Cold Storage Preservation Research Articles

Improvement of endothelial cell viability at 4 degrees C by addition of lazaroid U74500A to preservation solutions.

Lazaroids are potent inhibitors of lipid peroxidation. Endothelial cell damage has been shown to occur during cold storage preservation of lung and liver. This study examines the effects on endothelial cell viability of the addition of four lazaroids, U74006F, U78518F, U74500A, or U75412E to preservation solutions. Human umbilical vein endothelial cell cultures were stored at 4 degrees C for 48 and 96 hr in EuroCollins or 5% polyethylene glycol in buffered saline (PEG). U78518F, U74500A, U74006F, U75412E, or dexamethasone (each 50 microM) was added to EC (n = 32) or PEG (n = 32) and compared with control solutions of EC or PEG alone. Endothelial cell viability was determined by measuring cellular reduction of 3-[4,5-dimethylthiazol-2-yl]-2,3-diphenyltetrazolium bromide to a purple formazan dye. The reduction occurs only in viable cells and requires mitochondrial dehydrogenase activity. Results were quantified by measuring dye absorbance (Ab) with a micro-ELISA spectrophotometer. Absorbance values were compared by ANOVA and reported as mean values +/- standard deviation. Addition of U74500A to EC (Ab = 0.474 +/- 0.055) and PEG (Ab = 0.462 +/- .005) improved viability at 48 hr when compared with EC (Ab = 0.289 +/- 0.069) and PEG (Ab = 0.287 +/- 0.052) alone (P less than 0.05). At 96 hr, addition of U74500A resulted in improved viability in both EC (Ab = 0.377 +/- 0.068) and PEG (Ab = 0.195 +/- 0.09) or PEG alone (Ab = 0.212 +/- 0.1) (P less than 0.05). Other lazaroids tested were also effective in improving cellular viability, but to a lesser degree than U74500A. This study demonstrates that the addition of lazaroids to organ preservation solutions improves endothelial cell viability.

Effects of method of preservation on functions of livers from fed and fasted rabbits

Livers from fed, fasted (48 h) and glucose-fed rabbits were preserved for 24 and 48 h by either simple cold storage (CS) or continuous machine perfusion (MP) with the University of Wisconsin preservation solutions. After preservation liver functions were measured by isolated perfusion of the liver (at 37 °C) for 2 h. Fasting caused an 85% reduction in the concentration of glycogen in the liver but no change in ATP or glutathione. Glucose feeding suppressed the loss of glycogen (39% loss). After 24 h preservation by CS livers from fed or fasted animals were similar including bile production (6.2 ± 0.5 and 5.6 ± 0.4 ml/2 h, 100 g, respectively), hepatocellular injury (LDH release = 965 ± 100 and 1049 ± 284 U/liter), and concentrations of ATP (1.17 ± 0.15 and 1.18 ± 0.04 μmol/g, glutathione (1.94 ± 0.51 and 2.35 ± 0.26 μmol/g, respectively), and K:Na ratio (6.7 ± 1.0 and 7.7 ± 0.5, respectively). After 48 h CS livers from fed animals were superior to livers from fasted animals including significantly more bile production (5.0 ± 0.9 vs 2.0 ± 0.3 ml/2 h, 100 g), less LDH release (1123 ± 98 vs 3701 ± 562 U/liter), higher concentration of ATP (0.50 ± 0.16 vs 0.33 ± 0.07 (μmol/g) and glutathione (0.93 ± 0.14 vs 0.30 ± 0.13μmol/g), and a larger K:Na ratio (7.4 vs 1.5). Livers from fed animals were also better preserved than livers from fasted animals when the method was machine perfusion. The decrease in liver functions in livers from fasted animals preserved for 48 h by CS or MP was prevented by feeding glucose. Glucose feeding increased bile formation after 48 h CS preservation from 2.0 ± 0.3 (fasted) to 6.9 ± 1.2 ml/2 h, 100 g; LDH release was reduced from 3701 ± 562 (fasted) to 1450 ± 154 U/liter; ATP was increased from 0.33 ± 0.07 (fasted) to 1.63 ± 0.18 μmol/g; glutathione was increased from 0.30 ± 0.01 (fasted) to 2.17 ± 0.30 (μmol/g; and K:Na ratio was increased from 1.5 ± 0.9 to 5.3 ± 1.0. This study shows that the nutritional status of the donor can affect the quality of liver preservation. The improvement in preservation by feeding rabbits only glucose suggests that glycogen is an important metabolite for successful liver preservation. Glycogen may be a source for ATP synthesis during the early period of reperfusion of preserved livers.

