Treatment of Staphylococcus aureus infection with sphingosine in ex vivo perfused and ventilated lungs.

BACKGROUND: Ex vivo lung perfusion (EVLP) has expanded the donor pool for lung transplantation. Pulmonary Staphylococcus aureus infection, especially that caused by multidrug-resistant strains, is a severe threat to posttransplantation outcomes. Sphingosine is a lipid compound that exhibits broad-spectrum antibacterial activity. Therefore, we aimed to evaluate the effects of S aureus infection on EVLP and whether sphingosine administration during EVLP prevents infection with S aureus. METHODS: Eighteen pigs were randomly assigned to 3 groups: uninfected, infected with S aureus with NaCl treatment, or infected with sphingosine treatment. Bacterial numbers were determined before and after treatment. Sphingosine concentrations in the lung tissues were determined using biochemical assays. Lung histology, lung physiological parameters, perfusate content, lung weight, and cell death were measured to analyze the effects of infection and sphingosine administration on EVLP. RESULTS: Sphingosine administration significantly reduced the bacterial load. The concentration of sphingosine in the bronchial epithelium was elevated after sphingosine administration. S aureus infection increased pulmonary artery pressure and pulmonary vascular resistance. Lung edema, histology scores, lactate and lactate dehydrogenase levels in the perfusate, ΔPO2 in the perfusate, static lung compliance, and lung peak airway pressure did not differ among the groups. CONCLUSIONS: Infection of S aureus did not affect the lung function during EVLP but induced higher pulmonary artery pressure and pulmonary vascular resistance. Administration of sphingosine effectively eliminated S aureus without side effects in isolated, perfused, and ventilated pig lungs.

Composition of ex vivo perfusion solutions and kinetics define differential cytokine/chemokine secretion in a porcine cardiac arrest model of lung preservation.

Background: Ex vivo lung perfusion (EVLP) uses continuous normothermic perfusion to reduce ischemic damage and to improve post-transplant outcomes, specifically for marginal donor lungs after the donation after circulatory death. Despite major efforts, the optimal perfusion protocol and the composition of the perfusate in clinical lung transplantation have not been identified. Our study aims to compare the concentration levels of cytokine/chemokine in different perfusion solutions during EVLP, after 1 and 9 h of cold static preservation (CSP) in a porcine cardiac arrest model, and to correlate inflammatory parameters to oxygenation capacities. Methods: Following cardiac arrest, the lungs were harvested and were categorized into two groups: immediate (I-EVLP) and delayed EVLP (D-EVLP), after 1 and 9 h of CSP, respectively. The D-EVLP lungs were perfused with either Steen or modified Custodiol-N solution containing only dextran (CD) or dextran and albumin (CDA). The cytokine/chemokine levels were analyzed at baseline (0 h) and after 1 and 4 h of EVLP using Luminex-based multiplex assays. Results: Within 4 h of EVLP, the concentration levels of TNF-α, IL-6, CXCL8, IFN-γ, IL-1α, and IL-1ß increased significantly (P < 0.05) in all experimental groups. The CD solution contained lower concentration levels of TNF-α, IL-6, CXCL8, IFN-γ, IL-2, IL-12, IL-10, IL-4, IL-1RA, and IL-18 (P < 0.05) compared with those of the Steen solution. The concentration levels of all experimental groups have correlated negatively with the oxygenation capacity values (P < 0.05). Protein concentration levels did not reach statistical significance for I-EVLP vs. D-EVLP and CD vs. CDA solutions. Conclusion: In a porcine cardiac arrest model, a longer period of CSP prior to EVLP did not result in an enhanced protein secretion into perfusates. The CD solution reduced the cytokine/chemokine secretion most probably by iron chelators and/or by the protecting effects of dextran. Supplementing with albumin did not further reduce the cytokine/chemokine secretion into perfusates. These findings may help in optimizing the preservation procedure of the lungs, thereby increasing the donor pool of organs.

Human HepaRG liver spheroids: cold storage protocol and study on pyridinium oxime-induced hepatotoxicity in vitro.

