raffinose and thiazolyl-blue

Celsior solution (CS), a new preservation solution in thoracic organ transplantation, was evaluated for its efficacy in cold preservation of human liver endothelial cells (HLEC) and was compared to University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). HLEC cultures were preserved at 4 degrees C in CS, UW, and HTK, for 2, 6, 12, 24, and 48 hours, with 6 hours of reperfusion. Levels of lactate dehydrogenase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and adenosine 5'-triphosphate (ATP) were measured after each interval of ischemia and the respective phase of reperfusion. Preservation injury of HLEC as measured by LDH release, intracellular ATP level, and MTT reduction were overall significantly (P > CS > HTK.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Cells, Cultured; Coloring Agents; Cryopreservation; Cytoprotection; Disaccharides; Electrolytes; Endothelial Cells; Glucose; Glutamates; Glutathione; Histidine; Humans; Insulin; L-Lactate Dehydrogenase; Liver; Mannitol; Organ Preservation Solutions; Potassium Chloride; Preservation, Biological; Procaine; Raffinose; Tetrazolium Salts; Thiazoles

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 superio

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Caspase 3; Caspases; Cell Count; Cell Culture Techniques; Cell Division; Cell Survival; Cells, Cultured; Cryopreservation; Cryoprotective Agents; Disaccharides; DNA Fragmentation; Electrolytes; Female; Glucose; Glutamates; Glutathione; Hepatocytes; Histidine; Hypothermia, Induced; Insulin; L-Lactate Dehydrogenase; Liver Diseases; Male; Mannitol; Models, Biological; Necrosis; Organ Preservation Solutions; Potassium Chloride; Procaine; Raffinose; Tetrazolium Salts; Thiazoles; Tissue Transplantation

Celsior, a new preservation solution in thoracic organ transplantation was evaluated for efficacy in cold preservation of human hepatocytes and compared with University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). Human hepatocyte cultures were preserved at 4 degrees C in Celsior, UW and HTK for 2, 6, 12, 24 and 48 h with 6 h of reperfusion. Levels of lactate dehydrogenase (LDH; cell necrosis), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; mitochondrial function), and adenosine 5'-triphosphate (ATP; loss of intracellular energy) were measured. Cell necrosis, mitochondrial dysfunction, and loss of ATP were significantly ( P<0.001, P<0.001, P<0.002, respectively) lower in Celsior than in HTK. The amount of cell necrosis and mitochondrial dysfunction in Celsior solution (CS) and UW was equal ( P=n.s.) up to 24 h and significantly lower in UW after 48 h ( P<0.001). Additionally, the intracellular level of ATP was significantly higher after ischemia ( P<0.001) and reperfusion from long-term ischemia (24, 48 h) ( P<0.002). We can conclude that Celsior was superior to HTK and equal to UW in the protection of human hepatocytes against cold preservation injury from ischemia and reperfusion. Furthermore, Celsior was effective in long-term preservation of human hepatocytes.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Cells, Cultured; Cryopreservation; Disaccharides; Electrolytes; Glucose; Glutamates; Glutathione; Hepatocytes; Histidine; Humans; Insulin; L-Lactate Dehydrogenase; Mannitol; Organ Preservation Solutions; Oxidation-Reduction; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Tetrazolium Salts; Thiazoles

The effects of a cryopreservation procedure on the biochemical, morphological and functional integrity of rat liver slices just after thawing and after 24 h culture were evaluated. Freshly prepared slices were incubated in modified University of Wisconsin solution containing 50% fetal calf serum and 10% dimethyl sulfoxide for 20 min on ice prior to a rapid cooling in liquid nitrogen. After 10-40 days, slices were thawed rapidly at 42 degrees C. Total protein content and (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) (MTT) reduction were well preserved at thawing, whereas ATP content was markedly decreased relative to freshly prepared slices (-83%). The major microscopic findings in sections of just-thawed liver slices consisted of hepatocellular dissociation and minimal apoptosis. The qualitative profile of antipyrine (AP) metabolism was well preserved in cryopreserved slices, but the amounts of phase I and phase II AP metabolites produced over a 3-h incubation period were markedly reduced relative to fresh slices (-58 to -71%). When cryopreserved slices were cultured for 24 h after thawing, the viability was markedly reduced, as reflected by the almost complete absence of MTT reduction and the loss of ATP content. Histological examinations showed extensive cellular necrosis. The amount of AP metabolites produced by cryopreserved slices was similar after a 3- or a 24-h culture period, indicating that AP metabolism capacities were lost at 24 h culture. In conclusion, our results suggest that cryopreserved rat liver slices may be a useful model for short-term in vitro determination of drug metabolism pathways. Further work is required to extend their use for toxicological studies.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Antipyrine; Apoptosis; Biotransformation; Cell Survival; Cryopreservation; Cryoprotective Agents; Dimethyl Sulfoxide; Glutathione; Hepatocytes; Insulin; Liver; Male; Organ Culture Techniques; Organ Preservation Solutions; Oxidation-Reduction; Proteins; Raffinose; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Thiazoles

Various cryopreservation techniques have been investigated to elongate preservation time, however, most have failed to be clinically induced because of damage due to ice crystal formation. Subzero nonfreezing conditions could theoretically reduce organ metabolism without damage due to ice crystal formation. We evaluated the superiority of subzero nonfreezing storage compared with conventional hypothermic storage using isolated rat hepatocytes stored in University of Wisconsin (UW) solution without cryoprotectants.. Hepatocytes of Wistar rats isolated by collagenase digestion were suspended in UW solution and divided into the following three groups: subzero nonfreezing group (-4 degrees C), zero nonfreezing group (0 degrees C), and control group (4 degrees C). They were stored for 48 hr at the temperatures indicated. After 24 and 48 hr of storage, we carried out a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and measured lactate dehydrogenase release, lactic acid, ATP content, and the ability of hepatocytes to synthesize urea. After 48 hr of storage, morphological differences between the control group and the subzero nonfreezing group were investigated by scanning and transmission electron microscopy.. Significant improvements of the trypan blue exclusion test and ATP contents in the subzero nonfreezing group were observed. Lactic acid production was also significantly suppressed in the subzero nonfreezing group compared with that in the control group. The MTT assay value was significantly better at -4 degrees C than at 4 degrees C. The rate of urea synthesis at -4 degrees C was higher than that at 4 degrees C. Electron microscopy revealed that subzero nonfreezing delayed the lethal bleb-forming process of stored hepatocytes, which was followed by mitochondrial swelling, compared with the control group.. Subzero nonfreezing storage (-4 degrees C) in UW solution could provide better preservability for isolated rat hepatocytes with protection against hypoxic cell injury compared with conventional hypothermic storage (4 degrees C).

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cell Separation; Cryopreservation; Glutathione; Hypoxia; Insulin; Lactic Acid; Liver; Male; Microscopy, Electron, Scanning; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Tetrazolium Salts; Thiazoles; Trypan Blue

Topics: Adenosine; Allopurinol; Animals; Cell Survival; Cold Temperature; Coloring Agents; Disaccharides; Glutathione; Histidine; Insulin; Islets of Langerhans; Isotonic Solutions; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Strains; Solutions; Tetrazolium Salts; Thiazoles; Time Factors; Tissue Preservation