Enhanced prevention of cell death by hypothermic storage with propyl gallate.

Evaluation of preservation solutions by ESR-spectroscopy: Superior effects of University of Wisconsin over Histidine–Tryptophan–Ketoglutarate in reducing renal reactive oxygen species

Donor cells can be preserved in University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), or Celsior solution. However, differences in efficacy and mode of action in preventing hypothermia-induced cell injury have not been unequivocally clarified. Therefore, we investigated and compared necrotic and apoptotic cell death of freshly isolated primary porcine hepatocytes after hypothermic preservation in UW, HTK, and Celsior solutions and subsequent normothermic culturing. Hepatocytes were isolated from porcine livers, divided in fractions, and hypothermically (4 degrees C) stored in phosphate-buffered saline (PBS), UW, HTK, or Celsior solution. Cell necrosis and apoptosis were assessed after 24- and 48-h hypothermic storage and after 24-h normothermic culturing following the hypothermic preservation periods. Necrosis was assessed by trypan blue exclusion, lactate dehydrogenase (LDH) release, and mitochondrial 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. Apoptosis was assessed by the induction of histone-associated DNA fragments and cellular caspase-3 activity. Trypan blue exclusion, LDH release, and MTT reduction of hypothermically preserved hepatocytes showed a decrease in cell viability of more than 50% during the first 24 h of hypothermic preservation. Cell viability was further decreased after 48-h preservation. DNA fragmentation was slightly enhanced in hepatocytes after preservation in all solutions, but caspase-3 activity was not significantly increased in these cells. Normothermic culturing of hypothermically preserved cells further decreased cell viability as assessed by LDH release and MTT reduction. Normothermic culturing of hypothermically preserved hepatocytes induced DNA fragmentation, but caspase-3 activity was not hanced in these cells. Trypan blue exclusion, LDH leakage, and MTT reduction demonstrated the highest cell viability after storage in Celsior, and DNA fragmentation was the lowest in cells that had been stored in PBS and UW solutions. None of the preservation solutions tested in this study was capable of adequately preventing cell death of isolated porcine hepatocytes after 24-h hypothermic preservation and subsequent 24-h normothermic culturing. Culturing of isolated and hypothermically preserved hepatocytes induces DNA fragmentation, but does not lead to caspase-3 activation. With respect to necrosis and DNA fragmentation of hypothermically preserved cells, UW and Celsior were superior to PBS and HTK solutions in this model of isolated porcine hepatocyte preservation.

A new platform of hypothermic solutions, the HypoThermosol® (HTS) series, has been developed for the improved hypothermic storage of cells, tissues, and organs. Cells and tissues cold-stored in HTS-FRS demonstrate improved viability when returned to normothermic temperatures in comparison with the parent solution, HTS-BASE, or University of Wisconsin (UW) solution (UW-ViaSpan®). While our group and others have implicated apoptosis as a major player in cell death initiated by extended hypothermic storage, it has been unclear if the improved performance of HTS-FRS as a hypothermic storage solution is due to its ability to inhibit apoptosis. Data reported herein show that human renal cells hypothermically stored in renal cell culture medium, HTS-FRS, HTS-BASE, or UW solution demonstrated improved survival in HTS-FRS. Following 5 days of hypothermic preservation and 1 day of recovery at 37°C, cells preserved in HTS-FRS exhibit 75% metabolic activity, whereas cells stored in HTS-BASE, UW, or culture media demonstrate 32%, 17%, and 6% recovery, respectively. In addition, cells stored in HTS-BASE supplemented with caspase inhibitor exhibit increased cell numbers in comparison to cells stored in HTS-BASE (72% vs. 30% after 7 days of cold storage and 2 days of recovery). Experiments with annexin and propidium iodide as well as assessment of caspase activities suggest that the improved performance of HTS-FRS as a preservation solution may be due to its ability to inhibit both apoptosis and necrosis.

