes, especially macrophages. This study defines conditions that either will provide effective cryopreservation of DCs for immunological purposes or are most likely to bring about their inactivation in cryobiological approaches to modulating tissue immunogenicity.     

Multifaceted freezing injury in human polymorphonuclear cells at high subfreezing temperatures

Extracellular freezing injury at high subzero temperatures in human polymorphonuclear cells (PMNs) was studied with a cryomicroscope, electron microscope, and functional assays (phagocytosis, microbicidal activity, and chemotaxis). There are at least four major factors in freezing injury: osmotic stress, chilling, cold shock, and dilution shock. Extracellularly frozen PMNs lose functions when cooled to -2 degrees C without a cryoprotectant. Cells lose volume on freezing to the same degree as in hypertonic exposure. PMNs have a minimum volume to which they can shrink without injury. Greater dehydration produces irreversible injury to cellular functions, and cells eventually collapse under high osmotic stress. Chilling sensitivity is seen in slowly chilled, supercooled PMNs below -5 degrees C; at -7 degrees C, functions are lost in 1 h. This injury can be prevented by the addition of Me2SO but not glycerol. Me2SO does not, however, prevent cold shock (injury due to rapid cooling), which is seen during cooling at 10 degrees C/min to -14 degrees C, but not during slow cooling at 0.5 degrees C/min. One of the problems of using glycerol as a cryoprotectant stems from the high sensitivity of PMNs to dilution shock during the dilution or removal of glycerol.     

A comparative study of mouse embryo freeze-preservation including the examination of a thermoelectric freezing device

In Study 1 over 2000 4- to 8-cell mouse embryos were randomly pooled and assigned to 1 of 12 treatment groups. A 2 X 2 X 3 factorial design was used to analyze two types of cryoprotectant/post-thaw (PT) dilutions (dimethyl sulfoxide [Me2SO]/stepwise dilution versus glycerol/sucrose dilution), two storage containers (glass ampoules versus plastic straws), and three cooling treatments. Two commercial, controlled-rate freezing machines were examined, employing either nitrogen gas (Planer) or thermoelectric (Glacier) cooling. Embryos were cooled slowly (0.5 degrees C/min) to -35 or -80 degrees C and then cooled rapidly by transfer into liquid nitrogen (LN2). Thawed embryos were cultured for 24 hr after which developmental stage, post-thaw survival (PTS), embryo degeneration rate (EDR), quality grade (QG), and fluorescein diacetate viability grade (VG) were assessed. Overall, PTS and EDR were similar (P greater than 0.05) among the three freezing unit/plunge temperature treatments. Cumulative results of container and cryoprotectant/PT dilution treatments consistently demonstrated greater PTS, QG, and VG ratings and lower EDR values when embryos were frozen in ampoules using glycerol/sucrose dilution. Embryos treated with Me2SO/stepwise dilution were particularly sensitive to freezing damage when stored in plastic straws and plunged into LN2 at -35 degrees C. Study 2 was directed at determining whether Study 1 methods for diluting Me2SO-protected embryos markedly affected PTS rates. Post-thaw culture percentages were no different (P greater than 0.05) for four- to eight-cell Me2SO-treated embryos frozen in ampoules (using the forced-LN2 device), thawed, and diluted either conventionally in reduced concentrations of Me2SO or in the sucrose treatment normally accorded glycerolated embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterizing the ability of an ice recrystallization inhibitor to improve platelet cryopreservation

Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5–6% dimethyl sulfoxide (Me₂SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at −80 °C. Alternatively, platelets were frozen with 5–6% Me₂SO at −30 °C or with 3% Me₂SO at −80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me₂SO (p = 0.0461) but not at −30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me₂SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at −30 °C or with 3% Me₂SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at −30 °C or with 3% Me₂SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage.     