The functional effects of suppression of hypothermia-induced cell swelling in liver preservation by cold storage

It is known that cellular edema and functional impairment develop during anaerobic cold storage of organs. The extent of both is related to the storage time and the composition of the preservation solution used. We studied hypothermia-induced cell swelling and its effect on liver function after cold storage preservation with either Eurocollins (EC), a number of modified EC solutions in which glucose was replaced by various concentrations of raffinose, or UW solution. After 24 h storage, tissue swelling as determined by total tissue water (TTW) in rat liver tissue slices was most pronounced in slices incubated in Eurocollins, whereas the TTW was only moderately increased in slices stored in modified Eurocollins containing 90 to 120 m M raffinose. In contrast, slices incubated in UW solution had a TTW equal to normal rat liver tissue. Furthermore, intact rabbit livers preserved with Eurocollins had an increase in the whole organ weight, while there was no weight change after preservation with the modified solution containing 120 m M raffinose (M120). In contrast, a pronounced weight loss was observed after preservation with UW solution. After cold storage, the livers were reperfused for 2 h at 38 °C in an isolated perfusion circuit (IPL) with an acellular perfusate. Bile flow was significantly greater in livers preserved in M120 than in those preserved with the conventional Eurocollins. However, the bile flow in the livers stored in M120 was inferior to that in the livers preserved with UW solution, which in turn was equal to that in control livers. The release of alanine-aspartate-amino-transferase into the perfusate was higher in livers preserved with Eurocollins, with or without modification, than in the livers preserved with UW solution. It is concluded that although the replacement of the glucose in Eurocollins with raffinose did indeed prevent hypothermia-induced cell swelling as evidenced by the absence of weight changes in the intact rabbit liver, these livers were still not as well preserved as those stored in UW. Therefore, the mechanism of action of UW is not solely based upon suppression of cell swelling nor upon the absence of glucose in this solution, but upon other factors which are discussed in this paper.

THE IMPACT OF EXTENDED PRESERVATION ON CLINICAL LIVER TRANSPLANTATION

The introduction of UW solution into clinical transplantation has permitted extended cold storage preservation of the liver. Over a 46-month period, we have performed 308 orthotopic liver transplants (266 primary, 42 retransplants) in 266 recipients. Our experience is divided into cold-storage preservation in Eurocollins (163 transplants in 140 recipients) and UW (145 transplants in 131 recipients) solutions. Donor and recipient factors were comparable between the two groups. The use of UW solution has permitted an increase in the mean preservation time from 5.2 +/- 1.0 [EC] to 12.8 +/- 4.3 [UW] hr (P less than 0.001). The mean total operating time was reduced but intraoperative blood loss was unchanged with UW preservation. The number of transplants performed during the daytime hours has increased dramatically (21.5% [EC] vs. 71% [UW], P less than 0.001). The incidence of primary nonfunction, hepatic artery thrombosis, 1-month graft survival, and early retransplantation were similar in the 2 groups. Initial allograft function as determined by bile production, histology, and clinical assessment were likewise similar. Mean serum bilirubin, transaminase, and prothrombin levels were virtually identical by 5 days posttransplant. The enhanced margin of safety afforded by extended preservation has increased the capability for distant organ procurement and sharing, minimized organ wastage, and improved the efficiency of organ retrieval. With the relaxation of logistical constraints, our rate of liver import has nearly doubled (20.9% [EC] vs. 39.3% [UW], P less than 0.001). Extended preservation has permitted the development of reduced-size liver grafting (n = 12), resulting in a significant reduction in the number of deaths occurring while awaiting transplantation. Therefore, we advocate the use of UW solution with selective extension of preservation based not only on donor and recipient factors but also on manpower, resource, and logistical considerations.

