Cold Storage Medium Research Articles

A comparative study of different methods for the generation of tetra-n-butyl ammonium bromide clathrate hydrate slurry in a cold storage air-conditioning system

A cold storage air-conditioning system using tetra-n-butyl ammonium bromide (TBAB) clathrate hydrate slurry (CHS) as cold storage medium was built to investigate the high-efficiency method of TBAB CHS generation. In the present study, four kinds of different TBAB CHS generation methods were experimentally investigated and compared, and these methods included continuously cooling, turning off refrigerator while crystals appearing, supercooling release and accession of TBAB CHS into supercooled TBAB aqueous solution. The results showed that continuously cooling would lead to severe adhesion of crystal to the heat exchanger wall, and supercooling release took place with a big stochastic characteristic, hence the first and third method were concluded not reliable. Both the second and fourth methods could maintain the temperature of heat exchanger wall at a relatively higher level, therefore the crystal adhesion to the heat exchanger wall would be reduced significantly, which led to higher coefficient of performance (COP). In addition, accession of TBAB CHS into TBAB supercooled solution could shorten the time of supercooling release, resulting in about 21.8–35.4% shorter generation time than other methods. Moreover, the influence of flow rate on the CHS generation process was investigated, and the results showed that higher flow rate generally resulted in higher system COP.

Desferrioxamine in warm reperfusion media decreases liver injury aggravated by cold storage

To evaluate whether desferrioxamine decreases ischemia and perfusion injury aggravated by cold storage (CS) in a rat liver perfusion model. Isolated rat livers were kept in CS in University of Wisconsin Solution for 20 h at 4 °C, then exposed to 25 min of warm ischemia (WI) at 37 °C followed by 2 h of warm perfusion (WP) at 37 °C with oxygenated (95% oxygen and 5% carbon dioxide) Krebs-Henseleit buffer. Desferrioxamine (DFO), an iron chelator, was added at different stages of storage, ischemia and perfusion: in CS only, in WI only, in WP only, in WI and perfusion, or in all stages. Effluent samples were collected after CS and after WI. Perfusate samples and bile were collected every 30 min (0, 0.5, 1, 1.5 and 2 h) during liver perfusion. Cellular injury was assessed by the determination of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in the effluent and perfusate samples. Total iron was analysed in the perfusate samples. After WP, the liver was collected for the determination of liver swelling (wet to dry ratio) and liver morphological examination (hematoxylin and eosin staining). Increased CS time caused increased liver dysfunction during WP. After 2 h of WP, liver injury was indicated by increased release of AST (0.5 h CS: 9.4 ± 2.2 U/g liver vs 20 h CS: 45.9 ± 10.8 U/g liver, P < 0.05) and LDH (0.5 h CS: 59 ± 14 U/g liver vs 20 h CS: 297 ± 71 U/g liver, P < 0.05). There was an associated increase in iron release into the perfusate (0.5 h CS: 0.11 ± 0.03 μmoL/g liver vs 20 h CS: 0.58 ± 0.10 μmoL/g liver, P < 0.05) and reduction in bile flow (0.5 h CS: 194 ± 12 μL/g vs 20 h CS: 71 ± 8 μL/g liver, P < 0.05). When DFO was added during WI and WP following 20 h of CS, release of iron into the perfusate was decreased (DFO absent 0.58 ± 0.10 μmoL/g liver vs DFO present 0.31 ± 0.06 μmoL/g liver, P < 0.05), and liver function substantially improved with decreased release of AST (DFO absent 45.9 ± 10.8 U/g liver vs DFO present 8.1 ± 0.9 U/g liver, P < 0.05) and LDH (DFO absent 297 ± 71 U/g liver vs DFO present 56 ± 7 U/g liver, P < 0.05), and increased bile flow (DFO absent 71 ± 8 μL/g liver vs DFO present 237 ± 36 μL/g liver, P < 0.05). DFO was also shown to improve liver morphology after WP. Cellular injury (the release of LDH and AST) was significantly reduced with the addition of DFO in CS medium but to a lesser extent compared to the addition of DFO in WP or WI and perfusion. There was no effect on liver swelling or bile flow when DFO was only added to the CS medium. DFO added during WI and perfusion decreased liver perfusion injury aggravated by extended CS.

