Comparison of three methods for human corneal cryopreservation that utilize dimethyl sulfoxide.

We compared endothelial cell survival in human corneas after cryopreservation by three methods that utilize dimethyl sulfoxide. Twenty-eight human cadaver corneas were cryopreserved by one of three methods, stored briefly over liquid nitrogen, thawed, cultured at 37 degrees C for 3 days, and fixed for scanning electron microscopy. Seventeen control corneas underwent identical cryoprotectant immersion and culture protocols but were not frozen. Endothelial photographs taken after 1 and 3 days of culture were analyzed. Endothelial cell losses in cryopreserved corneas by Methods 1, 2, and 3, respectively, were 36, 22, and 10% after 1 day of culture and 57, 36, and 27% after 3 days of culture. Cryopreservation by Method 3 had less cell loss than Methods 1 or 2 (P<0.02) but greater cell loss than the control corneas for Method 3 (P<0.001). No loss of cells occurred in the control corneas for Methods 1 and 3 but substantial cell loss (26%) occurred in the control corneas for Method 2. Polymegethism and pleomorphism of the endothelial cells were seen in the corneas that lost cells. The endothelial cell loss of 10% seen after 1 day of culture in human corneas cryopreserved by Method 3 is similar to the loss that occurs during organ culture storage as currently used clinically and therefore would be acceptable for clinical use. After 3 days of culture, however, the cell loss had increased significantly to 27%. This additional decrease in cell number that occurs in culture may represent latent cryodamage and must be understood and overcome in vivo before the technique can be used clinically.     

Transplantation of cryopreserved human corneas in a xenograft model

An ideal model to test methods of corneal storage for transplantation would simulate the environment of the grafted human cornea and predict the success of clinical corneal transplants (human to human). In this study, we tested such a model, the corneal xenograft (human to cat). Nine pairs of human corneas were transplanted into both eyes of nine recipient cats. One cornea of each pair was cryopreserved at -196 degrees C in 2.5 M dimethyl sulfoxide while the other was stored in preservative medium at 4 degrees C (control) for 6 +/- 2 (mean +/- SD) days before transplantation. One week after transplantation, the cats were euthanized and the eyes were examined. Three of the grafts (all cryopreserved) were clinical failures and showed no survival of donor corneal endothelial cells on scanning electron microscopy. The remaining six pairs of grafts were examined with a specular microscope and showed endothelial cell losses of 48 +/- 16% in cryopreserved and 8 +/- 16% in control corneas (p < 0.05). This survival is similar to survival in an earlier corneal perfusion model. The nine cryopreserved grafts were thicker than the control grafts, had fewer surviving keratocytes in the central stroma, and had more apoptotic central keratocytes (TUNEL assay). This failure rate in cryopreserved corneas clearly shows that this technique of cryopreservation was not adequate for clinical use. The corneal xenograft model can be used to study cellular survival and apoptosis in vivo after preservation as well as to test new methods of corneal preservation before initiating clinical trials.     

Transient outwardly rectifying potassium channel in the rabbit corneal endothelium

Summary Ionic currents from freshly dissociated rabbit corneal endothelial cells were examined using patch-clamp technology and a perforated patch technique. Whole-cell current recordings revealed a transient outward K⁺-selective current that was blockable in a dose-dependent manner by 4-aminopyridine (4-AP) and quinidine. This current is similar to the A-type current present in many excitable cells and is the first reported instance of such a current in any epithelial cell type. In addition to the transient current, an outwardly rectifying nonselective cation current was also observed. This current is also blocked by quinidine.To examine the possible role of these currents in the stromal volume regulatory function of the endothelium, corneas were perfused under a specular microscope with a glutathionebicarbonate Ringer's solution (GBR) or GBR plus either 1 mM quinidine or 10 mM 4-AP. For quinidine perfusions, control corneas swelled at a rate of 6 m/hr, while quinidine-perfused corneas swelled at a rate of 48 m/hr. For 4-AP perfusions, control corneas deswelled at a rate of –2 m/hr, while 4-AP perfused corneas swelled at a rate of 24 m/hr. One possible mechanism of the stromal swelling induced by these K⁺ channel blockers may be the result of loss of the K⁺ recycling pathway necessary for proper Na⁺/K⁺ ATPase function.We would like to thank Dr. William Bourne for the use of his specular microscopy corneal perfusion apparatus and Helen Hendrickson for her technical assistance. This work was supported by NIH grants EY06206, EY03282, EY06005, and an unrestricted award from Research to Prevent Blindness.     