<p>Introduction. The amniotic membrane is used in transplant surgery, ophthalmology and dermatology. Various methods have been developed to preserve amniotic membrane: hypothermic storage, cryopreservation, lyophilization. Transplantation of fresh amniotic membrane showed low inflammatory response. The efficient antibiotic solutions are carefully chosen for the hypothermic storage of amniotic membranes. The aim of this study was to compare the efficacy of two antibiotic solutions for the hypothermic amniotic membrane preservation and the structure of the amniotic membrane after the preservation process. Methods. Fifteen amniotic membranes were prepared and hypothermically stored in penicillin solution in BSS (2000 IU/ml) while the remaining fifteen in an antibiotic solution containing: benzylpenicillin (50 µg/ml), gentamicin (100 µg/ml), ciprofloxacin (200 µg/ml) and fluconazole (100 µg/ml). All amniotic membranes were microbiologically tested after preparation and after hypothermic storage for two weeks. Histological analysis of thirty amniotic membranes was performed after the process of preservation. Results. Fifteen amniotic membranes were sterile after hypothermic preservation in the penicillin solution. Also, fifteen amniotic membranes were sterile after hypothermic preservation in the solution of antibacterial agents (penicillin, gentamicin, ciprofloxacin) and antifungal agent fluconazole. The amniotic membrane had a normal structure and thickness of 35.33±11.03 mm. Conclusion. Both antibiotic solutions, one that contains only penicillin and one that contains multiple antibacterial agents and fluconazole, provide sterility of fresh hypothermically stored amniotic membranes for two weeks. In the preparation of fresh hypothermically stored amniotic membrane, a solution with multiple antibiotics is preferred. The normal tissue structure of the amniotic membrane was histologically confirmed after the process of preservation.</p>

PII: S0003-4975(99)00714-6

Cold ischemia and reperfusion during renal transplantation result in release of reactive oxygen species. The aim of this study is to examine whether cold storage induced cell injury can be ameliorated by adding flavonoids directly to preservation solutions. Cultured renal tubular epithelial cells (LLC-PK1) were stored in University of Wisconsin (UW) or Euro-Collins (EC) solution at 4 degrees C for 20 hours. Preservation solutions were supplemented with various flavonoids. After rewarming, structural and metabolic cell integrity was measured by lactate dehydrogenase (LDH) release and MTT-test, and lipid peroxidation was assessed from generation of thiobarbituric acid-reactive substances (TBARS). Twenty hours of cold storage resulted in a substantial loss of cell viability in both preservation solutions (in EC: LDH release 92.4+/-2.7%; MTT-test 0.5+/-0.7%). Addition of luteolin, quercetin, kempferol, fisetin, myricetin, morin, catechin, and silibinin significantly reduced cell injury (for luteolin in EC: LDH release 2.4+/-1.6%; MTT-test 110.3+/-10.4%, P<0.01; TBARS-production (related to cold stored control cells) 8.9+/-2.6%). No cytoprotection was found for apigenin, naringenin, and rutin. Protective potency of flavonoids depends on number of hydroxyl-substituents and lipophilicity of the diphenylpyran compounds. Cold storage induced injury of renal tubular cells was substantially ameliorated by adding selected flavonoids directly to preservation solutions.

To evaluate the reparative capacity of the mechanically injured endothelium of corneas stored under organ culture (OC) or hypothermic conditions. The central endothelium of 12 pairs of human corneas with similar endothelial parameters was damaged to create a 1 mm(2) lesion. One cornea from each pair was stored under OC and one under hypothermic conditions. The endothelial cell density (ECD), coefficient of variation, hexagonality and percentage of dead cells were assessed before and after damage and on days 7, 14, 21 and 28 of storage. The mean ECD of corneas subsequently stored under OC or hypothermic conditions was 2764/mm(2). Immediately after damage, a denuded Descemet's membrane with a few remaining dead cells was observed at the injured area. After 7 days of storage under OC conditions, almost no dead cells were observed at the place of injury. A non-significant worsening of the qualitative parameters (polymegatism and pleomorphism) was found. After 14 days, ECD was 1933/mm(2) and 2478/mm(2) centrally and pericentrally, respectively. Similar values were found after 21 and 28 days of storage. The lesions with remnant dead cells persisted throughout hypothermic preservation. From day 14 the corneas became cloudy and in poor condition, while the pericentral ECD was 2523/mm(2). The reparative capacity of the cornea is maintained under OC but not under hypothermic conditions. For corneas containing dead endothelial cells, OC is therefore the method of choice because it may improve the quality of the stored tissue.