INFLUENCE OF PRESERVATION TIME ON OUTCOME AND METABOLIC FUNCTION OF BLADDER-DRAINED PANCREAS TRANSPLANTS

The influence of cold storage preservation time on graft survival and metabolic function of pancreatic transplants was studied in 130 recipients of bladder-drained grafts (47 simultaneous with, 33 after, and 50 without a kidney transplant) between October 1, 1984 and May 1, 1989. The recipients were divided into four groups according to the preservation time: less than 6 hr (n = 11), 6-12 hr (n = 24), 12-24 hr (n = 75), and greater than 24 hr (n = 20). Twenty-six grafts were procured by other transplant teams and sent to us. Silica gel fractionated plasma was used for preservation in 104 cases and the University of Wisconsin solution in 25 (1 in the less than 6 hr, 2 in the 6-12 hr, 16 in the 12-24 hr, and 6 in the greater than 24 hr groups). The technical failure rate at 1 month was 13% (17 grafts), 1 (9%) in the less than 6 hr, 5 (21%) in the 6-12 hr, 9 (12%) in the 12-24 hr, and 2 (10%) in the greater than 24 hr groups. At 1 month, 107 (82%) of the grafts were functioning, 10 (91%) in the less than 6 hr, 18 (75%) in the 6-12 hr, 62 (83%) in the 12-24 hr and 17 (85%) in the greater than 24 hr groups, the longest preserved for 30 hr. The respective 1-year graft survival rates were 51%, 50%, 57%, and 70%. Ninety patients (10 in the less than 6 hr, 16 in the 6-12 hr, 51 in the 12-24 hr, and 13 in the greater than 24 hr groups) had metabolic studies between 2 and 6 weeks postransplant. The results of 24-hour profiles (14 blood glucose determinations) were similar in each preservation time group; the means of the mean (+/- SD) profile glucose (mg/dl) values were 130 +/- 19, 126 +/- 31, 130 +/- 24, and 129 +/- 30, respectively (P greater than 0.6). Mean plasma glucose levels at 2 hr during OGTT were 141 +/- 32, 145 +/- 43. 163 +/- 49, and 184 +/- 100 in the respective preservation groups (P greater than or equal to 0.064). According to the National Diabetes Data Group classification, 75% of recipients in the less than 6 hr, 50% in the 6-12 hr, 44% in the 12-24 hr, and 33% in the greater than 24 hr groups had normal OGTT results.(ABSTRACT TRUNCATED AT 400 WORDS)

Cold storage preservation of pancreatic tissue prior to and after islet preparation in a dog autotransplantation model

Utilizing an intrasplenic canine islet autotransplant model, the effects of cold storage preservation on pancreatic tissue prior to and after collagenase dispersion were examined. A control series, in which freshly retrieved and prepared tissue was transplanted, yielded a 75% success rate (6/8). In contrast, when the pancreas was stored in modified silica gel filtered plasma (SGF I) for 24 hr, no autotransplant was successful (0/6). However, when the islet tissue was prepared following pancreatectomy and then stored in a mannitol-containing modification of SGF (SGF III), autotransplantation was successful in 83% (5/6) after 24 hr of preservation and in 60% (3/5) after 48 hr of preservation. Similarly, the islet tissue was stored in a hyperkalemic hydroxyethyl starch solution (HES) and this was successful in 20% (1/5) after 24 hr of preservation and in 50% (1/2) after 48 hr of preservation. Cold storage preservation techniques for the pancreas prior to islet isolation need to be refined, but dispersed islet-enriched pancreatic tissue can be successfully maintained at 4°C for up to 48 hr prior to transplantation in dogs using established pancreas preservation solutions.

