Effects of antibiotics on the endothelium of fresh and cryopreserved canine saphenous veins.

To determine if antibiotic solutions for potential use in antimicrobial treatment of tissues were detrimental to cell viability, canine saphenous veins were exposed to three different antibiotic solutions at 37 degrees C for 12 h. Viability was determined by tissue culture, utilizing a limiting dilution assay. The antibiotic series included two formulations currently used for the preservation of heart valves and a new formulation containing imipenem, a broad spectrum beta-lactam. Currently used antibiotic regimes and imipenem with gentamicin resulted in a decrease of 30 to 50% of the viable endothelial cells within 12 h. Exposure of the saphenous veins to imipenem or imipenem combined with amphotericin B had no adverse effects on the viability of the endothelial cells with 12 h exposure. However, veins exposed to amphotericin B were more susceptible to subsequent damage during freezing and thawing than veins frozen after incubation with either imipenem alone or imipenem combined with flucytosine. These studies indicate that imipenem combined with flucytosine is suitable for use with vein grafts.

Transplantation of cryopreserved canine venous allografts.

Local vascular reconstructions frequently require the use of vein grafts to bridge arterial or venous defects. Most previous studies on the use of cryopreserved veins have used relatively large caliber vessels. There have been few studies on the effectiveness of cryopreserved micro- or small-venous allografts. Here, we tested two types of cryopreserved venous allografts: (1) 1.5- to 1.9-mm diameter microvenous grafts (MVG); and (2) 4- to 5-mm diameter small venous grafts (SVG). Cryopreserved MVG allografts were placed into saphenous arteries of six experimental dogs and SVG cryopreserved allografts were placed into femoral arteries of six experimental dogs for 3 to 6 weeks. Two fresh MVG autografts were also transplanted into experimental dogs as controls and autografts were transferred to the contralateral side in SVG dogs as controls. None of the six cryopreserved MVG grafts retained patency but three/six cryopreserved SVG allografts were patent at harvest. Histological examination of grfts revealed control autografts were undergoing arterialization with an intact intima. Experimental cryopreserved allografts showed extensive medial fibrosis, significant lymphocytic infiltrates, and sporadic areas of intact intima for both patent and nonpatent grafts.

Bactericidal effectiveness of modulated UV light.

Studies were designed to evaluate the effectiveness of pulsed modulated UV light waveforms for killing bacteria. Exposure of five strains of bacteria to the modulated information encoded in the light decreased the colony population from a confluent lawn to less than 20 colonies. However, approximately 2,000 colonies survived treatment with the same intensity and time of exposure to UV light lacking the modulated information.

Freeze-dried microarterial replacement allografts in rabbits.

Microvascular techniques offer important alternatives for reconstructive head and neck surgery. To test the viability of freeze-dried allografts, a pilot experimental study was performed using the rabbit model. Freeze-dried preserved arterial allografts were implanted into femoral artery defects in eight subjects. After 6 weeks, all grafts were harvested and prepared for histological and electron microscopic analysis. Immediate patency was 100%. One subject was excluded on the third postoperative day. Of the seven remaining grafts, three (43%) were patent at 6 weeks. These results are comparable to previous data obtained using freeze-dried arterial allografts in the same animal model. Although further investigation is required, this pilot study suggests possible future application of cryopreserved vascular micrografts.

Parameters for evaluation of viability assays: accuracy, precision, specificity, sensitivity, and standardization.

The selection of appropriate viability assays is critical in evaluating the efficacy of any cryopreservation procedure. The appropriateness of a given assay depends on the specific tissue and the function which is being optimized. Although a broad range of "viability" assays have been used, these assays can be classified in seven principle groups: (i) Morphological procedures, including routine histology, surface antigen localization, and transmission electron or scanning microscopy; (ii) proliferation studies; (iii) metabolic assays; (iv) implantation; (v) mechanical assays; (vi) motility; and (vii) DNA or RNA synthetic assays. Regardless of the class of assay, each assay may be further characterized as qualitative, quantitative, or quantal and each type may vary in the degree of subjectivity. In selecting a specific viability assay, biological variability, assay bias, and the statistical probability of both Type I and Type II errors should be considered crucial. Here we discuss a number of critical factors involved in validating viability assays, including accuracy, precision, standardization, specificity, sensitivity, selection of statistical methodology, and range of the assay.

Power analysis of statistical methods for comparing treatment differences from limiting dilution assays.

Six different statistical methods for comparing limiting dilution assays were evaluated, using both real data and a power analysis of simulated data. Simulated data consisted of a series of 12 dilutions for two treatment groups with 24 cultures per dilution and 1,000 independent replications of each experiment. Data within each replication were generated by Monte Carlo simulation, based on a probability model of the experiment. Analyses of the simulated data revealed that the type I error rates for the six methods differed substantially, with only likelihood ratio and Taswell's weighted mean methods approximating the nominal 5% significance level. Of the six methods, likelihood ratio and Taswell's minimum Chi-square exhibited the best power (least probability of type II errors). Taswell's weighted mean test yielded acceptable type I and type II error rates, whereas the regression method was judged unacceptable for scientific work.