Cell cultures from cryopreserved human lung tissue.

To assess gene induction in primary human fibroblasts, we have developed a method for cryopreservation of lung biopsies in liquid nitrogen. Fresh biopsies (n = 10) were chopped into 5 x 5 mm pieces and transferred into an ice-cold freezing medium. Biopsies were kept on ice for 15 min, followed by further cooling of the tissue to -70 degrees C. With this method, lung biopsies were preserved for more than 1 year before they were used for generating cell cultures. There was no significant difference in the biological responsiveness of fibroblasts generated from immediately cultured lung biopsies compared with those from cryopreserved tissue. The doubling rate of fibroblasts from fresh tissue was 23.6 +/- 1.1 hr; compared to 23.5 +/- 1.5 hr for fibroblasts generated from cryopreserved tissue. PDGF-BB enhanced de novo synthesis of DNA 100 times, in both the immediately cultured fibroblasts and those generated from cryopreserved biopsies. Macrophages, dendritic cells and endothelial cells could also be recovered from cryopreserved lung tissue. This method permits long-term storage of lung tissue and the possibility of establishing primary cell lines from the same tissue at later times without appreciable changes in their cellular biological characteristics.     

Postthaw viability of precultured hepatocytes

Hepatocytes are being studied for a wide variety of applications, including drug metabolism studies, gene therapy, and use in liver-assist devices for temporary liver support. The ability to cryopreserve isolated hepatocytes would permit the pooling of cells to reach the required therapeutic coordination of the cell supply with patient care regimes and the completion of safety and quality-control testing. The objective of this investigation was to develop a method of cryopreserving isolated hepatocytes that will retain high levels of function and facilitate the use of the cells in different applications. Freshly isolated hepatocytes were cultured in a spinner flask for different periods of time, up to 48 h. The cells were cryopreserved by use of a range of solution concentrations and cooling rates. For fresh, nonfrozen hepatocytes precultured for 24 h prior to being plated on collagen, the albumin secretion rate was 0.88 +/- 0.62 mg/ml/h. When the cells were precultured for 24 h, frozen in a solution containing 10% Me2SO with a cooling rate of 1 degrees C/min, thawed, plated on collagen, and cultured, the albumin secretion rate was 0.21 +/- 0.24 microg/ml/h. In contrast, freshly isolated hepatocytes cryopreserved without preculture and cultured on collagen had an albumin secretion rate of 0.07 +/- 0.08 mg/ml/h. The influences of different solution compositions and cooling rates on postthaw function of precultured hepatocytes were also determined. These results indicate that the use of a preliminary culture step prior to cryopreservation can enhance the postthaw function of hepatocytes.     

Sperm cryopreservation affects postthaw motility, but not embryogenesis or larval growth in the Brazilian fish Brycon insignis (Characiformes)

Sperm cryopreservation is an important method for preserving genetic information and facilitating artificial reproduction. The objective was to investigate whether the cryopreservation process affects postthaw sperm motility, embryogenesis, and larval growth in the fish Brycon insignis. Sperm was diluted in methyl glycol and Beltsville Thawing solution, frozen in a nitrogen vapor vessel (dry shipper) and stored in liquid nitrogen. Half of the samples were evaluated both subjectively (% of motile sperm and motility quality score—arbitrary grading system from 0 [no movement] to 5 [rapidly swimming sperm]) and in a computer-assisted sperm analyzer (CASA; percentage of motile sperm and velocity). The other half was used for fertilization and the evaluation of embryogenesis (cleavage and gastrula stages), hatching rate, percentage of larvae with normal development and larval growth up to 112 days posthatching (dph). Fresh sperm was analyzed subjectively (percentage of motile sperm and motility quality score) and used as the control. In the subjective analysis, sperm motility significantly decreased from 100% motile sperm and quality score of 5 in fresh sperm to 54% motile sperm and quality score of 3 after thawing. Under computer-assisted sperm analyzer evaluation, postthaw sperm had 67% motile sperm, 122 μm/sec of curvilinear velocity, 87 μm/sec of straight-line velocity and 103 μm/sec of average path velocity. There were no significant differences between progenies (pooled data) for the percentage of viable embryos in cleavage (62%) or gastrula stages (24%) or in the hatching rate (24%), percentage of normal hatched larvae (93%), larval body weight (39.8 g), or standard length (12.7 cm) at 112 days posthatching. Based on these findings, cryopreserved sperm can be used as a tool to restore the population of endangered species, such as B. insignis, as well as for aquaculture purposes, without any concern regarding quality of the offspring.     