Drug metabolism and viability studies in cryopreserved rat hepatocytes

Rat hepatocytes were cryopreserved optimally by freezing them at 1 degrees C/min to -80 degrees C in cryoprotectant medium containing either 20% (v/v) dimethylsulfoxide (Me2SO) and 25% (v/v) fetal calf serum in Leibowitz L15 medium (Me2SO cryoprotectant) or 25% (v/v) vitrification solution (containing Me2SO, acetamide, propylene glycol and polyethylene glycol) in Leibowitz L15 medium (VS25). The VS25 solution was superior for maintaining viability during short-term storage (24-48 hr) but was slightly toxic during longer storage periods (7 days). Although thawed cells were 40-50% viable on ice after cryopreservation, their viability fell rapidly during incubation in suspension at 37 degrees C. This decline in viability occurred more rapidly after freezing in Me2SO cryoprotectant than in VS25 and was associated with extensive intracellular damage and cell swelling. The loss in viability at 37 degrees C does not appear to be due to ice-crystal damage as it occurred in cells stored at -10 degrees C (above the freezing point of the cryoprotectants) and it may be due to temperature/osmotic shock. Both cryoprotectant media were equally efficient at preserving enzyme activities in the hepatocytes over 7 days at -80 degrees C. Cytochrome P450 and reduced glutathione content and the activities of the microsomal enzymes responsible for aminopyrine N-demethylation and epoxide hydrolysis were well maintained over 7 days storage. In contrast, the cytosolic enzymes glutathione-S-transferase and glutathione reductase were markedly labile during cryopreservation. Cytosolic enzymes may be more susceptible to ice-crystal damage, whereas the microsomal membrane may protect the enzymes which are embedded in it.     

Retention of endothelium-dependent vasodilatory responses in canine coronary arteries following cryopreservation

In the present study, the cryoprotective effect of dimethyl sulfoxide (Me2SO) and fetal calf serum (FCS) on coronary endothelium and endothelium-dependent relaxation (EDR) responses was studied in isolated canine coronary arteries following cryostorage at -75 degrees C. Compared to the freshly isolated coronary arteries, the EDR responses to acetylcholine, thrombin, and calcium ionophore were not significantly altered following 1 day storage at -75 degrees C in the presence of 1.8 M Me2SO and 20% FCS. Prolonged cold storage to 7 days, however, resulted in a slight, but significant, rightward shift of the concentration-response curves of acetylcholine and thrombin, but not calcium ionophore. The maximum relaxant response after 7-day cryostorage was 80 to 85% of fresh controls. Omission of FCS from the cryostorage incubation medium further accentuated the loss of EDR responses to all three endothelium-dependent vasodilators tested. Scanning electron microscopic examinations of the intimal surface of the Me2SO and FCS cryostored canine coronary arteries confirmed the preservation of intimal endothelial cells following 1 or 7 days of storage at -75 degrees C, while significant patches of loss of endothelial cells were observed in the arteries cryostored only in the presence of Me2SO. No significant inhibitory effect of cryostorage was observed for the direct, endothelium-independent relaxation induced by isoproterenol, regardless of the presence or absence of FCS. These results demonstrate that slow freezing of canine coronary arteries to -75 degrees C in Krebs-Henseleit solution containing Me2SO and FCS provides good preservation of the vascular smooth muscle function and endothelium-dependent vasodilatory responses.     

Intracellular ice formation during the freezing of hepatocytes cultured in a double collagen gel.

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

Freezing of stored, chilled dog spermatozoa

The aims of this study were to find out if dog spermatozoa can be stored chilled for 1 or 2 days prior to freezing without a deterioration in post-thaw vitality and longevity, and to compare two extenders; the Uppsala Equex-2 (UE-2) and a TRIS egg yolk extender (EYT). Pooled dog semen was frozen immediately after collection, or was extended and stored at 4 degrees C for 1 or 2 days before freezing. Sperm motility and acrosome integrity were evaluated before freezing and for 6h post thaw at 38 degrees C, while sperm plasma membrane integrity was evaluated post thaw. There were no effects of pre-freeze storage time or extender on post-thaw motility or plasma membrane integrity, but a significant effect of extender (P < 0.0153) on post-thaw acrosomal integrity was found, UE-2 being better than EYT. There was a significant (P < 0.0001) negative effect of post-thaw storage time on acrosome integrity, but this was not influenced by pre-freeze storage time or extender. In conclusion, we found that dog spermatozoa can be frozen after 1 or 2 days of cold storage without significant deterioration in post-thaw motility, acrosome integrity or sperm plasma membrane integrity compared to when frozen immediately after collection. The UE-2 extender was superior to the EYT extender for freezing of cold stored dog spermatozoa.     

