Water crystallization within rat precision-cut liver slices in relation to their viability

This study examined whether tissue vitrification, promoted by partitioning within the tissue, could be the mechanism explaining the high viability of rat liver slices, rapidly frozen after preincubation with 18% Me2SO or VS4 (a 7.5 M mixture of Me2SO, 1,2-propanediol, and formamide with weight ratio 21.5:15:2.4). To achieve this, we first determined the extent to which crystallization or vitrification occurred in cryoprotectant solutions (Me2SO and VS4) and within liver slices impregnated with these solutions. Second, we determined how these events were related to survival of slices after thawing. Water crystallization was evaluated by differential scanning calorimetry and viability was determined by histomorphological examination of the slices after culturing at 37 degrees C for 4 h. VS4-preincubated liver slices indeed behaved differently from bulk VS4 solution, because, when vitrified, they had a lower tendency to devitrify. Vitrified VS4-preincubated slices that were warmed sufficiently rapid to prevent devitrification had a high viability. When VS4 was diluted (to 75%) or if warming was not fast enough to prevent ice formation, slices had a low viability. With 45% Me2SO, low viability of cryopreserved slices was caused by cryoprotectant toxicity. Surprisingly, liver slices preincubated with 18% Me2SO or 50% VS4 had a high viability despite the formation of ice within the slice. In conclusion, tissue vitrification provides a mechanism that explains the high viability of VS4-preincubated slices after ultrarapid freezing and thawing (>800 degrees C/min). Slices that are preincubated with moderately concentrated cryoprotectant solutions (18% Me2SO, 50% VS4) and cooled rapidly (100 degrees C/min) survive cryopreservation despite the formation of ice crystals within the slice.     

Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification

Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200 degrees C/min) using 18% Me(2)SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3-4h of incubation at 37 degrees C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me(2)SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50-80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.     

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.     

Cryopreservation of Holospora undulata, a bacterial parasite of the ciliate Paramecium caudatum

We investigated cryopreservation of horizontal transmission stages of Holospora undulata, a micronucleus-specific bacterial parasite of Paramecium caudatum. Unlike in previous studies on related Holospora species, protocols using glycerol as cryoprotectant failed entirely. In contrast, freezing with dimethyl sulfoxide (Me2SO) conserved infectiousness of nearly all replicate inocula, although infection success was considerably lower than that of fresh inocula. Infection probability was enhanced by increasing the Me2SO concentration from 5 to 10%, and by freezing at -196 degrees C rather than -80 degrees C. Prolonged storage of up to 3 months had no significant effect on the viability of the inocula.     

Cryopreservation of tissue-engineered dermal replacement in Me2SO: Toxicity study and effects of concentration and cooling rates on cell viability

Cryopreservation of tissue-engineered human dermal replacement plays an important role in skin tissue engineering and skin banking. With the inspection of electronic scanning microscope and viability evaluation by Trypan Blue staining assay and the tetrazolium salt, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, this study investigated the toxicity of Me(2)SO to dermal fibroblasts and effects of cryoprotectant concentration and cooling rate on the viability of dermal replacement. The results demonstrated that the Me(2)SO toxicity to fibroblasts was affected by the exposure time, temperature, and concentration. Furthermore adding cryoprotectant solution at low temperature of 4 degrees C significantly reduced the toxic effect on the tissue-engineered dermal equivalent. An optimal cryopreservation protocol consisting of cooling rate at 1 degrees Cmin(-1) in 10% (V/V) Me(2)SO was derived, with the viability of studied dermal equivalent treated by this protocol being 75% of that of fresh control. The micrograph obtained by electronic scanning microscope also confirmed this result.     