Oximes play an essential role in the therapy of organophosphorus compound (OP) poisoning by reactivating inhibited acetylcholinesterase. Impairment of liver function was observed in OP poisoning and associated with obidoxime treatment by some reports. In this study human three-dimensional HepaRG spheroids were used as complex in vitro model to investigate oxime-induced liver toxicity. In this context, cold storage of liver spheroids at 4 °C in standard culture medium and in optimized tissue preservation solutions of up to 72 h was assessed. Cold storage in standard culture medium resulted in a complete loss of viability whereas an optimized tissue preservation solution preserved viability. Separately from that liver spheroids were exposed to the four oximes pralidoxime, obidoxime, HI-6, MMB-4 and cytotoxicity (effective concentration, EC50) was determined with an ATP-based assay at several time points. The release of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin secretion was measured in supernatants. The same parameters were assessed with diclofenac as positive hepatotoxic control and with the OP pesticides malathion and malaoxon alone or in the presence of obidoxime. All individual tested oximes and OP showed a low cytotoxicity with effective concentrations mostly >2,000 µM. In contrast, the exposure to malaoxon in the presence of 1,000 µM obidoxime resulted in a marked decrease of viability and an increased release of AST indicating risk of liver injury only if oxime antidotes are strongly overdosed.

Antimicrobial effects of inhaled sphingosine against Pseudomonas aeruginosa in isolated ventilated and perfused pig lungs.

BACKGROUND: Ex-vivo lung perfusion (EVLP) is a save way to verify performance of donor lungs prior to implantation. A major problem of lung transplantation is a donor-to-recipient-transmission of bacterial cultures. Thus, a broadspectrum anti-infective treatment with sphingosine in EVLP might be a novel way to prevent such infections. Sphingosine inhalation might provide a reliable anti-infective treatment option in EVLP. Here, antimicrobial potency of inhalative sphingosine in an infection EVLP model was tested. METHODS: A 3-hour EVLP run using pig lungs was performed. Bacterial infection was initiated 1-hour before sphingosine inhalation. Biopsies were obtained 60 and 120 min after infection with Pseudomonas aeruginosa. Aliquots of broncho-alveolar lavage (BAL) before and after inhalation of sphingosine were plated and counted, tissue samples were fixed in paraformaldehyde, embedded in paraffin and sectioned. Immunostainings were performed. RESULTS: Sphingosine inhalation in the setting of EVLP rapidly resulted in a 6-fold decrease of P. aeruginosa CFU in the lung (p = 0.016). We did not observe any negative side effects of sphingosine. CONCLUSION: Inhalation of sphingosine induced a significant decrease of Pseudomonas aeruginosa at the epithelial layer of tracheal and bronchial cells. The inhalation has no local side effects in ex-vivo perfused and ventilated pig lungs.

Characterisation of cold-induced mitochondrial fission in porcine aortic endothelial cells.

BACKGROUND: Previously, we observed that hypothermia, widely used for organ preservation, elicits mitochondrial fission in different cell types. However, temperature dependence, mechanisms and consequences of this cold-induced mitochondrial fission are unknown. Therefore, we here study cold-induced mitochondrial fission in endothelial cells, a cell type generally displaying a high sensitivity to cold-induced injury. METHODS: Porcine aortic endothelial cells were incubated at 4-25 °C in modified Krebs-Henseleit buffer (plus glucose to provide substrate and deferoxamine to prevent iron-dependent hypothermic injury). RESULTS: Cold-induced mitochondrial fission occurred as early as after 3 h at 4 °C and at temperatures below 21 °C, and was more marked after longer cold incubation periods. It was accompanied by the formation of unusual mitochondrial morphologies such as donuts, blobs, and lassos. Under all conditions, re-fusion was observed after rewarming. Cellular ATP content dropped to 33% after 48 h incubation at 4 °C, recovering after rewarming. Drp1 protein levels showed no significant change during cold incubation, but increased phosphorylation at both phosphorylation sites, activating S616 and inactivating S637. Drp1 receptor protein levels were unchanged. Instead of increased mitochondrial accumulation of Drp1 decreased mitochondrial localization was observed during hypothermia. Moreover, the well-known Drp1 inhibitor Mdivi-1 showed only partial protection against cold-induced mitochondrial fission. The inner membrane fusion-mediating protein Opa1 showed a late shift from the long to the fusion-incompetent short isoform during prolonged cold incubation. Oma1 cleavage was not observed. CONCLUSIONS: Cold-induced mitochondrial fission appears to occur over almost the whole temperature range relevant for organ preservation. Unusual morphologies appear to be related to fission/auto-fusion. Fission appears to be associated with lower mitochondrial function/ATP decline, mechanistically unusual, and after cold incubation in physiological solutions reversible at 37 °C.