Altered expression of aquaporins in bullous keratopathy and Fuchs' dystrophy corneas.

Corneas with edema-related diseases lose transparency, which causes significant vision loss. This study analyzed seven aquaporins (AQPs) in normal corneas, pseudophakic/aphakic bullous keratopathy (PBK/ABK) corneas, Fuchs' dystrophy corneas, keratoconus corneas, post-cataract surgery (PCS) corneas, and normal organ-cultured corneas. RNA levels for AQP1, AQP4, and beta2-microglobulin were measured by RT-PCR. AQP1 antibody localized to stromal cells of all corneas. PBK/ABK and Fuchs' dystrophy corneas had decreased endothelial cell staining compared with normal. AQP1 mRNA was found in whole corneas and cultured stromal fibroblasts but not in isolated epithelial cells. AQP3 staining was found in basal epithelial cells of the normal, Fuchs' dystrophy, and keratoconus corneas but throughout the entire epithelium of PBK/ABK corneas. AQP4 antibody localized to endothelial cells of all corneas and in stromal cells of PBK/ABK corneas. AQP4 mRNA was identified in whole human corneas. AQP5 was found in epithelial cells of all corneas. AQP0, AQP2, and AQP9 were not found in any corneas. Normal AQP distributions were found in PCS and organ-cultured corneas, although they showed signs of swelling. Our study demonstrates that AQP abnormalities are found in PBK/ABK corneas (decreased AQP1, increased AQP3 and AQP4) and Fuchs' dystrophy corneas (decreased AQP1). Although both have vision-disrupting corneal edema, the mechanisms of fluid accumulation may be different in each disease.     

Cryopreservation of rhesus macaque (Macaca mulatta) spermatozoa and their functional assessment by in vitro fertilization

Although spermatozoa from several species of nonhuman primates have been cryopreserved, there has been no report of success with rhesus macaque spermatozoa as judged by functional assays. Two Tris--egg yolk freezing media, TEST and TTE, which have been successfully used for cynomolgus macaque (Macaca fascicularis) spermatozoa, were compared for cryopreservation of spermatozoa from four rhesus macaques (Macaca mulatta). The postthaw motility (percentage and duration) of spermatozoa cryopreserved in TTE was much higher than that for spermatozoa cryopreserved in TEST. The function of sperm cryopreserved in TTE was evaluated by in vitro fertilization of oocytes collected from gonadotropin-stimulated prepubertal rhesus macaques. Of the inseminated oocytes, 82 +/- 13% were fertilized and 63 +/- 22 and 39 +/- 21% of the resulting zygotes developed into morulae and blastocysts, respectively. These results indicate that rhesus macaque spermatozoa can be effectively cryopreserved in TTE medium. This finding will facilitate the application of in vivo and in vitro assisted reproductive technologies in this species.     