1. Hypothermic storage of whole organs flushed with a preservation solution is common practice in clinical transplantation. This procedure leaves vascular endothelial cells in direct contact with the preservation solution during the length of the cold ischaemic period. 2. Aiming to study the effects of organ preservation on vascular endothelium, we subjected cultures of human umbilical vein endothelial cells to hypoxic and hypothermic storage conditions in vitro for 3 or 16 h. Four preservation solutions with different levels of sodium and potassium were tested. Morphometric analysis and 51Cr leakage index were used to assess monolayer continuity, cell viability and membrane integrity. 3. Hypothermic storage resulted in severe changes in endothelial cell morphology with formation of intercellular gaps that destroyed monolayer continuity after only 3h. Cellular blebbing was a common feature in seriously damaged cells. 4. Morphometric analysis and 51Cr leakage results correlated well. No significant differences between the solutions tested were found after 3h of hypothermic hypoxic storage. After 16h, viability and monolayer continuity were significantly better preserved (Mann-Whitney, P < 0.01) in cells stored in lactobionate-based solutions than in hypertonic citrate solutions. No significant differences were found between endothelial cells stored in extracellular versus intracellular types of solutions for the lactobionate-based solutions. 5. The results of the present experiment showed that after a period of hypothermic hypoxic storage, vascular endothelial cells appeared morphologically deformed and poorly attached in vitro. Lactobionate-based preservation solutions were more effective in preserving viability and continuity. Protection of vascular endothelium under cold hypoxic conditions could be a critical factor in successfully preserving organs for transplantation.

Intracellular Volumes and Membrane Permeability in Rat Hearts During Prolonged Hypothermic Preservation with St Thomas and University of Wisconsin Solutions

Isolated hepatocytes, suspended in an organ preservation solution, can be preserved at 4°C for up to 6 days. After preservation, normothermic-normoxic incubation causes loss of hepatocyte viability. The addition of 3 mmol/L glycine to the rewarming medium prevents the loss of viability. In this study we investigated the cytoprotective effects of glycine under many conditions known to cause hepatocellular injury to understand the mechanism of cold-induced injury in the liver. Hepatocytes were suspended in modified Krebs-Henseleit buffer with or without 3 mmol/L glycine and exposed to agents or conditions known to induce cell death. Hepatocyte viability was assessed by measuring the percentage of lactate dehydrogenase leakage from the cells and the concentration of ATP during incubation at 37°C under room air for up to 90 min. Mitochondrial inhibitors (potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone); calcium ionophores (ionomycin and A23187); an oxidizing agent, tert-butyl hydroperoxide; and anoxia were all used to cause cell injury. Hepatocytes were also isolated from fasted rats and hypothermically preserved as another model of cell death. Other amino acids were also tested in the hypothermic preservation model to study the specificity of the amino acid requirement for prevention of lactate dehydrogenase leakage. Of the amino acids tested, only alanine (10 mmol/L) and the combination of alanine (3 mmol/L) and serine (3 mmol/L) were as effective as glycine in preventing lactate dehydrogenase release in the hypothermic preservation model. Anoxia, potassium cyanide (3 and 5 mmol/L) and carbonyl cyanide m-chlorophenylhydrazone (5 and 10 μmol/L) all induced cell injury, as indicated by release by the cell membrane of lactate dehydrogenase (60% to 90% release) and severe depletion of ATP content (less than 10% of freshly isolated cells). In this model of cell death, glycine suppressed lactate dehydrogenase release (25% to 35% release) but had no effect on ATP content. A23187 (10 to 50 μmol/L) and ionomycin (5 to 15 μmol/L) (calcium-induced cell death) caused lactate dehydrogenase leakage that was not prevented by glycine. Nor was induction of cell death by tert-butylhydroperoxide (oxidative stress) suppressed by glycine. Fasting the rat before hepatocyte isolation sensitized the cells to hypothermic preservation injury (80% lactate dehydrogenase release in fasted cells vs. 35% in fed cells) after 1 day of cold storage followed by rewarming. Glycine suppressed lactate dehydrogenase release from hepatocytes in fasted rats. Glycine was effective in suppressing hepatocellular injury caused by cold storage, anoxia, fasting and mitochondrial poisons. However, neither calcium-induced nor oxidative stress–induced cell death was prevented by glycine. This study suggests that hepatocellular injury caused by hypothermia (and suppressed by glycine) may not, therefore, be caused by calcium or oxidative stress (injuries not suppressed by glycine). However, in this hepatocyte model, the methods used to induce calcium or oxidative stress–dependent cell death may have resulted in effects more severe than those obtained in hepatocytes exposed to hypothermia, and these methods may have overwhelmed the cytoprotective effects of glycine against calcium-induced or oxidative stress–induced cell death. The beneficial effect of some amino acids (glycine, alanine and serine) in hypothermic preservation of hepatocytes suggest that these agents may be useful in improving liver preservation for transplantation. (HEPATOLOGY 1993;17:91–98.)