Custodiol-MP for ex vivo lung perfusion - A comparison in a porcine model of donation after circulatory determination of death.

INTRODUCTION: Ex vivo lung perfusion (EVLP) is an established technique to evaluate and eventually recondition lungs prior to transplantation. Custodiol-MP (C-MP) solution is a new solution, designed for clinical machine perfusion, that has been used for kidneys. The aim of this study was to compare the effects of EVLP with Custodiol-MP on lung functional outcomes to the gold standard of EVLP with Steen Solution™. MATERIAL AND METHODS: In a porcine EVLP model of DCDD (Donation after Circulatory Determination of Death), lungs were perfused with Steen Solution™ (SS, n = 7) or Custodiol-MP solution supplemented with 55 g/l albumin (C-MP, n = 8). Lungs were stored cold for 4 h in low potassium dextran solution and subsequently perfused ex vivo for 4 h. During EVLP pulmonary gas exchange, activities of lactate dehydrogenase (LDH) and alkaline phosphatase (AP) as well as levels of lactate in the perfusate were recorded hourly. RESULTS: Oxygenation capacity differed significantly between groups (averaged over 4 h: SS 274 ± 178 mmHg; C-MP 284 ± 151 mmHg p = 0.025). Lactate dehydrogenase activities and lactate concentrations were significantly lower in Custodiol-MP perfused lungs.In a porcine model of DCDD with 4 h of EVLP the use of modified Custodiol-MP as perfusion solution was feasible. The use of C-MP showed at least comparable lung functional outcomes to the use of Steen SolutionTM. Furthermore C-MP perfusion resulted in significantly lower lactate dehydrogenase activity and lactate levels in the perfusate and higher oxygenation capacity.

HTK-N as a new preservation solution for human kidney preservation: Results of a pilot randomized controlled clinical phase II trial in living donor transplantation.

BACKGROUND: HTK-N was developed based on the traditional HTK preservation solution, resulting in stronger protection against reactive oxygen species as well as better tolerance to hypothermia and ischemia. Aim of the present study was to compare HTK-N to HTK in clinical kidney transplantation demonstrating safety and non-inferiority. METHODS: We performed a randomized controlled single blinded clinical phase II trial in patients undergoing living donor kidney transplantation. After retroperitoneoscopic nephrectomy kidneys were either perfused and stored with classical HTK solution or the new HTK-N solution. Primary endpoint was the glomerular filtration rate (eGFR according to CKD EPI) 3 months after transplantation. Secondary endpoints included graft and patient survival beside others. RESULTS: The study included 42 patients, of which 22 were randomized in the HTK-N group and 20 in the HTK group. The primary end point showed a mean eGFR of 55.4 ± 14.0 ml/min/1.73 m2 in the HTK group compared to a GFR of 57.2 ± 16.7 ml/min/m2 in the HTK-N group (P = .72). Regarding secondary endpoints, there were no apparent differences. Posttransplant graft and patient survival was 100%. CONCLUSION: This study is the first clinical application of HTK-N for kidney preservation and demonstrates non-inferiority compared to HTK in the setting of living donor kidney transplantation.

Recovery from cold-induced mitochondrial fission in endothelial cells requires reconditioning temperatures of ≥ 25◦C.

