Twenty-four-hour normothermic perfusion of discarded human kidneys with urine recirculation.

Transportable normothermic kidney perfusion for 24 hours or longer could enable viability assessment of marginal grafts, increased organ use, and improved transplant logistics. Eleven clinically declined kidneys were perfused normothermically, with 6 being from donors after brain death (median cold ischemia time 33 ± 36.9 hours) and 5 being from donors after circulatory death (36.2 ± 38.3 hours). Three kidneys were perfused using Ringer's lactate to replace excreted urine volume, and 8 kidneys were perfused using urine recirculation to maintain perfusate volume without fluid replenishment. In all cases, normothermic perfusion either maintained or slightly improved the histopathologically assessed tubular condition, and there was effective urine production in kidneys from both donors after brain death and donors after circulatory death (2367 ± 1798 mL vs 744.4 ± 198.4 mL, respectively; P = .44). Biomarkers, neutrophil gelatinase-associated lipocalin, and kidney injury molecule-1 were successfully detected and quantified in the perfusate. All kidneys with urine recirculation were readily perfused for 24 hours (n = 8) and exhibited physiological perfusate sodium levels (140.7 ± 1.2 mmol/L), while kidneys without urine recirculation (n = 3) achieved a reduced normothermic perfusion time of 7.7 ± 1.5 hours and significantly higher perfusate sodium levels (159.6 ± 4.63 mmol/:, P < .01). Normothermic machine perfusion of human kidneys for 24 hours appears to be feasible, and urine recirculation was found to facilitate the maintenance of perfusate volume and homeostasis.

The effect of the serum corona on interactions between a single nano-object and a living cell.

Nanoparticles (NPs) which enter physiological fluids are rapidly coated by proteins, forming a so-called corona which may strongly modify their interaction with tissues and cells relative to the bare NPs. In this work the interactions between a living cell and a nano-object, and in particular the effect on this of the adsorption of serum proteins, are directly examined by measuring the forces arising as an Atomic Force Microscope tip (diameter 20 nm) - simulating a nano-object - approaches and contacts a cell. We find that the presence of a serum protein corona on the tip strongly modifies the interaction as indicated by pronounced increase in the indentation, hysteresis and work of adhesion compared to a bare tip. Classically one expects an AFM tip interacting with a cell surface to be repelled due to cell elastic distortion, offset by tip-cell adhesion, and indeed such a model fits the bare-tip/cell interaction, in agreement with earlier work. However, the force plots obtained with serum-modified tips are very different, indicating that the cell is much more compliant to the approaching tip. The insights obtained in this work may promote better design of NPs for drug delivery and other nano-medical applications.

Factors regulating viable cell density in the intervertebral disc: blood supply in relation to disc height.

The intervertebral disc is an avascular tissue, maintained by a small population of cells that obtain nutrients mainly by diffusion from capillaries at the disc-vertebral body interface. Loss of this nutrient supply is thought to lead to disc degeneration, but how nutrient supply influences viable cell density is unclear. We investigated two factors that influence nutrient delivery to disc cells and hence cell viability: disc height and blood supply. We used bovine caudal discs as our model as these show a gradation in disc height. We found that although disc height varied twofold from the largest to the smallest disc studied, it had no significant effect on cell density, unlike the situation found in articular cartilage. The density of blood vessels supplying the discs was markedly greater for the largest disc than the smallest disc, as was the density of pores allowing capillary penetration through the bony endplate. Results indicate that changes in blood vessels in the vertebral bodies supplying the disc, as well as changes in endplate architecture appear to influence density of cells in intervertebral discs.

Hepatotoxicity assessment of the azo dyes disperse orange 1 (DO1), disperse red 1 (DR1) and disperse red 13 (DR13) in HEPG2 cells.

During the dyeing process in baths approximately 10 to 15% of the dyes used are lost and reach industrial effluents, thus polluting the environment. Studies showed that some classes of dyes, mainly azo dyes and their by-products, exert adverse effects on humans and local biota, since the wastewater treatment systems and water treatment plants were found to be ineffective in removing the color and reducing toxicity of some dyes. In the present study, the toxicity of the azo dyes disperse orange 1 (DO1), disperse red 1 (DR1), and disperse red 13 (DR13) was evaluated in HepG2 cells grown in monolayers or in three dimensional (3D) culture. Hepatotoxicity of the dyes was measured using 3-(4,5-dimethylthiazol-2yl)2,5-diphenyltetrazolium (MTT) and cell counting kit 8 (CCK-8) assays after 24, 48, and 72 h of incubation of cells with 3 different concentrations of the azo dyes. The dye DO1 only reduced the mitochondrial activity in HepG2 cells grown in a monolayer after 72 h incubation, while the dye DR1 showed this deleterious effect in both monolayer and 3D culture. In contrast, dye DR13 decreased the mitochondrial activity after 24, 48, and 72 h of exposure in both monolayer and 3D culture. With respect to dehydrogenase activity, only the dye DR13 diminished the activity of this enzyme after 72 h of exposure in both monolayer and 3D culture. Our results clearly demonstrated that exposure to the studied dyes induced cytotoxicity in HepG2 cells.

Monitoring of metabolite gradients in tissue-engineered constructs.

At present, the assessment of developing tissue-engineered constructs is almost always carried out destructively using biochemical or histological methods to determine cell number, viability and tissue growth throughout the construct. Since many of these experiments are long, taking weeks or even months to complete, simple and readily applicable non-destructive methods of monitoring changes in cell metabolism, viability and tissue deposition within the construct would be invaluable; such methods could point out adverse responses during the early stages of culture. Here, we describe the use of microdialysis for detecting local changes in cellular metabolism within a tissue-engineered construct. Three-dimensional constructs consisting of bovine articular chondrocytes entrapped in an alginate gel were cultured in a bioreactor for two weeks. Glucose and lactate were monitored by microdialysis, as the major nutrient and metabolite, respectively. Concentration gradients within the construct were evident, with the highest lactate concentrations in the construct centre. The local lactate concentration was a measure of cellular metabolic activity, decreasing as cellular activity fell and increasing as cellular activity was stimulated. Nutrient starvation and cell death in the construct centre could be readily detected in constructs deliberately cultured under adverse conditions. The results show that probe measurements can give an early warning of inappropriate local metabolic changes. Such information during the growth of tissue-engineered constructs would allow either corrective action or else an early end to an unsuccessful test.