Comparison of statistical methods for the analysis of limiting dilution assays.

This study reports the results of a critical comparison of five statistical methods for estimating the density of viable cells in a limiting dilution assay (LDA). Artificial data were generated using Monte Carlo simulation. The performance of each statistical method was examined with respect to the accuracy of its estimator and, most importantly, the accuracy of its associated estimated standard error (SE). The regression method was found to perform at a level that is unacceptable for scientific research, due primarily to gross underestimation of the SE. The maximum likelihood method exhibited the best overall performance. A corrected version of Taswell's weighted-mean method, which provides the best performance among all noniterative methods examined, is also presented.

Rapid assessment of islet viability with acridine orange and propidium iodide.

A simple, rapid method for estimating the viability of isolated islets of Langerhans with fluorescent dyes is described. Low concentrations of acridine orange and propidium iodide (AO/PI) were used to visualize living and dead islet cells simultaneously. AO/PI-stained islets can be divided into three distinct groups. Group A islets fluoresce green, contain insulin, and have normal ultrastructure; group C islets fluoresce primarily red, contain little or no insulin, and have cells with disrupted cellular membranes. Group B islets fluoresce red, green, and yellow. The yellow color is due to the addition of two primary colors from the superimposed red and green fluorescing cells. In this assay, the interpretation that red islet cells are dead and green islet cells are alive was confirmed by sequentially staining single islet cells with AO/PI and trypan blue. The observation that red islets are dead was confirmed by heat-killing, enzymatically damaging, treating with ethanol, or depriving islets of nutrients and observing the red fluorescence. This assay should be useful in studies where the assessment of islet viability is essential.

Function of lymphocytes and macrophages after cryopreservation by procedures for pancreatic islets: potential for reducing tissue immunogenicity.

The survival of tissue allografts can be extended by pretreating the tissue to remove the stimulatory leucocytes that populate the graft; with this in mind, we have recently begun to explore a cryobiological approach to modulating tissue immunogenicity by using the differential susceptibility of different cells to freezing injury. The sensitivity of leucocytes to fast cooling rates, which were used in procedures that have been reported to yield viable pancreatic islets of Langerhans, was examined. The loss of both cell numbers and the ability of peripheral blood lymphocytes to undergo blastogenic transformation in response to the mitogen concanavalin A after freezing and thawing was determined over a range of cell concentrations using the "curve-shift" method. Lymphocytes frozen at 1 degree C/min by a control procedure that was designed to yield maximum survival of lymphocytes showed that although there was a decrease in the number of responding cells, there was no reduction in the ability of the recovered cells to undergo blastogenesis when compared with the response of nonfrozen cells. However, cooling at 1 degree C/min in the experimental procedures resulted in both the loss of cells as well as a marked reduction in the ability of recovered cells to incorporate 125I-deoxyuridine into nucleic acid. Cells cooled at either 20 or 75 degrees C/min by any of the procedures showed total inability to respond to stimulation. Lysozyme is produced continuously by all types of macrophages in culture. The large net increase in total lysozyme content of macrophage cultures is therefore a useful measure of the viability of these accessory cells. Cooling at 1 degree C/min by a control, optimized procedure yielded 91% survival of viable peritoneal exudate cells. Cooling at either 1 or 20 degrees C/min in the experimental procedures resulted in 72-75% survival of cells frozen by one method and 33% survival when frozen by an alternative procedure. Negligible recovery of viable cells was obtained after cooling at 75 degree C/min. The preservation protocols employed in this study differ significantly in the variables known to influence the survival of the cells; these include the concentration of cryoprotectant (CPA), the length and temperature of exposure to CPA, the dilution regimen, and the optimum cooling rate for survival of pancreatic islets. This study therefore defines clearly those conditions most likely to effect a depletion of "passenger" lymphoid cells by freezing during the cryopreservation of islets of Langerhans.

A quantitative enzyme-linked immunosorbent assay for rat insulin.