Viability of limbal epithelium after anterior lamellar harvesting using a microkeratome

Objective To determine the viability in cold eye bank storage of different layers of central and limbal corneal epithelium, including the limbal basal stem cell population, on days 0, 3, 6, and 9 after harvest using a large diameter microkeratome system. Methods Twenty-two human whole globes not suitable for transplantation were obtained from an eye bank (San Diego Eye Bank, San Diego, California) and used for study. Large-diameter anterior corneal discs were prepared using a large diameter microkeratome and stained with calcein AM and an ethidium homodimer to differentiate live from dead cells, respectively. A laser confocal microscope and digital imaging were used to distinguish live (green) from dead (red) cells. Central and limbal epithelial regions were isolated and the middle and basal epithelial sections were cell counted by 3 independent observers. These sections were stored up to 9 days at 4° C in an eye bank corneal storage medium. Differences were tested using nonparametric methods. Main outcome measures The percentage of live cells in each of these epithelial layers was determined for up to 9 days in cold eye bank corneal storage medium. Results At all time points studied, the better protected basal epithelial layers displayed greater mean viability than the overlying middle epithelial layers. However, the difference was not statistically significant on all days. When comparing the basal epithelial viability of the limbal and central regions, after day 0 in 4° C cold organ culture, the observed viability of the limbal basal epithelium, the purported location of the limbal epithelial stem cell region, was significantly greater than that of the central epithelium. On day 0, median limbal basal versus central basal epithelial viability were 100% (range, 71.7–100%) versus 98.4% (range, 88.9–100%) ( P>0.05); on day 3, 100% (range, 64.3–100%) versus 63.4% (range, 13.6–95.5%) ( P<0.0005); on day 6, 95.0% (range, 35.0–100%) versus 28.0% (range, 0–92.0%) ( P<0.0005); and on day 9, 95.0% (range, 3.7–100%) versus 68.6% (range, 0–100%) ( P<0.0005). Conclusions After microkeratome harvesting, the limbal basal epithelium is significantly longer lived in cold eye bank storage than central basal epithelium and the middle layers of limbal and central epithelium. This longevity not only bodes well for organ storage of limbal grafts, but also confirms the hardiness of the stem cell region.

The caspase inhibitor IDN-6556 prevents caspase activation and apoptosis in sinusoidal endothelial cells during liver preservation injury

Cold ischemia (CI)–warm reperfusion (WR) liver injury remains a problem in liver transplantation. CI-WR initially causes sinusoidal endothelial cell (SEC) apoptosis through a caspase-dependent mechanism. We previously showed that the caspase inhibitor IDN-1965 prevents CI-WR–induced SEC apoptosis. However, this agent required to be administered to the donor, preservation solution, and recipient for efficacy. Here, we show that a second-generation caspase inhibitor, IDN-6556, effectively prevents CI-WR–induced SEC injury when added only to University of Wisconsin (UW) cold storage media. Rat livers were stored in UW solution for 24 hours at 4°C and reperfused for 1 hour at 37°C. Apoptosis was quantitated using terminal deoxynucleotide transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and caspase 3 activation determined by biochemical measurement and immunohistochemical analysis. Pan-caspase inhibitors (IDN-8066, IDN-7503, IDN-7436, IDN-1965, and IDN-6556) were applied at preischemic, cold preservation, or reperfusion periods. TUNEL-positive SEC and caspase 3–like activity in the liver was increased by CI-WR. Three caspase inhibitors (IDN-8066, IDN-1965, and IDN-6556) effectively attenuated SEC apoptosis and caspase 3 activation. The most potent inhibitor, IDN-6556, reduced SEC apoptosis and caspase 3 activity by 55% and 94%, respectively. Prevention of SEC apoptosis by IDN-6556 was not reduced when this agent was administered only during the cold preservation period. When added to the preservation solution, the caspase inhibitor IDN-6556 appears to be a feasible therapeutic agent against ischemia-reperfusion injury in liver transplantation. (Liver Transpl 2003;9:278-284.)