Cell viability improves following inhibition of cryopreservation-induced apoptosis

A new concept in cryopreservation solution design was developed that focuses on the use of an intracellular-type, hypothermic maintenance medium coupled with additives that inhibit cryopreservation-induced apoptosis. HypoThermosol' (HTS), a hypothermic (4 degrees C) maintenance medium utilized in the long-term storage of cell, tissue, and organ systems, was tested for cryoprotective capability on a renal cell line (Madin-Darby Canine Kidney cells). HTS and HTS derivatives were tested against conventional cell culture medium (Dulbecco's Minimal Essential medium, DME) as the cryoprotectant carrier solution because (1) cells are exposed to an extended state of hypothermia during the freeze-thaw process, and (2) HTS is designed to protect cells exposed to a hypothermic state. Cells separately cryopreserved in either HTS or DME + 5% dimethyl sulfoxide (DMSO) yielded equivalent 24-h postthaw survival (approximately 30%) and 5-d recovery (approximately 90%). Cells cryopreserved in CryoStor CS 5, a HTS derivative containing 5% DMSO, yielded approximately 75% 24-h postthaw survival and recovery to 100% within 3 d. DNA gel electrophoresis was performed to determine the mechanisms of cell death contributing to cryopreservation failure. Cells preserved in DME (DMSO-free) died primarily through necrosis, whereas cells preserved in either DME + 5% DMSO, HTS, or CryoStor CS 5 died through a combination of apoptosis and necrosis. This observation led to the inclusion of an apoptotic inhibitor designed to improve cryopreservation outcome. MDCK cells cryopreserved in CryoStor CS 5 supplemented with an apoptotic inhibitor (Caspase I Inhibitor V), hereafter termed CryoStor CS 5N, resulted in a 24-h postthaw survival and recovery rate exceeding that of any other cryoprotective solution tested (85%). We conclude that: (1) the use of HTS (a dextran-based, intracellular-type solution) without DMSO can yield postthaw viability equivalent to that of standard DMSO-based cryopreservation methods, (2) postthaw viability can be significantly increased through the use of an intracellular-type solution in conjunction with DMSO, (3) the use of HTS allows for cryopreservation to be accomplished with reduced levels of cryoprotectants, and (4) the regulation of apoptosis is essential for the improvement of cryopreservation outcome.     

Optimization of a Method for the Cryopreservation of Rabbit Corneas: Attempted Application to Human Corneas

The purpose of the present study was to set up and test a cryopreservation method for long-term storage of human corneas. Therefore the freezing solution was optimized in 264 rabbit corneas by testing the type of cryoprotectant, its concentration, addition and dilution pattern and exposure temperature. Then rabbit corneas were frozen in the optimum solution at different cooling rates and thawed in a water bath at different temperatures. Eight human corneas were cryopreserved with the method showing optimum results in rabbit corneas and four additional corneas were used as controls. Endothelial viability was assessed after each step by vital staining and scanning electron microscopy. Best results after exposure of rabbit corneas to the freezing solution were achieved when using a 10% cryoprotectant concentration, with direct addition/dilution and exposure at room temperature (3512 ±300 viable cellsmm² when using dimethylsulfoxide; 3403 ± 245 viable cellsmm² when using 1,2-propanediol). Cryopreserved rabbit corneas had the highest endothelial cell survival when frozen at 1°C/min and thawed at 37°C (2003 ± 372 viable cells/mm² when using dimethylsulfoxide and 1357 ± 667 viable cells/mm² when using 1,2-propanediol). Cryopreserved human corneas had 753 ± 542 viable cells/mm² when using dimethylsulfoxide and 56 ± 56 viable cells/mm² when using 1,2-propanediol. We can conclude that the method developed is easy to handle and shows optimum results in rabbit corneas, with an endothelial cell survival that is consistent with transplant acceptability criteria. The results obtained in human corneas are below prediction and are still unsatisfactory for successful use in eye banking.     

Expansion and cryopreservation of porcine and human corneal endothelial cells

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me₂SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me₂SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine.     

Corneal cryopreservation with dextran.