Ultrastructural changes in the MP3 neuron of the mollusk Lymnaea stagnalis after cryopreservation of the isolated brain

Investigation of a possibility of long-term storage of frozen (-196 degrees C) viable neurons and nervous tissue is one of the central present day problems. In this study ultrastructural changes in neurons of frozen-thawed snail brain were examined as a function of time. We studied the influence of cryopreservation, cryoprotectant (Me2SO), cooling to 4-6 degrees C, and a prolonged incubation in physiological solution at 4-6 degrees C on dictyosomes of Golgi apparatus, endoplasmic reticulum (ER) cisternae and mitochondria. It has been found that responses of these intracellular structures of cryopreserved neurons to the above influences are similar: dissociation of Golgi dictyosomes, swelling of endoplasmic reticulum cisternae and mitochondrial cristae. Both freezing-thawing and cryoprotectant were seen to cause an increase in the number of lysosomes, liposomes, myelin-like structures, and to form large vacuoles. The structural changes in molluscan neurons caused by cryopreservation with Me2SO (2 M) were reversible.     

Survival of corneal endothelium following exposure to a vitrification solution

Corneal endothelium, a monolayer of cells lining the inner surface of the cornea, is particularly susceptible to freezing injury. Ice formation damages the structural and functional integrity of the endothelium, and this results in a loss of corneal transparency. Instead of freezing, an alternative method of cryopreservation is vitrification, which avoids damage associated with ice formation. Vitrification at practicable cooling rates, however, requires exposure of tissues to very high concentrations of cryoprotectants, and this can cause damage through chemical toxicity and osmotic stress. The effects of a vitrification solution (VS1) containing 2.62 mol/liter (20.5%, w/v) dimethyl sulfoxide, 2.62 mol/liter (15.5%, w/v) acetamide, 1.32 mol/liter (10%, w/v) propane-1,2-diol, and 6% (w/v) polyethylene glycol were studied on corneal endothelium. Endothelial function was assessed by monitoring corneal thickness during 6 hr of perfusion at 35 degrees C with a Ringer solution supplemented with glutathione and adenosine. Various dilutions of the vitrification solution were introduced and removed in a stepwise manner to mitigate osmotic stress. Survival of endothelium after exposure to VS1 or a solution containing 90% of the cryoprotectant concentrations in VS1 (90% VS1) was dependent on the duration of exposure, the temperature of exposure, and the dilution protocol. The basic dilution protocol was performed at 25 degrees C: corneas were transferred from 90% VS1 or VS1 into 50% VS1 for 15 min, followed by 25% VS1 for 15 min and finally into isosmotic Ringer solution. Using this protocol, corneal endothelium survived exposure to 90% VS1 for 15 min at -5 degrees C, but 5 min in VS1 at -5 degrees C was harmful and resulted in some very large and misshapen endothelial cells. This damage was not ameliorated by using a sucrose dilution technique; but endothelial function was improved when the temperature of exposure to VS1 was reduced from -5 to -10 degrees C. Exposure to VS1 for 5 min at -5 degrees C was well tolerated, however, when the temperature of the first dilution step into 50% VS1 was reduced from 25 to 0 degree C. The large, misshapen cells were not observed under these conditions nor after exposure to VS1 at -10 degrees C. These results suggested that damage was the result of cryoprotectant toxicity rather than osmotic stress. Thus, corneal endothelium survived exposure to two solutions of cryoprotectants, namely, 90% VS1 and VS1, that were sufficiently concentrated to vitrify. Whether corneas can be cooled fast enough in these solutions to achieve vitrification and warmed fast enough to avoid devitrification remains to be determined.     

Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy

Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 degree C, and super-cooling in the absence of ice. Cryoinjuries were however induced by: osmotic shock resulting from excessive cryodehydration, intracellular ice, and fracturing of the frozen medium on thawing. Suboptimal cooling at -0.3 degrees C min(-1) to -60 degrees C and osmotic shock invariably resulted in damage to the organism's pellicle and osmoregulatory system causing, a significant (P > 0.005) increase in cell size. Cell damage was not repairable and led to death. The responses of E. gracilis to cryopreservation as visualised by flow-cytometry and cryomicroscopy assisted the development of an improved storage protocol. This comprised: cryoprotection with methanol [10%(v/v)] at 0 degree C, cooling at 0.5 degrees C min(-1) to -60 degrees C, isothermal hold for 30 min, and direct immersion in liquid nitrogen. Highest post-thaw viability (>60%) was obtained using two-step thawing, which involved initial slow warming to -130 degrees C followed by relatively rapid warming (approximately 90 degrees C min(-1)) to ambient temperature (ca. 25 degrees C).     