Mitochondrial integrity and function constitute a prerequisite for cellular function and repair processes. We have previously shown that mitochondria of different cell types exhibit pronounced fragmentation under hypothermic conditions. This fission, accompanied by a decline of cellular ATP content, showed reversibility at 37◦C. However, it is unclear whether other temperatures as currently discussed for reconditioning of organs allow this reconstitution of mitochondria. Therefore, we here study in a model of cultured porcine aortic endothelial cells how different rewarming temperatures affect mitochondrial re-fusion and function. After 48 h cold incubation of endothelial cells in Krebs-Henseleit buffer with glucose (5 mM) and deferoxamine (1 mM) at 4◦C pronounced mitochondrial fission was observed. Following 2 h rewarming in cell culture medium, marked fission was still present after rewarming at 10◦ or 15◦C. At 21◦C some re-fusion was visible, which became more marked at 25◦C. Networks of tubular mitochondria similar to control cells only re-appeared at 37◦C. ATP content decreased at 4◦C from 3.6 ± 0.4 to 1.6 ± 0.4 nmol/106 cells and decreased even further when rewarming cells to 10◦ and 15◦C. Values after rewarming at 21◦C were similar to the values before rewarming while ATP gradually increased at higher rewarming temperatures. Metabolic activity dropped to 5 ± 11% of control values during 4◦C incubation and recovered with increasing temperatures to 36 ± 10% at 25◦C and 78 ± 17% at 37◦C. Integrity of monolayers, largely disturbed at 4◦C (large gaps between endothelial cells; cell injury ≤ 1%), showed partial recovery from 15◦C upwards, complete recovery at 37◦C. Endothelial repair processes (scratch assay) at 25◦C were clearly inferior to those at 37◦C. These data suggest that reconditioning temperatures below 21◦C are not optimal with regard to reconstitution of mitochondrial integrity and function. For this goal, temperatures of at least 25◦C appear required, with 30◦C being superior and 37◦C yielding the best results.

Optimization of long-term cold storage of rat precision-cut lung slices with a tissue preservation solution.

Precision-cut lung slices (PCLS) are used as ex vivo model of the lung to fill the gap between in vitro and in vivo experiments. To allow optimal utilization of PCLS, possibilities to prolong slice viability via cold storage using optimized storage solutions were evaluated. Rat PCLS were cold stored in DMEM/F-12 or two different preservation solutions for up to 28 days at 4°C. After rewarming in DMEM/F-12, metabolic activity, live/dead staining, and mitochondrial membrane potential was assessed to analyze overall tissue viability. Single-cell suspensions were prepared and proportions of CD45+, EpCAM+, CD31+, and CD90+ cells were analyzed. As functional parameters, TNF-α expression was analyzed to detect inflammatory activity and bronchoconstriction was evaluated after acetylcholine stimulus. After 14 days of cold storage, viability and mitochondrial membrane potential were significantly better preserved after storage in solution 1 (potassium chloride rich) and solution 2 (potassium- and lactobionate-rich analog) compared with DMEM/F-12. Analysis of cell populations revealed efficient preservation of EpCAM+, CD31+, and CD90+ cells. Proportion of CD45+ cells decreased during cold storage but was better preserved by both modified solutions than by DMEM/F-12. PCLS stored in solution 1 responded substantially longer to inflammatory stimulation than those stored in DMEM/F-12 or solution 2. Analysis of bronchoconstriction revealed total loss of function after 14 days of storage in DMEM/F-12 but, in contrast, a good response in PCLS stored in the optimized solutions. An improved base solution with a high potassium chloride concentration optimizes cold storage of PCLS and allows shipment between laboratories and stockpiling of tissue samples.

Inhaled sphingosine has no adverse side effects in isolated ventilated and perfused pig lungs.