Manifestations of cell damage after freezing and thawing

The nature of the primary lesions suffered by cells during freezing and thawing is unclear, although the plasma membrane is often considered the primary site for freezing injury. This study was designed to investigate the nature of damage immediately after thawing, by monitoring several functional tests of the cell and the plasma membrane. Hamster fibroblasts, human lymphocytes, and human granulocytes were subjected to a graded freeze-thaw stress in the absence of cryoprotective compound by cooling at -1 degree C/min to a temperature between -10 and -40 degrees C, and then were either warmed directly in water at 37 degrees C or cooled rapidly to -196 degrees C before rapid warming. Mitochondrial function in the cells was then assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), fluorescein diacetate (FDA), colony growth, and osmometric response in a hypertonic solution. Cells behaved as osmometers after cooling at -1 degree C/min to low temperatures at which there were no responses measured by other assays, indicating that the plasma membrane is not a primary site for injury sustained during slow cooling. These results also indicate that the FDA test does not measure membrane integrity, but reflects the permeability of the channels through which fluorescein leaves the cells. Fewer cells could respond osmotically after cooling under conditions where intracellular freezing was likely, implying that the plasma membrane is directly damaged by the conditions leading to intracellular freezing. A general model of freezing injury to nucleated mammalian cells is proposed in which disruption of the lysosomes constitutes the primary lesion in cells cooled under conditions where the cells are dehydrated at low temperatures.     

Effects of dimethyl sulfoxide and glycine on cryopreservation induced damage of plasma membranes and mitochondria to striped bass (Morone saxatilis) sperm

Intact plasma membrane and functional mitochondria are important attributes for the fertilization capacity of fish sperm. In the present study, dimethyl sulfoxide (Me(2)SO) and glycine were investigated in an effort to improve plasma membrane integrity and mitochondrial function in cryopreserved striped bass (Morone saxatilis) sperm. Prior to freezing, no concentration of Me(2)SO (2.5, 5 or 10%) was found to affect (P>0.05) the integrity of plasma membranes after sperm were exposed for 10 min. However, mitochondrial function decreased (P>0.05) with increasing Me(2)SO concentration. Both fluorescent staining and microscopic examination of the ultrastructure of post-thaw plasma membranes indicated that with increasing Me(2)SO concentration, plasma membranes were better protected, and 10% Me(2)SO had the highest percentage of sperm with plasma membranes intact. However, sperm mitochondrial function decreased (P>0.05) with increasing Me(2)SO concentration. The inverse relationship between plasma membrane integrity and mitochondrial function, given the Me(2)SO concentration, suggests that care must be taken to select Me(2)SO concentration that will maximize the protection of both plasma membranes and mitochondrial function. The addition of glycine to the cryomedia increased (P<0.05) the percentage of sperm with post-thaw functional mitochondria and ATP content. However glycine did not provide (P<0.05) protection to post-thaw plasma membrane integrity. The highest percentage of sperm with both intact plasma membranes and functional mitochondria was obtained with 7.5% Me(2)SO and 75 mM glycine.     

Factors to consider in the assessment of viability of cryopreserved islets of Langerhans

With the development of techniques for the isolation and transplantation of pancreatic islets of Langerhans, research has been directed toward low-temperature storage of islets as a means of preservation. For successful islet cryopreservation several factors must be considered. In these studies we have investigated the effects of the cryoprotectant dimethyl sulfoxide (Me2SO) on islet function in the absence of freezing. We have found that Me2SO pretreatment can inhibit subsequent glucose-induced insulin release, but this effect can be minimized by hypothermic exposure to the cryoprotectant using a stepwise addition and dilution protocol for treatment. By studying islet function after freezing and thawing, we have found also that a slow cooling rate (0.3 degrees C/min) results in optimal survival and that islet function can be significantly improved by increasing the duration of post-thaw culture. The results of these studies address only a few of the many questions that need to be answered before clinical application of cryopreserved islet transplantation occurs.     