A simple cryopreservation method for the maintenance of cell viability and mechanical integrity of a cultured cartilage analog

A method for cryopreserving a 100-microm-thick sheet of tissue produced by cultured rabbit chondrocytes has been developed. The method maintains cell viability and avoids tissue fracture and degradation of mechanical properties. A slow-freeze, fast-thaw procedure with 2 M Me(2)SO as the cryoprotectant resulted in no tissue fracture and approximately 90% viable cells after storage in culture flasks at -80 degrees C. The cells in the retrieved tissue remained responsive to IL-1beta, and tensile and fracture toughness properties of the tissue were not degraded by cryopreservation.     

Preservation of taenia coli by freezing and storage at -196 degrees C

Some damaging effects that occur during cryopreservation by freezing to -196 degrees C have been evaluated in rabbit taenia coli by analyzing the proportional recovery of acetylcholine- and histamine-induced maximal contractions. Dimethyl sulfoxide (Me2SO) 10 v/v% was used as the cryoprotectant; it reversibly abolishes spontaneous contractility even after incubation at 37 degrees C during 2 hr. Programmed freezing at 0.6 degrees C/min with compensation for the latent heat of fusion and warming at 35 degrees C/min proved to be slightly superior to programmed cooling without compensation and slower warming. The degree of functional recovery was comparable after either abrupt or stepwise removal of Me2SO. Freeze-thawing resulted in a significant reduction of contractile force in each buffer solution tested, and acetylcholine-induced contractility was always better preserved than histamine-induced contractility. The best preservation (approximately 65%) was obtained in a potassium-rich buffer solution. The absence of calcium and magnesium from the incubating medium had no influence, whereas the presence of EDTA significantly affected functional recovery. It is difficult to compare our results with those reported by others because of multiple methodological differences. However, it seems that previous results can be improved by changing the freezing rate and the composition of the incubating and cryoprotecting medium.     

Cryopreservation of chum salmon blastomeres by the straw method

In order to preserve genetic resources of chum salmon, Oncorhynchus keta, optimum conditions for cryopreservation of isolated blastomeres were investigated. Survival rates under various conditions were compared: the nature and the concentration of cryoprotectants before and after freezing, the seeding temperature, and the developmental stages of donor embryos. Isolated blastomeres immersed for 30 min in Eagle's MEM containing both a cryoprotectant and 10% fetal bovine serum (FBS) at 10 degrees C were transferred into a straw and frozen at 1 degrees C/min to -30 degrees C by a programmable freezer before being plunged into liquid nitrogen. Ice seeding was carried out at -5 to -15 degrees C. Frozen blastomeres were thawed in water at 15 degrees C. Blastomeres cryopreserved with MEM containing 10% dimethyl sulfoxide (Me(2)SO) and 10% FBS (10% Me(2)SO/MEM10) showed higher survival rates than those cryopreserved with MEM containing 10% FBS and 10% glycerol, ethyleneglycol, 1, 2-propanediol, or sucrose. Blastomeres treated with 10% Me(2)SO/MEM10 showed higher survival rates than those treated with MEM containing only 10% Me(2)SO. Blastomeres seeded above -10 degrees C showed higher survival rates than non-seeded ones. Frozen blastomeres at advanced stages demonstrated high survival rates. Blastomeres cryopreserved under optimum conditions showed survival rates of 59.3+/-2.8%. These results indicate that 10% Me(2)SO/MEM10 is a suitable cryoprotectant medium to cryopreserve chum salmon blastomeres, that seeding should be carried out above -10 degrees C on pre-freezing, and that blastomeres at the blastula stage should be used as material.     

Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae)

The cryopreservation of algae could prevent genetic drift and minimize labor costs compared to the current method of maintenance and subculturing. Clear, simple protocols for cryopreservation of marine microalga, Nannochloropsis oculata were developed and cryoprotectant choice and concentration optimized. The viability of the microalga was assessed directly after thawing, and algal concentration was measured after 2-30 days of growth. Five cryoprotectants (dimethyl sulphoxide, Me2SO; ethylene glycol, EG; glycerol, Gly; methanol, MeOH; and propylene glycol, PG) at five concentrations (10, 20, 30, 40, and 50%; v/v) were evaluated to determine the toxicity of various cryoprotectants to N. oculata. The toxicity of cryoprotectant (Me2SO, EG, MeOH, and PG) was observed only at higher concentrations of CPAs: > 20% for EG, > 30% for Me2SO and methanol, and > 40% for PG. Direct freezing of algae in liquid nitrogen resulted in a severe loss of viability and a modified cryopreservation protocol proved to be more appropriate for the preservation of N. oculata. Cryopreservation protocols developed and tested in the present study might be applied to cryopreserving other strains, or species, in this genus.     

Transplantation and in vitro perifusion of rat islets of Langerhans after slow cooling and warming in the presence of either glycerol or dimethyl sulfoxide

The cryoprotectants dimethyl sulfoxide (Me2SO) and glycerol have been used for the cryopreservation of fetal rat pancreases but only Me2SO has been reported for the cryopreservation of adult rat islets. Since glycerol may be preferred to Me2SO for clinical use, this study was undertaken to compare the effectiveness of these cryoprotectants during the slow cooling of isolated adult rat islets. Islets of Langerhans prepared from the pancreases of WAG rats by collagenase digestion were stored at -196 degrees C after slow cooling (0.3 degrees C/min) to -70 degrees C in the presence of multimolar concentrations of either Me2SO or glycerol. Samples were rewarmed slowly (approximately 10 degrees C/min) and dilution of the cryoprotectant was achieved using medium containing sucrose. Function was assessed by determination of the time course of the glucose-induced insulin release during in vitro perifusion at 37 degrees C and also by isograft transplantation. Transplants were carried out by intraportal injection of a minimum of 1700 frozen and thawed islets into streptozotocin-induced diabetic recipients and tissue function was assessed by monitoring blood glucose levels and body weight changes. Without exception the islets frozen and thawed in the presence of glycerol failed to reduce high serum glucose levels of recipient rats and in vitro dynamic release curves showed to demonstrate a glucose-sensitive insulin release pattern. Reversal of the diabetic conditions was achieved in two of five animals receiving islets which had been frozen and thawed with 2 M Me2SO; and in one of three animals receiving islets cryopreserved with 3 M Me2SO. Nevertheless, perifusion studies showed that the pattern of insulin secretion from groups of cryopreserved islets which did show an ability to secrete insulin was atypical compared with that of untreated controls, suggesting that the tissue was altered or damaged in some way.     

Cryopreservation of cultured periosteum: effect of different cryoprotectants and pre-incubation protocols on cell viability and osteogenic potential

Evidence has accumulated that periosteal cells have a great potential to regenerate bone. We have demonstrated that cultured periosteum (CP) in membrane form is an effective device to regenerate alveolar bone. To increase the availability of CP in a clinical environment, an effective cryopreservation protocol for CP has been developed. In this study, three different cryoprotectants (Me(2)SO, glycerol, and ethylene glycol) were used. The effect on cell viability of pre-incubation temperature, pre-incubation time, and agitation during incubation was investigated. Samples were stored at -196 degrees C for 10 days. Cell viability was assessed by a colorimetric cell viability assay using a tetrazolium salt, and the assay results were confirmed by confocal laser scanning microscopy after staining with a combination of calcein AM and ethidium homodimer-1. The activity of the cells after thawing was assessed by alkaline phosphatase assay. To assess the osteogenic potential of cryopreserved CP, the CP was grafted to calvarial defects in athymic rats. The greatest cell viability was obtained in the group equilibrated at 37 degrees C for 30 min with Me(2)SO, under agitation, showing 63.3 +/- 10.5% recovery. After cryopreservation, the cell growth of surviving cells was identical when Me(2)SO was used as a cryoprotectant. Alkaline phosphatase (ALP) activity was maintained in the groups cryopreserved with Me(2)SO and glycerol. The transplantation experiment showed that the calvarial defects were completely closed by grafting cryopreserved CP, which demonstrates that the osteogenic property of CP was well maintained. An efficient cryopreservation protocol for CP has been developed and this will provide a convenient and effective treatment option for bone regeneration in clinics.     