Ex-vivo lung perfusion (EVLP) systems like XVIVO are more and more common in the setting of lung transplantation, since marginal donor-lungs can easily be subjected to a performance test or be treated with corticosteroids or antibiotics in high dose regimes. Donor lungs are frequently positive in bronchoalveolar lavage (BAL) bacterial cultures (46-89%) which leads to a donor-to-recipient transmission and after a higher risk of lung infection with reduced posttransplant outcome. We have previously shown that sphingosine very efficiently kills a variety of pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus and epidermidis, Escherichia coli or Haemophilus influenzae. Thus, sphingosine could be a new treatment option with broadspectrum antiinfective potential, which may improve outcome after lung transplantation when administered prior to lung re-implantation. Here, we tested whether sphingosine has any adverse effects in the respiratory tract when applied into isolated ventilated and perfused lungs. A 4-h EVLP run using minipig lungs was performed. Functional parameters as well as perfusate measurements where obtained. Biopsies were obtained 30 min and 150 min after inhalation of sphingosine. Tissue samples were fixed in paraformaldehyde, embedded in paraffin and sectioned. Hemalaun, TUNEL as well as stainings with Cy3-coupled anti-sphingosine or anti-ceramide antibodies were implemented. We demonstrate that tube-inhalation of sphingosine into ex-vivo perfused and ventilated minipig lungs results in increased levels of sphingosine in the luminal membrane of bronchi and the trachea without morphological side effects up to very high doses of sphingosine. Sphingosine also did not affect functional lung performance. In summary, the inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no local side effects in ex-vivo perfused and ventilated minipig lungs.

Use of the new preservation solution Custodiol-MP for ex vivo reconditioning of kidney grafts.

Organ shortage and the increasing use of extended criteria donor grafts for transplantation drives efforts for more efficient organ preservation strategies from simple cold storage toward dynamic organ reconditioning. The choice of a suitable preservation solution is of high relevance in different organ preservation or reconditioning situations. Custodiol-MP is a new machine perfusion solution giving the opportunity to add colloids according to organ requirements. The present study aimed to compare new Custodiol-MP with clinically established Belzer MPS solution. Porcine kidneys were ischemically predamaged and cold stored for 20 hours. Ex vivo machine reconditioning was performed either with Custodiol-MP (n = 6) or with Belzer MPS solution (n = 6) for 90 minutes with controlled oxygenated rewarming up to 20°C. Kidney function was evaluated using an established ex vivo reperfusion model. In this experimental setting, differences between both types of perfusion solutions could not be observed. Machine perfusion with Custodiol-MP resulted in higher creatinine clearance (7.4 ± 8.6 mL/min vs. 2.8 ± 2.5 mL/min) and less TNC perfusate levels (0.22 ± 0.25 ng/mL vs. 0.09 ± 0.08 ng/mL), although differences did not reach significance. For short-term kidney perfusion Custodiol-MP is safe and applicable. Particularly, the unique feature of flexible colloid supplementation makes the solution attractive in specific experimental and clinical settings.

A novel histidine-tryptophan-ketoglutarate formulation ameliorates intestinal injury in a cold storage and ex vivo warm oxygenated reperfusion model in rats.

AIM: The present study aims to evaluate protective effects of a novel histidine-tryptophan-ketoglutarate solution (HTK-N) and to investigate positive impacts of an additional luminal preservation route in cold storage-induced injury on rat small bowels. METHODS: Male Lewis rats were utilized as donors of small bowel grafts. Vascular or vascular plus luminal preservation were conducted with HTK or HTK-N and grafts were stored at 4°C for 8 h followed by ex vivo warm oxygenated reperfusion with Krebs-Henseleit buffer for 30 min. Afterwards, intestinal tissue and portal vein effluent samples were collected for evaluation of morphological alterations, mucosal permeability and graft vitality. RESULTS: The novel HTK-N decreased ultrastructural alterations but otherwise presented limited effect on protecting small bowel from ischemia-reperfusion injury in vascular route. However, the additional luminal preservation led to positive impacts on the integrity of intestinal mucosa and vitality of goblet cells. In addition, vascular plus luminal preservation route with HTK significantly protected the intestinal tissue from edema. CONCLUSION: HTK-N protected the intestinal mucosal structure and graft vitality as a luminal preservation solution. Additional luminal preservation route in cold storage was shown to be promising.