A simple, quantitative micro-ELISA (Enzyme-Linked Immunosorbent Assay) has been developed for rat insulin. The micro-ELISA is a solid phase, indirect, competitive immunoassay. The useful range of the micro-ELISA was superior to that of a commercial RIA for rat insulin (e.g. 0.4 to 46.0 ng/ml for the ELISA; 0.2-8.6 ng/ml for the RIA). The ELISA's sensitivity (the lower limit of detection) was 1.0 ng/ml +/- 0.13 ng/ml, (mean, +/- SEm; 9 assays) or 20 +/- 2.6 pg/determination, and compared favorably with the sensitivity of a radioimmunoassay (RIA) for rat insulin (0.38 +/- 0.10 ng/ml; 4 assays). The ELISA measured pure rat insulin standards accurately. Correlation experiments showed that the results of the ELISA agreed with those of the RIA (r = 0.91), when rat insulin was assayed in crude extracts of isolated pancreatic Islets of Langerhans. When the standard curve was plotted as a log of the dose response curve, a sigmoidally shaped curve was obtained which could be transformed into a straight line relationship with a logit-log program. The goodness of fit of the transformed standard curve to a straight line relationship was excellent (r = 0.97 to 0.99: n = 4 ELISAs). The transform facilitated dose interpolation, tests of parallelism, and quality control. Tests of parallelism showed that the ELISA was specific for rat insulin. The precision of the ELISA was superior to the precision of the rat insulin RIA tested. The intraassay precision of the ELISA was always less than 10% (CV%), and its interassay precision was always less than or equal to 15% (CV%). The micro-ELISA is versatile, since it can be used to measure human, porcine, rat, and probably mouse insulin.

Videoanalysis of chemokinesis: characterization of speed, persistence and orientation in an agarose assay.

Time-lapse video recording and off-line computer analysis were used to characterize the chemokinetic behavior of individual human neutrophils migrating in an agarose assay. When neutrophils were stimulated with an isotropic concentration of formyl-methionyl-leucyl-phenylalanine (fMLP), they migrated with a mean speed of 9.6 micron per min and oriented at random. The ratio of net displacement to total distance travelled (persistence of locomotion) was 0.66, indicating that neutrophils maintained some directional persistence even in the absence of a gradient of fMLP. The speed and persistence of locomotion index were correlated because both faster and slower cells had high persistence, while only slower cells had low persistence. The orientation angle was independent of both speed and persistence of locomotion. These are the first reported direct measurements of the chemokinetic locomotion of neutrophils using the agarose assay.

Kinematic analysis of chemotaxis of fresh and stored neutrophils.

When neutrophils are isolated from the circulation the first function to begin to deteriorate is chemotaxis. To characterize the loss of chemotaxis that occurs during storage, a computer-assisted video motion analysis of neutrophils responding to formyl-methionyl-leucyl-phenylalanine (FMLP) was used in an agarose assay. The chemotactic speed, velocity, and orientation angle were measured, and a persistence of locomotion index (velocity/speed) and chemotropic index (cosine of the orientation angle) were calculated for fresh neutrophils and neutrophils stored in plasma at 20 to 22 degrees C for 24 hours. The data reveal that: (1) the frequency distribution of speed for individual stored cells had a different shape than that of fresh cells owing to a subpopulation of stored cells (approximately 35 percent) which migrated at a slower mean speed; (2) the frequency distribution of orientation for fresh cells is not normally distributed and contains a subpopulation (approximately nine percent of the total) of cells which orient at random in a gradient; (3) the precision of orientation of the majority of stored cells is comparable to that of fresh cells, but approximately 35 percent of the stored cells orient at random in a chemoattractant gradient; (4) neither the persistence index nor the orientation of both fresh and stored cells were correlated with speed; (5) the chemotropic index and persistence index are correlated, and this correlation is not altered by storage suggesting that stored cells which show decreased persistence also show a decreased chemotropic index. It is proposed that neutrophils respond to a gradient of fMLP with either fast, persistent, accurately oriented locomotion or slower, less persistent, randomly oriented locomotion. In addition to those neutrophils which do not migrate in response to fMLP, it is proposed that there are two subpopulations of motile neutrophils. Storage at 20 to 22 degrees C induces shifts between these three modes of behavior.

Assessment of islet cell viability using fluorescent dyes.

A rapid fluorometric method has been developed to evaluate the viability of isolated islet cells. The assay differentiates between viable and nonviable cells by the simultaneous use of the inclusion and exclusion dyes acridine orange and propidium iodide. When viewed by fluorescent microscopy, viable cells fluoresce green, while nonviable cells fluoresce bright red. Although the acridine orange and propidium iodide assay measures membrane integrity, the results of this assay correlate with other measures of cell viability. Compared to trypan blue exclusion, this assay is easier to read, more stable, and has fewer staining artifacts. The assay enables the rapid estimation of the viability of a population of islet cells prior to time-consuming experiments rather than retrospectively. This assay can also be used with intact islets. Stained islets can be divided into three distinct groups: green fluorescing islets contain insulin, red fluorescing islets contain little or no insulin and a third class of islets containing some non-viable cells fluoresce red, green, and yellow. The yellow colour is due to the superimposition of red and green fluorescing cells.

Selective killing of leucocytes by freezing: potential for reducing the immunogenicity of pancreatic islets.