COLD-STORAGE PRESERVATION OF THE CANINE AND RAT PANCREAS PRIOR TO ISLET ISOLATION

Cold-storage preservation of the canine pancreas prior to islet isolation has previously been noted to reduce the intrasplenic islet autograft success rate; but the mechanism of this deleterious effect has not been determined. We undertook a study in both outbred dogs and Lewis (RT1-1) rats to determine the influence of cold-storage preservation interval, preservation solution, and flushing technique on islet yield and islet viability. The preservation solutions used were those that had proved most efficacious in preserving segmental canine pancreases--namely, the modifications of silica gel fractionated plasma (SGF-III and SGF-IV) and an hydroxyethylstarch/lactobionate solution (UW-1). In the first set of experiments, the traditional vascular flush was used; this was followed by storage at 4 degrees C. After brief periods of preservation (3 hr in the rat, 12 hr in the dog) there was a significant (P less than 0.006) reduction in islet yield. The reduced yields were similar with each solution tested, were made worse with increasing intervals of storage, and resulted in a significant reduction in autograft success rate. The second set of experiments examined the effect of using an intraductal flush prior to preservation, along with the effect of adding collagenase to the preservation fluid. Islet yields were maintained at control values in both animal models using preservation intervals of up to 24 hr. These islet yields produced auto- or isograft success rates similar to those obtained by transplanting freshly obtained tissue; verifying adequate islet viability. We recommend that a pre-storage ductal flush technique be used for cold-storage preservation of the pancreas prior to islet isolation and transplantation.

Organ flush out solutions and cold storage preservation solutions: effect on organ cooling and post ischemic erythrocyte trapping in kidney grafts. An experimental study in the rat.

Three flush out and cold storage solutions were tested for flow rate during flush out, ability to decrease organ temperature, degree of tissue edema and degree of preservation damage of the kidney as measured by the amount of post transplantation erythrocyte trapping in the renal medulla. The solutions tested were a modification of a new perfusion solution (University of Wisconsin; mUW), a standard preservation solution (Sacks') and an extracellular histidine solution (Frödin-Wolgast; FW). The flow rate was significantly higher for FW compared to mUW and Sacks' and consequently a more rapid decrease of organ temperature was achieved. The results also show that edema formation as well as the amount of erythrocyte trapping was lower in kidneys stored in mUW, indicating that mUW is better cold storage solution than Sacks' or FW.

An experimental evaluation of nucleotide enhancement techniques for kidney transplantation

The effect of various nucleotide-enhancing agents on renal function and intracellular nucleotide levels was evaluated in a canine autotransplant model. Thirty-five dogs (18–28 kg) underwent left nephrectomy and 30 min of warm ischemia followed by Collins C-4 flush and 24 hr of cold-storage preservation. Heterotopic autotransplantation and immediate contralateral nephrectomy was then performed. Seven equal groups were evaluated: group A—controls, group B—adenosine pretreatment (1.0 g), group C—dipyridamole pretreatment (10 mg), group D—adenosine (1.0 g), and dipyridamole (10 mg) pretreatment, group E—adenosine (200 mg) and EHNA (2.5 mg/kg) pretreatment, group F—adenosine (200 mg) and EHNA (2.5 mg/kg) in the Collins C-4 flush, and group G—adenosine (200 mg) and EHNA (2.5 mg/kg) at the time of autotransplantation. All kidneys underwent cortical biopsies at the end of preservation and 1 hr after restoration of blood flow for determinations of AMP, ADP, and ATP. In the pretreatment groups (groups B through E) there was 60% graft survival whereas the controls (group A) and the groups treated after ischemia (groups F and G) had 0, 0, and 20% graft survival, respectively. In groups B and E, ATP levels were greater than controls after preservation and 1 hr after restoration of blood flow. Group C AMP and ADP levels and group D energy charge were greater than controls in the post-transplantation biopsies. Administration of adenosine and EHNA after ischemia was not associated with increased intracellular nucleotide levels. One hour post-transplantation biopsies demonstrated greater ability to regenerate cortical nucleotides in the surviving animals but no absolute value could be identified as a predictor of viability. In conclusion, pretreatment with adenosine, dipyridamole, and EHNA alone and in combination is beneficial in ischemically injured kidneys undergoing cold-storage preservation.

The effect of warm ischemia and cold-storage preservation on rat pancreas transplantation

Pancreatic isografts subjected to preharvest warm ischemia as well as cold-storage preservation in Collins' solution were studied after transplantation into diabetic rats to determine whether warm ischemia will limit the ability to preserve pancreas grafts for transplantation. Warm ischemic periods of up to 2 hr did not alter islet function as measured by daily glucose levels and response to intravenous glucose challenge. Likewise, hypothermic preservation of nonischemic pancreata was also well tolerated for up to 24 hr. However, the combination of preharvest warm ischemia and cold storage was deleterious. Whereas 60 min of warm injury coupled with 12 hr of cold storage resulted in successful transplantation in 86% of recipients, lengthening the duration of either warm or cold ischemia uniformly resulted in nonfunctioning grafts. Thus while islet function in the transplanted pancreas is very tolerant of warm ischemia alone, these studies suggest that it should be kept to a minimum if cold storage preservation is to be used.