Use of modified Custodiol-N as perfusion solution in ex vivo lung perfusion.

OBJECTIVES: Ex vivo Lung Perfusion (EVLP) is a promising tool to increase the donor pool for lung transplantation. Custodiol-N solution was originally designed for organ preservation during cold static preservation (CSP) and was successfully used for machine perfusion in kidneys. It was the aim of this study to compare the lung functional outcomes after 4 hours of EVLP using modified Custodiol-N or STEEN SolutionTM as perfusion solution. METHODS: In a porcine DCD model, lungs were perfused either with STEEN SolutionTM (Standard SS, n=8) or modified Custodiol-N with added 1.1 g/l glucose monohydrate and 50 g/l dextran 40 (CD, n=8). For a third group 7 g/l albumin was supplemented to modified Custodiol-N (CDA, n=8). During four hours of EVLP pulmonary gas exchange and activities of lactate dehydrogenase (LDH) and alkaline phosphatase (AP) in perfusate were recorded. RESULTS: Lungs that underwent EVLP with modified Custodiol-N showed significantly higher oxygen capacity (ΔpO2 averaged over four hours of EVLP: SS: 236.28 ± 47.26 mmHg, CD: 402.79 ± 30.33 mmHg, CDA: 414.86 ± 9.77 mmHg) than lungs perfused with STEEN SolutionTM. The addition of albumin did not have a significant effect on lung function but these lungs showed lower wet/dry ratio. CONCLUSION: In a porcine DCD model of 9 hours CSP followed by four hours of EVLP the use of modified Custodiol-N as perfusion solution was feasible and associated with higher oxygen capacity than STEEN SolutionTM. The addition of albumin seems to further stabilize lung function.

Effects of immediate versus delayed ex-vivo lung perfusion in a porcine cardiac arrest donation model.

OBJECTIVE: Ex-vivo lung perfusion is a promising tool to evaluate and recondition marginal donor lungs usually after a cold static preservation. The concept of continuous organ perfusion is supposed to reduce ischemic damage; however, the optimal perfusion protocol has not been established yet. The aim of this study was to compare immediate ex-vivo lung perfusion (I-EVLP) to delayed ex-vivo lung perfusion (D-EVLP) after a certain cold static preservation period on lung function in a large animal model. METHODS: In a porcine model, lungs were procured after circulatory death and 60 min of no-touch warm ischemia. Lungs were preserved with single-flush cold low potassium dextran solution and prepared either for I-EVLP (n = 8) or stored cold for 9 h with subsequent D-EVLP (n = 8). Functional outcomes and morphology were compared during 4 h of ex-vivo lung perfusion, using STEEN SolutionTM as perfusion solution. RESULTS: Pulmonary functional data, perfusate activities of lactate dehydrogenase, alkaline phosphatase, and products of lipid peroxidation did not differ significantly. There was a trend toward lower wet-dry ratio (I-EVLP: 13.4 ± 2.9; D-EVLP: 9.1 ± 2.5) and higher ΔpO2 in D-EVLP group (I-EVLP: 209 ± 51.6 mmHg; D-EVLP: 236.3 ± 47.3 mmHg). CONCLUSION: In this donation-after-circulatory-death model, 9 h of cold static preservation followed by ex-vivo lung perfusion results in comparable pulmonary function to I-EVLP as indicated by oxygenation capacities and wet-dry ratio. Our findings indicate that prolonged cold static preservation prior to ex-vivo lung perfusion is as safe and effective as I-EVLP in the procurement of donor lungs.

Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution.