Optimization of a culture technique for the isolation of Listeria monocytogenes from faecal samples.

A culture technique employing cold enrichment at 4 degrees C followed by selective enrichment and plating at higher temperatures (30 degrees C) was used to isolate Listeria monocytogenes from faecal samples. The samples were held at 4 degrees C for 15 weeks and cultured weekly to assess the sensitivity of the culture after cold storage for different lengths of time. No media, Listeria selective enrichment broth (LSEB), nutrient broth (NB) and saline were used as cold storage medium. Cold storage increased the frequency of Listeria positive samples. The sensitivity of the culture for Listeria spp. and L. monocytogenes was 72 and 94%, and 56 and 61% after third and seventh week of cold storage, respectively. When the results of third and seventh week of cold storage were combined, the sensitivity was 100% for Listeria spp. and 94% for L. monocytogenes. LSEB and NB as storage medium increased Listeria positive samples after the first week of cold storage but did not maintain the increase thereafter while saline had an adverse effect on the growth of the bacteria. However, samples held in no media in a pilot study involving monthly sampling of a herd revealed better results. Detection limit of the culture media was also investigated. The lowest concentration detected by culture media was 3.17 organisms/ml. This was seven organisms/g for known Listeria positive sample. The faecal samples spiked with 10-fold dilutions of L. monocytogenes and held at 4 degrees C revealed that the sample spiked with 3.17 x 10-1 cfu/ml organisms resulted in growth after the second week of cold storage. The results suggest that the culture technique employing cold enrichment followed by selective enrichment and plating is more sensitive, the storage of faecal samples in no media when compared with the samples in storage medium, LSEB, NB and saline, during cold enrichment is a better application and culture of faeces, immediately after collection, at third and seventh week of cold enrichment produce more satisfactory results.

INFERIOR OUTCOME OF CADAVERIC KIDNEYS PRESERVED ???24 HRS IN HISTIDINE-TRYPTOPHAN-KETOGLUTARATE (HTK) SOLUTION

360 During recent years, an increasing number of transplant centers within the Eurotransplant organization started using the Histidine-Tryptophan-Ketoglutarate (HTK) solution instead of University of Wisconsin (UW) solution as their preferred cold storage medium for abdominal organ preservation. We report on our single center experience on the outcome of imported kidneys preserved with either HTK or UW in relation to cold ischemia times (CIT) of < or ≥24 hours. Between July 1989 and July 1997, 323 cadaveric kidneys preserved with UW or HTK and imported as a result of an exchange within the Eurotransplant organization were transplanted at our institution. CIT was <24 hrs in 216 kidneys (UW: n=174, HTK: n=42) and ≥24 hrs in 107 kidneys (UW: n=67, HTK: n=41). Renal functional outcome was evaluated by comparing delayed graft function (DGF) rates, daily urinary output, the evolution of serum creatinine(SCr) and creatinine clearance (CrCl) at 1, 3, 5, 7, 14 days and 1, 3, 6, and 12 months postoperatively, and 1 yr graft survival, in relation to the cold storage solution and a CIT of < or ≥24 hrs. Whereas the incidence of DGF did not differ significantly between kidneys preserved for <24 hrs in UW (18.6%) or HTK (26.2%), this rate increased to 50% (HTK) compared to only 23.9% (UW) when kidneys were preserved for ≥24 hrs (P=0.006). Mean SCr and CrCl values were better at 1 and 5 days postoperatively in kidneys preserved for <24 hrs with UW(P<0.05). After 24 hrs of CIT, HTK preserved kidneys showed a more prominently impaired renal function, not only in the immediate postoperative phase, but lasting even significantly different at 1, 3, 6 and 12 months after transplantation (Table, *P<0.05,**P<0.01 UW vs HTK). Graft survival at 1 year was 92.9% in UW vs 87.5% in HTK kidneys preserved for <24 hrs, and 91% vs 77.4% in kidneys stored for ≥24 hrs (P=0.059).From these single center findings we conclude that UW solution remains superior to HTK in kidney preservation, particularly when ischemia times of≥24 hrs can be expected.