Recent developments in transplantation immunobiology, concerning the mechanism of tissue rejection, clearly indicate that antigen recognition alone is not sufficient for lymphocyte activation. "Passenger" leucocytes (antigen presenting cells) carried in the donor tissue are now recognized as the major immunogenic stimulus, such that removal of these contaminating leucocytes, using a variety of procedures, has enabled the immunogenicity of allografts to be reduced and the survival of the graft to be significantly extended. Remarkable advances have been made in recent years in preventing rejection of islet allografts, and even xenografts, in experimental animals by using procedures which do not involve continuous immunosuppressive therapy. Cryopreservation offers not only the means by which donor tissue can be stored effectively during such procedures but also the possibility that, under appropriate conditions, the freezing and thawing process itself could modulate tissue immunogenicity by allowing the selective killing of immunocompetent leucocytes whilst preserving the function of parenchymal cells in the graft. In this preliminary study we have characterized the survival of leucocytes and islets from the same species (rat) after cryopreservation by the same technique using dimethyl sulphoxide (Me2SO) as the cryoprotectant. Optimum survival of rat lymphocytes and macrophages was found at cooling rates in the range of 0.3-5 degrees C/min, after cooling at rates greater than 75 degrees C/min, survival was reduced to a negligible level. On the other hand, recovery of islets was 73 +/- 9% at 75 degrees C/min, indicating that depletion of lymphoid cells, with satisfactory preservation of endocrine cells, should be obtainable at this cooling rate.

Selective destruction of leucocytes by freezing as a potential means of modulating tissue immunogenicity: membrane integrity of lymphocytes and macrophages.

It is now known, when a tissue allograft is transplanted, that antigen recognition alone is not sufficient for lymphocyte activation in the host. "Passenger" leucocytes (antigen-presenting cells) present in the donor tissue are now recognized as a major immunogenic stimulus. Removal of these contaminating leucocytes, using a variety of procedures, has enabled the immunogenicity of allografts to be reduced, thus enhancing the survival of tissue allografts. This initial study explores the possibility of using a cryobiological approach to modulating the immunogenicity of tissues by virtue of the well-recognized differential susceptibility of different cell types to freezing injury. The investigation was prompted by demonstrations that pancreatic islets can secrete insulin in response to a graded glucose challenge after cryopreservation using relatively fast cooling rates which would be expected to be suboptimal for leucocyte survival. Batches of rat peripheral blood lymphocytes, or peritoneal exudate cells (macrophages) were cooled at 0.3, 1, 5, 20, 75, or 200 degrees C/min using three different cryopreservation protocols reported to yield viable pancreatic islets. Cell survival was evaluated in terms of the numbers of cells recovered after freezing as well as a fluorometric viability assay which assessed the membrane integrity of cells. Optimum survival of both lymphocytes and macrophages after freezing and thawing was found at cooling rates in the range of 0.3 to 5 degrees C/min. A significant number (10-40%) of these lymphoid cells survived freezing at 20 degrees C/min and only after cooling at rates greater than 75 degrees C/min was survival reduced to a negligible level.

Basic principles of cryobiology.

The ultimate aim of most cryopreservation procedures is the retention of the structural and functional integrity of the frozen cells or tissue. Designing strategies for achieving high "viability" of the frozen tissue requires an appreciation of the complex physical-chemical events which occur during freezing and thawing, as well as the importance of other processing steps including tissue procurement and post-thaw processing of the tissue, all of which can adversely affect the tissue. Here we have provided a general overview of cryobiology with special emphasis on techniques for organ preservation.

Measurement of postcryopreservation viability.

For any tissue, there is a cell viability threshold below which the ability of the tissue to maintain itself and function will eventually be compromised. During cryopreservation and subsequent thawing of tissues there are many steps involved, each with attendant potential risks for reduction of viability. To determine the effectiveness of any cryopreservation procedure it is important to select appropriate assays. In this manuscript viability assays, in general, are reviewed from a biological viewpoint prior to review of methods employed for assessment of heart valve viability. Both in situ and in vitro assays of heart valve viability indicate that valve mechanical properties and the majority of fibroblasts, which are responsible for maintenance of the valve connective tissue, are retained after cryopreservation.

Analog enhancement of videomicroscope images.

A simple, inexpensive technique for enhancing the contrast and resolution of videomicroscope images has been developed. The system has manual controls for gain and pedestal (black level) which permit expansion of low contrast images to the full white-to-black video range. Analog delay-line based circuits are used to sharpen the edges and enhance fine details in the image. These circuits also produce an effective increase in the information content of the image by selectively amplifying low amplitude, high frequency components of the video signal. When live, unstained cells were examined at high magnifications, cytoplasmic structures which were only faintly visible in the unenhanced image became clear. The images of fluorescent objects appear in pseudo-relief, which improves visibility even in the presence of background fluorescence. The system enhances images by performing signal processing functions that otherwise require expensive digital image processing equipment.