Cryopreservation is the only long-term storage option for the storage of vessels and vascular constructs. However, endothelial barrier function is almost completely lost after cryopreservation in most established cryopreservation solutions. We here aimed to improve endothelial function after cryopreservation using the 2D-model of porcine aortic endothelial cell monolayers. The monolayers were cryopreserved in cell culture medium or cold storage solutions based on the 4°C vascular preservation solution TiProtec®, all supplemented with 10% DMSO, using different temperature gradients. After short-term storage at -80°C, monolayers were rapidly thawed and re-cultured in cell culture medium. Thawing after cryopreservation in cell culture medium caused both immediate and delayed cell death, resulting in 11 ± 5% living cells after 24 h of re-culture. After cryopreservation in TiProtec and chloride-poor modifications thereof, the proportion of adherent viable cells was markedly increased compared to cryopreservation in cell culture medium (TiProtec: 38 ± 11%, modified TiProtec solutions ≥ 50%). Using these solutions, cells cryopreserved in a sub-confluent state were able to proliferate during re-culture. Mitochondrial fragmentation was observed in all solutions, but was partially reversible after cryopreservation in TiProtec and almost completely reversible in modified solutions within 3 h of re-culture. The superior protection of TiProtec and its modifications was apparent at all temperature gradients; however, best results were achieved with a cooling rate of -1°C/min. In conclusion, the use of TiProtec or modifications thereof as base solution for cryopreservation greatly improved cryopreservation results for endothelial monolayers in terms of survival and of monolayer and mitochondrial integrity.

Serum-Free Cryopreservation of Primary Rat Hepatocytes in a Modified Cold Storage Solution: Improvement of Cell Attachment and Function.

AIMS: The use of primary hepatocytes in pharmacological and toxicological research as well as for clinical and biotechnological applications requires adequate storage options for these cells. However, hepatocytes are very susceptible to cryopreservation injury. Based on experience in hypothermic storage of hepatocytes, we, in this study, aimed to optimize hepatocyte cryopreservation. MATERIALS AND METHODS: Rat hepatocytes were cryopreserved in serum-containing cell culture medium or in serum-free solutions optimized for hypothermic storage, all with 10% dimethyl sulfoxide, using a standard protocol (-1°C/min in a controlled-rate freezer). After rapid thawing, cells were seeded in supplemented Leibovitz-15 cell culture medium without further purification steps. Cell attachment and metabolic activity were assessed. RESULTS: Cell attachment (37% ± 15% vs. 9% ± 7% of noncryopreserved control cells) and metabolic activity (resazurin reduction: 47% ± 23% vs. 25% ± 8%; glucose release: 44% ± 6% vs. 15% ± 7%; and urea production: 31% ± 16% vs. 5% ± 8%) were significantly higher after cryopreservation in the new solution compared to cryopreservation in cell culture medium. Experiments with modified solutions suggested that the protective effect of the new solution is multifactorial. CONCLUSIONS: In summary, significant improvement of cell attachment and function compared to cell culture medium was achieved after cryopreservation in serum-free hepatocyte cold storage solution.

Cold Storage Injury to Rat Small-bowel Transplants-Beneficial Effect of a Modified HTK Solution.

BACKGROUND: The small bowel is prone to ischemic injury during transport before transplantation, an injury that endangers the recipient patient. The small-bowel mucosal microcirculation in particular appears to be highly sensitive to injury. Current preservation solutions such as histidine-tryptophan-ketoglutarate (HTK) solution provide some protection to the graft. However, these were developed decades ago and do not address several critical processes, such as hypoxia-induced membrane pores and free radical-mediated hypothermic injury. METHODS: To protect the graft from cold ischemic injury, we implemented a modified HTK solution here, including glycine, alanine, and iron chelators in a heterotopic, syngeneic small-bowel transplantation model of the rat. The effects of the modified solution and its major components were compared against the conventional HTK solution using intravital microscopy in the early reperfusion period. RESULTS: The amino acid glycine, added to HTK solution, slightly improved mucosal perfusion. Both, the modified base solution (without iron chelators) and iron chelators increased functional capillary density of the mucosa during the early reperfusion period. The complete modified solution (with glycine, alanine, and iron chelators) significantly increased the perfusion index, functional capillary density of the mucosa, and red blood cell velocity in the grafts after reperfusion in comparison with the grafts preserved with HTK. CONCLUSIONS: The modified preservation solution improved the microcirculation of the transplants and needs detailed evaluation in further models of small-bowel transplantation.