Different methods of corneal cryopreservation have been introduced, those employing intracellular cryoprotectants such as Me2SO or glycerol being the most widely favored. We investigated the influence of several freeze-thaw trauma variables on the survival of porcine endothelial monolayers when employing the extracellular cryoprotective agent dextran. We first examined the effects of various dextran concentrations and then, having ascertained the optimal concentration, further investigated the influence of fetal calf serum (FCS) concentration in the cryopreservation medium, the cooling rate, the thawing temperature, and the length of the preincubation in the freezing medium prior to cryopreservation. The numerical densities of endothelial cells were determined at dissection in hypoosmotic balanced salt solution and after organ culture by staining with alizarin red S and trypan blue. Morphological evaluation was not performed directly after thawing but after a subsequent organ culture at 37 degrees C to detect latent cell damage after freeze-thaw trauma. Our data revealed that corneas cryopreserved in minimal essential medium containing 10% dextran but lacking FCS, preincubated for 3 h, frozen at a cooling rate of 1 degrees C/min, and thawed at 37 degrees C incurred the lowest cell losses (22.4%, SD +/- 3.8). We conclude that dextran is an effective cryoprotectant for freezing of porcine corneas. However, variations between species in the results of cryopreservation require further investigation of an in vivo animal model and studies with human corneas before its clinical use can be recommended.     

Equine sperm membrane phase behavior: the effects of lipid-based cryoprotectants

The plasma membrane of sperm can undergo lipid phase separation during freezing, resulting in irreversible damage to the cell. The objective of our study was to examine the membrane phase behavior of equine spermatozoa in the absence and presence of lipid-based cryoprotectants. Biophysical properties of sperm membranes were investigated with Fourier-transform infrared spectroscopy. Compared to fresh untreated sperm, postthaw untreated sperm showed extensive lipid phase separation and rearrangement. In contrast, postthaw sperm that were cryopreserved in egg phosphatidylcholine (egg PC)- or soy phosphatidylcholine (soy PC)-based diluents showed similar lipid phase behavior to that of fresh, untreated sperm. Studies with a deuterium-labeled PC lipid (POPCd-31) suggest that exogenous lipid from the diluents are strongly associated with the sperm membrane, and scanning electron microscopy images of treated sperm show the presence of lipid aggregates on the membrane surface. Thus, the exogenous lipid does not appear to be integrated into the sperm membrane after cryopreservation. When compared to a standard egg-yolk-based diluent (INRA 82), the soy and egg PC media preserved viability and motility equally well in postthaw sperm. A preliminary fertility study determined that sperm cryopreserved in the soy PC-based medium were capable of fertilization at the same rate as sperm frozen in the conventional INRA 82 medium. Our results show that pure lipid-based diluents can prevent membrane damage during cryopreservation and perform as well as a standard egg-yolk-based diluent in preserving sperm viability, motility, and fertility.     

In vitro fertility evaluation of cryopreserved ram semen and its correlation with relative in vivo fertility

The present study was designed to evaluate zona-free hamster ova assay conditions for cryopreserved ram semen and to investigate the correlation between ability to penetrate zona-free hamster ova and in vivo fertility. In vivo fertility was estimated for cryopreserved semen from 5 Merino rams using heterospermic insemination. Equal numbers of postthaw motile spermatozoa from a Merino ejaculate and pooled Suffolk ejaculates were mixed prior to insemination. Each Merino ejaculate was paired with the same pool of cryopreserved Suffolk semen. Relative in vivo fertility for each Merino ram was calculated as the proportion of offspring that were sired by the Merino (range 42 to 100%). These ejaculates also differed in their ability to penetrate zona-free-hamster ova (3.6 to 9.0 penetrated spermatozoa per ovum). Differences in penetration rate were correlated with in vivo fertility (P < 0.002, R2 = 0.69). Results of these studies suggest that the zona-free hamster ova bioassay may be a useful test in the assessment of cryopreserved ram sperm fertility.     

Epithelial sheet movement: effects of tunicamycin on migration and glycoprotein synthesis

Corneas with central epithelial wounds, 3 mm in diameter, were organ cultured in the presence of tunicamycin (TM) (1 microgram/ml), an antibiotic that inhibits glycosylation of asparagine-linked glycoproteins. Compared with control corneas, which healed in 22 hr, corneas cultured in the presence of TM for the entire culture time or for only the first 6 hr displayed a progressively slower epithelial healing rate that essentially dropped to zero by 24 hr of culture time. At 24 hr, approximately 75% of the wound was covered. After repeated washings with TM-free culture media (6X, 10 min each), this effect could consistently be reversed in corneas exposed to TM for 6 hr. Incorporation of [3H]glucosamine into trichloroacetic acid-precipitable proteins of migrating epithelial sheets was reduced to 14% that of controls after 12 hr of culture with TM, whereas [14C]leucine incorporation was not significantly affected. The decreased glycosylation was reflected on the cell surface after 12 and 20 hr culture in the presence of TM: apical cell membranes of the first six cells of the leading edge of the migrating sheet bound significantly fewer ferritin-concanavalin A particles per micrometer of membrane than did controls. These results indicate that synthesis of asparagine-linked glycoproteins is required for continued migration of corneal epithelial sheets. The asparagine-linked glycoproteins that are required for migration probably include cell-surface glycoproteins.     