Freezing without surrounding cryomedium preserves the endothelium and its function in human internal mammary arteries

PURPOSE: Cryopreserved human blood vessels may become important tools in bypass surgery. Optimal cryopreservation of an arterial graft should, therefore, preserve both histological and physiological characteristics of smooth muscle and endothelium comparable to the unfrozen artery. METHODS: Rings from human internal mammary arteries (IMA) were investigated in vitro either unfrozen or after immersion into a cryomedium (RPMI 1640 containing 1.8M Me2SO and 0.1M sucrose) and cryostorage with and without surrounding medium. RESULTS: In unfrozen IMA, neither contractile responses to noradrenaline (NA) nor endothelium-dependent relaxant responses to acetylcholine (ACH) was modified after exposure of the IMA to cryomedium or during activation of protein kinase C by phorbol-12,13-dibutyrate (PDBu). Exposure to cryomedium with gradually increasing Me2SO content before starting the cooling process did not improve the post-thaw functional activity of the artery. Optimal post-thaw recovery of contractile responses to NA and PGF(2alpha) was observed after freezing at a speed of -1.2 and -3 degrees C/min in arteries stored with and without surrounding cryomedium. Compared to unfrozen controls, the ACH-induced endothelium-dependent relaxation during active tone induced by 3 microM PGF(2alpha) reached 16 and 56% after freezing with and without surrounding medium. All functional data were reflected by electron microscopy images showing considerably better preservation of the endothelial layer after freezing without medium. CONCLUSION: Freezing of human arteries at a mean cooling rate of -3 degrees C/min and storage without surrounding medium offers the prospect of optimal preservation of both smooth muscle and endothelial function in cryopreserved human IMA.     

Frozen/thawed platelets: importance of osmotic tolerance and platelet subpopulations.

Me2SO cryopreserved platelets circulate in 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 PGI2 is 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.     

The potential for cryopreserving larvae of the sea urchin, Evechinus chloroticus

Larvae of the sea urchin, Evechinus chloroticus, at varying stages of development, were assessed for their potential to survive cryopreservation. Ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), at concentrations of 1-2 M, were evaluated as cryoprotectants (CPAs) in freezing regimes initially based on methods established for freezing larvae of other sea urchin species. Subsequent work varied cooling rate, holding temperature, holding time, and plunge temperature. Ethylene glycol was less toxic to larvae than Me2SO. However, no larvae survived freezing and thawing in EG. Larvae frozen in Me2SO at the gastrula stage and 4-armed pluteus stage regained motility post-thawing. The most successful freezing regime cooled straws containing larvae in 1.5 M Me2SO from 0 to -35 degrees C at 2.5 degrees C min(-1), held at -35 degrees C for 5 min, then plunged straws into liquid nitrogen. Motility was high 2-4 h post-thawing using this regime but decreased markedly within 24 h. Some 4-armed pluteus larvae that survived beyond this time developed through to metamorphosis and settled. Different Me2SO concentrations and supplementary trehalose did not improve long-term survival. Large variation in post-thaw survival was observed among batches of larvae produced from different females.     

Determination of human platelet membrane permeability coefficients using the Kedem-Katchalsky formalism: estimates from two- vs three-parameter fits.

Attempts to cryopreserve human blood platelets have resulted in poor postthaw survival rates and have been inadequate for routine clinical application. As a result, most blood banks maintain platelets in nonfrozen solutions. Using this approach, platelets can be stored for only about 5 days and are then discarded. This situation greatly limits the use of platelet transfusion in clinical practice. Information regarding fundamental cryobiological characteristics can be applied to predict platelet response to cryoprotective agent (CPA) addition/removal and to cooling/warming. Methods can then be engineered to optimize cryopreservation procedures, thereby minimizing platelet damage and maximizing postthaw recovery. It was therefore the purpose of this study to determine some of the necessary biophysical parameters required for this process: (i) plasma membrane hydraulic conductivity (Lp), (ii) cryoprotectant solute permeability coefficient (Ps), (iii) the associated reflection coefficient (sigma), and (iv) their activation energies. The CPAs studied included dimethyl sulfoxide (Me2SO) and propylene glycol at 1.5 M concentration. Permeability was measured at 22, 10, and 4 degrees C using a modified Coulter counter in conjunction with a water-jacketed beaker system for temperature regulation. The Kedem-Katchalsky formalism was used to estimate the parameters using: (1) a three-parameter fit and (2) a two-parameter fit in which a noninteracting value of sigma was calculated. Two-parameter estimates were in closer agreement with previously published values, and these were used in a model to simulate addition and removal of 0.64 M (5%) and 1.0 M (7.8%) Me2SO, the most common CPA currently used in empirically determined platelet cryopreservation protocols.