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.     

Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation

Little work in the literature focuses on the cryopreservation of primary hepatocytes as monolayer cultures, yet this technique offers many distinct advantages over other cryopreservation systems, including high recovery, high post-thaw nutrient penetration, and low numbers of trapped dead cells. This article investigates the cryopreservation of primary rat hepatocytes at -78 degrees C attached as monolayers to collagen coated culture dishes, and describes efforts to increase post-thaw viability and function through manipulation of the freeze/thaw protocol. Different concentrations of foetal calf serum (FCS) with 10% (v/v) dimethyl sulphoxide (ME2SO) were tested as cryopreservation media, and high cryoprotectant serum levels were found to be important in maintaining membrane integrity and function in the cryopreserved rat hepatocyte monolayer cultures. Cultures cryopreserved with 90% (v/v) FCS plus 10% (v/v) ME2SO maintain 79.7+/-6.5% of the monolayer area as viable cells with normal morphology (by image analysis), 112.7+/-14.2% protein concentration, 55.4+/-4.2% carboxyfluorescein diacetate de-acetylation, 27.2+/-7.5% kaempherol glucuronidation (a measure of UDP-glucuronosyl transferase activity), and 39.3+/-7.3% testosterone hydroxylation (a measure of cytochrome P-450 activity) compared with non-cryopreserved controls. This method of cryopreservation may provide a simple, convenient means of long-term storage of hepatocytes for in vitro metabolism studies.     

Exposure to dimethyl sulfoxide at 37 degrees C prior to freezing significantly improves the recovery of cryopreserved hybridoma cells

Standard cryopreservation protocols recommend the use of dimethyl sulfoxide (Me2SO) at moderate temperatures only (room temperature, 4 degrees C) due to its toxicity which appears to be potentiated by warm temperatures. In the present study, we asked whether a transient increase in temperature during membrane sealing of cryovials affects the cell viability. We show here that the cell viability of hybridoma cells and Schwann cells was not reduced following membrane sealing of cryotubes. On the contrary, incubation of cells at 37 degrees C in Me2SO-containing medium prior to freezing significantly stimulated the viability of cryopreserved hybridoma cells, whereas the viability of Schwann cells remained unaltered. We conclude that the exposure of cells to Me2SO at elevated temperatures does not necessarily reduce cell viability and that contrary to this, cell type-specific, beneficial effects of Me2SO could be observed.     

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 fetal skin is improved by extracellular trehalose

In this study, we tested a non-permeating cryoprotectant, trehalose, in combination with dimethyl sulfoxide (Me(2)SO) in the cryopreservation of human fetal skin and compared it to Me(2)SO and glycerol, protocols that are routinely used by skin banks. The viability of fetal skin from four groups (fresh, and cryopreserved with glycerol, Me(2)SO, or trehalose/Me(2)SO) were evaluated using an in vitro membrane integrity assay and by transplantation to immunodeficient mice. The membrane integrity assay showed a 90% integrity in fresh, unfrozen fetal skin. The number of intact cells dropped to 23 and 44% in fetal skin cryopreserved with glycerol and Me(2)SO, respectively. When trehalose was added to the cryopreservation medium containing Me(2)SO, the membrane integrity rose to 65%. When transplanted to immunodeficient mice, fetal skin cryopreserved with trehalose/Me(2)SO showed a graft performance indistinguishable from fresh unfrozen fetal skin and strikingly better graft take than that of fetal skin cryopreserved with Me(2)SO or glycerol only. These results suggest that cryopreservation protocols routinely used the skin banks can be improved by combining sugars such as trehalose with a permeating cryoprotectant.     

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)