Effects of osmotic stress on rabbit corneal endothelium

The effects of osmotic stress on corneal endothelium were investigated by exposing rabbit corneas to anisosmotic conditions, and then perfusing the corneas with isosmotic glutathione bicarbonate Ringer solution for 4 hr at 35 degrees C. During the perfusion, endothelial function was assessed by measuring corneal thickness with a specular microscope. After perfusion, the corneas were prepared for scanning and transmission electron microscopy. Endothelial ultrastructure and function were well maintained after exposure to a wide range of osmolality (0.12-2.7 osmol/kg), but this tolerance of osmotic stress was dependent both on the duration and the temperature of exposure to the anisosmotic conditions. Exposure to an osmolality of 2.7 osmol/kg for 15 min at 23 or 37 degrees C resulted in gross damage to the endothelium when the hyperosmotic agent was sodium chloride. This damage was not the result of increased osmolality per se nor cellular shrinkage because the endothelium tolerated exposure to a sucrose solution of the same osmolality for 15 min at 37 degrees C. The detrimental effect of sodium chloride, however, was mitigated by reducing the temperature of exposure to 0 degrees C or reducing the duration of exposure to 5 min. These results suggest that endothelial cells become more tolerant of high electrolyte concentrations with reducing temperature, and this could be an important factor in the survival of the endothelium in corneal cryopreservation. The results also help to define the limits of osmotic shrinkage and swelling tolerated by endothelial cells. This will be of value in overcoming the detrimental osmotic effects associated with the addition and, in particular, the removal of cryoprotectants.     

Cell adhesion molecules in stromal corneal dystrophies

The aim of the present study was to investigate the expression pattern of different cell adhesion molecules in corneal stromal dystrophies. Fifteen corneal buttons from patients diagnosed with three different types of stromal corneal dystrophies and healthy corneas were investigated. Paraffin embedded sections were stained immunohistochemically with monoclonal antibodies against human intercellular adhesion molecule-1 (ICAM-1), endothelial selectin (E-selectin) and endothelial cadherin (E-cadherin) using the avidin-biotin-peroxidase-complex technique. The sections were compared to normal eye bank controls. In corneas from granular dystrophy patients ICAM-1 was expressed focally in epithelial cells and in keratocytes, and expressed diffusely in endothelial cells. In corneas from macular dystrophy patients diffuse epithelial staining was observed and the stromal and endothelial expression was found to be similar to that of granular dystrophy. In lattice dystrophy, only the epithelial cells and endothelium were intensively positive for ICAM-1. E-selectin was not present on any layer of the corneal specimens. E-cadherin was observed only in the epithelium of all three types of corneal dystrophies. Normal corneas did not express any of the investigated adhesion molecules. We found different expression patterns of adhesion molecules in corneas from stromal dystrophies. Our results suggest that adhesion molecules may be involved in the pathogenesis of corneal stromal dystrophies.     

Restoring the endothelium of cryopreserved arterial grafts: co-culture of venous and arterial endothelial cells