Methylene Blue Treatment of Grafts During Cold Ischemia Time Reduces the Risk of Hepatitis C Virus Transmission.

Background: Although organ shortage is a rising problem, organs from hepatitis C virus (HCV) ribonucleic acid (RNA)-positive donors are not routinely transplanted in HCV-negative individuals. Because HCV only infects hepatocytes, other organs such as kidneys are merely contaminated with HCV via the blood. In this study, we established a protocol to reduce HCV virions during the cold ischemic time. Methods: Standard virological assays were used to investigate the effect of antivirals, including methylene blue (MB), in different preservation solutions. Kidneys from mini pigs were contaminated with Jc1 or HCV RNA-positive human serum. Afterwards, organs were flushed with MB. Hypothermic machine perfusion was used to optimize reduction of HCV. Results: Three different antivirals were investigated for their ability to inactivate HCV in vitro. Only MB completely inactivated HCV in the presence of all perfusion solutions. Hepatitis C virus-contaminated kidneys from mini pigs were treated with MB and hypothermic machine perfusion without any negative effect on the graft. Human liver-uPA-SCID mice did not establish HCV infection after inoculation with flow through from these kidneys. Conclusions: This proof-of-concept study is a first step to reduce transmission of infectious HCV particles in the transplant setting and might serve as a model for other relevant pathogens.

Preservation of Cell Structure, Metabolism, and Biotransformation Activity of Liver-On-Chip Organ Models by Hypothermic Storage.

The liver is a central organ in the metabolization of nutrition, endogenous and exogenous substances, and xenobiotic drugs. The emerging organ-on-chip technology has paved the way to model essential liver functions as well as certain aspects of liver disease in vitro in liver-on-chip models. However, a broader use of this technology in biomedical research is limited by a lack of protocols that enable the short-term preservation of preassembled liver-on-chip models for stocking or delivery to researchers outside the bioengineering community. For the first time, this study tested the ability of hypothermic storage of liver-on-chip models to preserve cell viability, tissue morphology, metabolism and biotransformation activity. In a systematic study with different preservation solutions, liver-on-chip function can be preserved for up to 2 d using a derivative of the tissue preservation solution TiProtec, containing high chloride ion concentrations and the iron chelators LK614 and deferoxamine, supplemented with polyethylene glycol (PEG). Hypothermic storage in this solution represents a promising method to preserve liver-on-chip function for at least 2 d and allows an easier access to liver-on-chip technology and its versatile and flexible use in biomedical research.

Resveratrol Does Not Protect from Ischemia-Induced Acute Kidney Injury in an in Vivo Rat Model.

BACKGROUND/AIMS: The natural polyphenol resveratrol (RSV) has been shown to ameliorate ischemia/reperfusion (I/R)-induced damage. Therefore, a rat model of I/R-induced AKI equipped with intensive monitoring was utilized to examine direct renal protection by RSV in vivo. METHODS: AKI was induced by bilateral renal clamping (45 min) followed by reperfusion (3 h). Solvent-free RSV was continuously infused intravenously (0.056 and 0.28 mg/kg) in a total volume of 7 ml/kg/h starting from 30 min before renal clamping. At a mean arterial blood pressure below 70 mmHg for more than 5 min, bolus injections of 0.5 ml 0.9% NaCl solution were administered repetitively (max. 5 ml/kg/h). RESULTS: No differences could be found between normoxic control groups with/without RSV. Bilateral renal clamping and subsequent reperfusion caused a progressive rise in creatinine, cystatin C, and CK, a decrease in cellular ATP content and diuresis. Infusion of RSV increased sirtuin 1 expression after ischemia/reperfusion and was associated with decreased blood pressure during ischemia and early reperfusion accompanied by an increased requirement of bolus injections as well as with increased expression of TNFα. CONCLUSION: RSV did not exert protective effects on I/R-induced AKI in the present short-term in vivo rat model. The lack of protection is potentially connected to aggravation of blood pressure instability.