The effects of ATP-MgCl 2 and dipyridamole in cold-storage preservation

The effects of ATP-MgCl 2 and dipyridamole were evaluated in a canine model. Twenty-five adult mongrel dogs were divided into five equal groups. All dogs underwent left nephrectomy and 30 min of warm ischemia followed by Collins' C-4 flush and 24 hr of cold-storage preservation. Heterotopic autotransplantation and immediate contralateral nephrectomy were then performed. Group A served as controls; Group B was pretreated with intravenous ATP-MgCl 2 (2.5 m M); Group C with intravenous dipyridamole (10 mg), and Group D with both ATP-MgCl 2 (2.5 m M) and dipyridamole (10 mg). Group E was treated with ATP-MgCl 2 (2.5 m M) at the time of transplantation. All kidneys underwent cortical biopsies at the end of preservation and 1 hr after restoration of blood flow for determinations of AMP, ADP, and ATP. In Groups A and E there were no survivors, whereas Groups B, C, and D had 40, 60, and 40% graft survival. In Groups B and D, ATP and energy charge (EC) were greater than that of controls after 24 hr of preservation, with Group D values being significantly greater ( P < 0.01). AMP and ADP levels were significantly greater ( P < 0.02) in Group C when compared to Group A. One hour posttransplantation biopsies demonstrated greater ability to regenerate cortical nucleotides in the surviving animals, but no absolute value could be identified as a predictor of viability. In conclusion, pretreatment with ATP-MgCl 2 or dipyridamole maintains intracellular nucleotide levels and has a beneficial effect on graft survival in ischemically injured kidneys undergoing cold-storage preservation.

Cold storage preservation of islet and pancreas grafts as assessed by in vivo function after transplantation to diabetic hosts

The major goal of hypothermic (4–8 °C) preservation of intact pancreases or isolated islets will be to provide sufficient time for HLA typing, cross matching, selection, and preparation of recipients—logistical efforts requiring 12–72 hr for clinical kidney transplantation, usually <48 hours. Some investigators have studied in vitro function of islets after cold storage, but the critical test of viability—permanent restoration of normoglycemia after transplantation to diabetic recipients—has been tested in only a few experiments. Reversal of hyperglycemia by syngeneic or autogenic transplants in diabetic animals has been achieved after CS of dispersed pancreatic tissue from neonatal rats in GIB media for ⩽ 146 hr, adult dogs in TCM 199 for ⩽24 hr, and adult DL-ethionine-treated rats in RPMI 1640 for ⩽72 hr. In the neonatal rat donor model, intravenous glucose tolerance test (IVGTT) results were similar in recipients of fresh or stored islets; in the dog model, IVGTT test results were variable, but generally inferior in recipients of stored as compared to fresh islets; in the adult rat donor model, recipients of ⩽24-hr coldstorage islets had insulin and IVGTT K values similar to those of recipients of fresh islets, but the success rate progressively declined for CS times >24 hr. Various agents were added to the media, but the need or the optimal concentrations were not critically determined by using different recipes for different groups of recipients. Cold storage of intact pancreas autografts has been tested in dogs; simple electrolyte solutions are satisfactory for 24 hr, but only a silica gel-filtered plasma-based solution has been reliable for 48 hr. Pulsatile machine perfusion (PMP) of canine pancreas grafts for 24 hr has had a success rate similar to CS in some experiments and lower in others. PMP has been almost totally unreliable for >24 hr. Further refinements are needed if preservation of islets for >24 hr and pancreases for >48 hr are to be consistently successful. If current experimental techniques are effective for human islets or pancreases, however, these times are sufficient to complete the logistical maneuvers required before transplantation.