The use of arterial homografts in clinical practice is becoming increasingly common, yet there is an urgent need to address one of the most well-established problems associated with their use: the loss of integrity of the endothelium following cryopreservation. The partial lack of endothelium causes contact between the extracellular matrix and blood flow, which, in turn, often gives rise to thrombosis and/or restenosis. Our objective was first to attempt to replace the arterial endothelial cells lost during the cryopreservation process by seeding autologous venous endothelial cells, and to evaluate the behaviour of venous and arterial endothelial cells in co-culture. The idea was to establish whether venous endothelial cells would be accepted by arterial endothelial cells and could therefore be used to restore the endothelial lining for the subsequent use of these vessels in in vivo grafting procedures. For the co-culture experiments, endothelial cells were obtained from the jugular vein and both iliac arteries of the minipig by treatment with 0.1% type I collagenase. The venous endothelial cells were fluorescently labelled with the membrane intercalating dye PKH26. Equal numbers of venous and arterial endothelial cells were mixed and co-cultured for 24h, 48h or 4 days. Cell viability, determined by 2% trypan blue staining and the TUNEL method, was established before and after fluorescence labelling. Cellular activity was determined by estimating PGI2 levels in the cultures. The proliferation index was established by [H(3)]thymidine (1muCi/ml) in the cell culture medium. For the in vivo tests, 5 cm length segments of minipig iliac artery were used to establish the groups: control (n = 6), fresh arterial segments; group I (n = 16), cryopreserved arterial segments and group II (n = 16), cryopreserved arterial segments seeded with autologous venous endothelial cells. The cryopreserved vessels in group II were seeded by flooding with a labelled venous endothelial cell suspension. Once seeded, the arterial segments were included in an in vitro flow circuit. All the specimens were processed for fluorescence and light microscopy, and scanning electron microscopy. The denuded endothelial surface was determined in each group. Cell death was evaluated by the TUNEL method. We confirmed the existence of intercellular PECAM1-type junctions between venous (PKH26+) and arterial cells in co-culture and the functional activity of the cells. The cryopreserved arterial segments showed a well-preserved wall structure. However, different size areas of marked endothelial denudation were detected. After seeding with labelled cells (PKH26+), these denuded areas of the cryopreserved artery were entirely covered by fluorescent cells. After seeding, a drop in the proportion of damaged endothelial cells was recorded. Despite some loss of seeded cells after inclusion in the in vitro flow circuit, the endothelial cell count was not significantly different to those recorded for control, non-cryopreserved specimens. In conclusion arterial and venous endothelial cells growing in co-culture modify their behaviour to form multilayers. The two cell populations form normal PECAM1 junctions and preserve their functional properties. Seeding autologous venous endothelial cells on the luminal surface of cryopreserved arterial segments serves to restore the integrity of the endothelial layer.     

The use of pentoxifylline to improve motility of cryopreserved equine spermatozoa

Pentoxifylline was evaluated as a method to increase motility of cryopreserved equine spermatozoa. In a preliminary experiment, pentoxifylline (3.5 mM or 7.0 mM) was added to extended semen that was chilled to 4 degrees C. Motility was evaluated at 8-h intervals for 48 h. The addition of 3.5 or 7.0 mM pentoxifylline appeared to increase the motility of chilled spermatozoa compared to controls. Based on these results, similar concentrations of pentoxifylline were added to semen either before or after cryopreservation. The addition of pentoxifylline (3.5 or 7.0 mM) to semen before cryopreservation significantly (P < 0.001) decreased total and progressive motility compared to controls. However, the addition of pentoxifylline (3.5 or 7.0 mM) to cryopreserved semen immediately after thawing significantly (P < 0.01) increased total and progressive motility compared to controls. These results indicate that pentoxifylline enhanced the postthaw motility of cryopreserved equine semen when added after thawing. Further research is required to evaluate the effect of pentoxifylline on the fertility of cryopreserved equine semen.     

Keratocyte injury in human corneas cryopreserved under standard conditions

This study was conducted to characterize ultrastructural damage to human corneas cryopreserved by a standard protocol. The materials used were seven human corneas that were unsuitable for transplantation due to the presence of positive bacteriological cultures; they were cryopreserved according the standard procedure. After freezing and thawing, samples were obtained for scanning and transmission electron microscopy studies. Marked damage was observed in keratocytes with signs of apoptotic cellular injury. However our observations have shown that apoptosis contribute less significantly than necrosis to cellular death in keratocytes of human corneas and although the control of apoptosis is clearly desirable, in order to improve the success of cryopreserved corneas for transplant, we need to continue our investigation to reduce the effects of the necrotic process. This revised version was published online in July 2006 with corrections to the Cover Date.