21. Supplemented oxygenation during hypothermic storage: Friend or foe

Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions

427 Curcumin (CUR), a component of the spice turmeric, enhances the liver preservation properties of Euro-Collin's (EC) solution, making EC storage for 24 hours at 4°C equivalent to University of Wisconsin (UW) solution or no hypothermic storage (NL) in an ex-vivo perfusion model. It is unclear whether this occurs because CUR replaces a critical component in UW missing from EC. Altematively, CUR might have an entirely novel effect and might be capable of enhancing UW preservation. To assess this, Sprague-Dawley rat livers underwent 2 hours' oxygenated perfusion after being flushed with different preservation solutions. The livers in the normal (NL) group (10) were immediately perfused. The UW group were flushed with UW solution + or − 100 μM CUR and stored for 24, 36, and 48 hours (10 each). During 120-minute periods, during which the liver was perfused via the portal vein with 37°C oxygenated Krebs-Henseleit solution at 18 cm H2O, flow rates, measured serially and shown here as the mean rates ± SE at 30 and 120 minutes. Bile production rates and liver enzyme (AST, ALT) release into the perfusate were also measured over the 120 minute perfusion. (Table)TableOur results indicate that as the duration of hypothermic storage increased, a distinct enhancement of UW's preservation of flow and bile production was seen with CUR. AST and ALT release, markers of hepatocellular injury, were not significantly changed, suggesting that they are less sensitive markers to preservation injury. CUR may contribute to liver preservation by a mechanism not covered by the components of UW.

The effect of hypothermic storage (HS) on viability and functional activity of human mesenchymal stromal cells (MSCs) in suspension and within alginate microspheres (AMSs) was studied. Cells were stored in sealed cryovials at 4°C in culture medium (CM), sucrose-based solution (SBS) and that of the University of Wisconsin (UW). After 3 and 7 days of storage the viability, metabolic activity and morphology of MSCs were assessed by MTT-assay, Alamar Blue test, adhesive activity, morphology and ability of multilineage differentiation at monolayer culture conditions. Hypothermic storage of MSCs suspension in CM for 3 days was established to result in a 3–4-fold decrease in their viability and adhesive ability. The encapsulation into AMSs partially prevented cell death. Hypothermic storage for 7 days caused death of almost all MSCs both in suspension and AMSs. The substitution of CM for UW and SBS preservation solutions maintained the MSCs viability and metabolic activity following HS within 3 days both in suspension and within AMSs. After 7 days of HS in UW and SBS solutions the MSCs in suspensions and within AMSs preserved their viability on the level of 60–80%, adhesive properties, metabolic activity, as well as the ability for induced adipogenic and osteogenic differentiation. The use of UW and SBS preservation solutions enabled to prolong the hypothermic storage terms for MSCs.Probl Cryobiol Cryomed 2015; 25(4):329-339.

The effects of low concentrations (10 and 100 microM) and high concentrations (1, 10, and 20 mM) of deferoxamine (DFO) on superoxide (O2-.) formation, lipid peroxidation, and cell injury were studied in freshly isolated perfused rat hepatocytes during a 2-h reoxygenation period after 2.5 h of anoxia. O2-. production was measured by lucigenin-enhanced chemiluminescence, lipid peroxidation by malondialdehyde (MDA) formation, and cell injury by lactate dehydrogenase (LDH) release. On reoxygenation and in the absence of DFO, O2-. generation increased 11-fold, MDA increased 3.7-fold, and LDH release practically doubled. Low concentrations of DFO had no effect on O2-. generation but decreased MDA and LDH release from 44 to 75%. High concentrations of DFO significantly depressed O2-. formation, with very little additional effect on MDA or LDH release. These experiments illustrate in a biological system the dual effect of DFO: 1) at low Concentrations, DFO acts as a specific iron chelator and inhibits lipid peroxidation and cell injury without preventing O2-. formation, and 2) at high concentrations, DFO acts as a nonspecific scavenger of oxygen free radicals such as O2-.