Current status of cold storage (CS) preservation of pancreas and islet grafts as assessed by in vivo function after transplantation (TX) to diabetic hosts

Current status of cold storage (CS) preservation of pancreas and islet grafts as assessed by in vivo function after transplantation (TX) to diabetic hosts

The effect of ATP-MgCl 2 and dipyridamole pretreatment in renal preservation

The effect of ATP-MgCl 2 alone and in combination with dipyridamole administered prior to ischemia and cold-storage preservation was evaluated in canine kidneys. Fifteen adult mongrel dogs were divided into three groups. All dogs underwent left nephrectomy and 30 min of in situ warm ischemia followed by Collin's C-4 flush and 24 hr of cold-storage preservation. Heterotopic allotransplantation and delayed (2 weeks) contralateral nephrectomy were performed in all dogs. Group A served as ischemic controls. Group B dogs were treated with intravenous ATP-MgCl 2 (2.5 m M) and Group C dogs with intravenous ATP-MgCl 2 (2.5 m M) and dipyridamole (10 mg) administered 10 min prior to the ischemic insult. All kidneys underwent cortical biopsies at 0, 4, and 24 hr of preservation for determination of ATP. Mean values for cortical ATP (μmole/g) were 0.165 ± 0.062, 0.046 ± 0.013 and 0.015 ± 0.007 in Group A, 1.101 ± 0.429, 0.352 ± 0.110, and 0.077 ± 0.033 in Group B and 2.296 ± 0.888, 0.496 ± 0.174 and 0.192 ± 0.064 in Group C. The cortical ATP levels were significantly greater at all time intervals in the kidneys pretreated with ATP-MgCl 2 and dipyridamole; however, no significant difference could be demonstrated in dog survival, peak, or final serum creatinine in the three groups. In conclusion, pretreatment with ATP-MgCl 2 with or without dipyridamole does not improve graft function in ischemically injured kidneys.

ADVANTAGE OF COLD STORAGE OVER MACHINE PERFUSION FOR PRESERVATION OF CADAVER KIDNEYS

Evidence is provided that, with cold ischemia up to 48 hr and warm ischemia up to 60 min, simple storage preservation results in equally good or better transplant success rates than pulsatile machine perfusion. In the critical 24- to 43-hr preservation period, 1-year survival rates were 53 +/- 3% for 282 kidneys preserved in Collins solution, as compared with 40 +/- 2% for 565 kidneys on the Belzer machine (P less than 0.0001) and 48 +/- 1% for 1441 kidneys perfused on the Waters machine (P = not significant). Warm ischemia of 40 to 60 min also yielded better results with simple cold storage than with machine preservation (P less than 0.05). Although it has been maintained that one of the functions of preservation machines is to select out kidneys of poor quality, we found that this preselection actually has resulted in lower transplant survival rates. Because storage in Collins solution is simpler, safer, and much less expensive, we conclude that there is no longer any reason to preserve kidneys by pulsatile perfusion on machines.

Physiologic aspects of intrasplenic autotransplantation of pancreatic fragments in the dog after 24 hours of cold storage

This report confirms the efficacy of using partially digested pancreatic fragments for endocrine replacement in the pancreatectomized dog and intrasplenic autotransplantation model. In addition, the feasibility of short-term cold storage preservation is demonstrated. Dogs transplanted with either freshly prepared or 24-hr cold storage preserved (4°C) islet grafts maintained nearly normal fasting glucose levels. In contrast, 48-hr preservation was unsuccessful in that recipients of these grafts became diabetic and had survival rates similar to pancreatectomized, nontransplanted controls. The successfully transplanted dogs have remained normoglycemic during a 5-month follow-up period and have had satisfactory glucose responses in IVGTTs. However, α- and β-cell reactivity as measured by insulin and glucagon determinations remains abnormal.

Hyperosmolar cold storage kidney preservative solution

A hyperosmolar kidney preservative solution which can maintain kidneys from experimental animals viable for up to 72 hours is described. Using the criterion of a one-month failure rate, the cold storage preservation method was found to be superior to machine preservation methods. Sachs' solution was found to be superior to all other cold storage solutions. The most important aspect of the hyperosmolar kidney preservative solution appears to be its ability to maintain normal intracellular electrolyte composition and to prevent cellular damage due to swelling. The present formula requires that the basic solution and a magnesium chloride additive solution be prepared separately and combined before use. A stable combined solution is proposed which can be sterilized by membrane filtration.