INFERIOR OUTCOME OF CADAVERIC KIDNEYS PRESERVED ≥24 HRS IN HISTIDINE-TRYPTOPHAN-KETOGLUTARATE (HTK) SOLUTION

360 During recent years, an increasing number of transplant centers within the Eurotransplant organization started using the Histidine-Tryptophan-Ketoglutarate (HTK) solution instead of University of Wisconsin (UW) solution as their preferred cold storage medium for abdominal organ preservation. We report on our single center experience on the outcome of imported kidneys preserved with either HTK or UW in relation to cold ischemia times (CIT) of < or ≥24 hours. Between July 1989 and July 1997, 323 cadaveric kidneys preserved with UW or HTK and imported as a result of an exchange within the Eurotransplant organization were transplanted at our institution. CIT was <24 hrs in 216 kidneys (UW: n=174, HTK: n=42) and ≥24 hrs in 107 kidneys (UW: n=67, HTK: n=41). Renal functional outcome was evaluated by comparing delayed graft function (DGF) rates, daily urinary output, the evolution of serum creatinine(SCr) and creatinine clearance (CrC1) at 1, 3, 5, 7, 14 days and 1, 3, 6, and 12 months postoperatively, and 1 yr graft survival, in relation to the cold storage solution and a CIT of < or ≥24 hrs. Whereas the incidence of DGF did not differ significantly between kidneys preserved for <24 hrs in UW (18.6%) or HTK (26.2%), this rate increased to 50% (HTK) compared to only 23.9% (UW) when kidneys were preserved for ≥24 hrs (P=0.006). Mean SCr and CrCl values were better at 1 and 5 days postoperatively in kidneys preserved for <24 hrs with UW(P<0.05). After 24 hrs of CIT, HTK preserved kidneys showed a more prominently impaired renal function, not only in the immediate postoperative phase, but lasting even significantly different at 1, 3, 6 and 12 months after transplantation (Table,*P<0.05, **P<0.01 UW vs HTK). Graft survival at 1 year was 92.9% in UW vs 87.5% in HTK kidneys preserved for <24 hrs, and 91% vs 77.4% in kidneys stored for ≥24 hrs (P=0.059).From these single center findings we conclude that UW solution remains superior to HTK in kidney preservation, particularly when ischemia times of≥24 hrs can be expected.

Pancreatic islet purification using bovine serum albumin: the importance of density gradient temperature and osmolality.

Euro-Ficoll and bovine serum albumin (BSA) are two of the most commonly used density gradient media for the purification of pancreatic islets. Euro-Ficoll is based upon Euro-Collins, a cold storage medium, and must, therefore, be used at 4 degrees C. The ionic composition of BSA, however, is likely to contribute to hypothermic cellular swelling, and this may influence the efficiency of islet purification using this medium at 4 degrees C. Experience in this laboratory also suggested that batch-to-batch variation in islet purity using BSA was related to differences in BSA osmolality. The aim of this study was to assess the effect of gradient medium temperature and osmolality on the purification of human and porcine islets using BSA. Pancreata were collagenase-digested, and islets were purified on continuous linear density gradients of BSA. The distribution of insulin and amylase in each gradient was assayed, and used to calculate the median density of islets and exocrine tissue, and the efficiency of islet purification (% amylase contamination at a fixed insulin yield), using: 1) gradient osmolalities of 300, 400, and 500 mOsm/kg H2O (seven porcine pancreata), and 2) gradients at 4 degrees C and at 22 degrees C (eight human and seven porcine pancreata). Increase in density gradient osmolality produced increases in porcine exocrine tissue density which exceeded changes in islet density, resulting in improved islet purity, maximal at a BSA osmolality of 400 mOsm/kg H2O. For human pancreata there was no significant change in pancreatic tissue densities nor islet purity with temperature.(ABSTRACT TRUNCATED AT 400 WORDS)