A comparison of a sucrose-based solution with other preservation media for cold storage of isolated hepatocytes

A sucrose-based solution has been compared with other preservation solutions (University of Wisconsin (UW) solution and Marshall's citrate solution, with Dulbecco's medium as control) during hypothermic preservation of isolated rat hepatocytes for up to 72 h. Studies on the stability of liver cells at low temperature by exclusion of trypan blue dye and morphological appearance were conducted. During storage beyond 24 h, there was a clear difference between cells stored in Dulbecco's medium and Marshall's citrate and those stored in sucrose-based solution and UW solution, with the former storage groups showing many cells developing large membrane "blebs" and the latter storage groups maintaining a more typical morphology and developing only small membrane protrusions. Dye exclusion was higher in sucrose-based solution (48 h, 75 +/- 7%; 72 h, 65 +/- 6%) and UW solution (48 h, 72 +/- 5%; 72 h, 63 +/- 4%) than in Marshall's citrate (48 h, 31 +/- 5%; 72 h, 10 +/- 1%) and Dulbecco's medium (48 h, 8 +/- 2%; 72 h, 5 +/- 1%). These data suggest that sucrose-based solution should be investigated further as a less complex alternative solution for storage of isolated hepatocytes.     

Forty-eight hours hypothermic storage of whole canine pancreas allografts. Improved preservation with a colloid hyperosmolar solution.

This study indicates that crystalloid (Sacks') and colloid (modified silica gel fraction of plasma, MSGF) hyperosmolar solutions are edequate means to preserve whole pancreas allografts under hypothermia (4 to 7 °C) for 24 hr. These groups of preserved canine pancreas allografts survived as long as the fresh transplanted pancreas. However, when the preservation was extended to 48 hr, the crystalloid hyperosmolar solution did not protect the pancreas allografts, but the colloid hyperosmolar solution gave similar results to fresh pancreas allografts.     

Liver preservation by cold storage with hyperosmolar solutions for twenty-four hours.

Canine livers were successfully preserved in an ischemic state for 24 hr under hypothermic storage with hyperosmolar colloid or crystalloid solutions. Livers preserved with a colloid hyperosmolar solution (MSGF) showed slightly better survival results than the ones preserved with a crystalloid hyperosmolar solution. It is possible that hyperosmolarity associated with hyperkalemia is an important factor for liver preservation for transplantation.     

A comparison of cold storage solutions for hepatic preservation using the isolated perfused rabbit liver

Rabbit livers were stored cold for periods of 6 or 24 hr and tested using the isolated perfused liver model. Five solutions were tested: Eurocollins (EC), Ross and Marshall's hypertonic citrate (HC), modified plasma protein fraction (Cambridge PPF), Ringer lactate, and the recently developed "University of Wisconsin" (UW) solution. After storage livers were perfused with an erythrocyte-free oxygenated Krebs-Henseleit solution containing 4% bovine serum albumin at 38 degrees C for 2 hr. Bile production proved to be the most sensitive index of liver function for discriminating between the various storage solutions and the different preservation times. After 6 hr of cold storage, bile production was similar to control liver bile production (9.8 +/- 2.4 ml/2 hr/100 g) in livers stored in HC (8.8 +/- 2 ml), PPF (9.9 +/- 2.2 ml), and UW (10.3 +/- 1.9 ml); it was slightly depressed in EC (6.7 +/- 2.5 ml, P = 0.06), and markedly depressed in Ringer lactate (4.3 +/- 0.8 ml, P less than 0.05). After 24 hr of cold storage bile production in UW-stored livers was near normal (9.3 +/- 0.7 ml) but significantly depressed (3.5-6.2 ml) in all other solutions tested. Release of enzymes into the normothermic perfusate was also measured (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase). In this small series the differences between cold storage solutions did not always reach statistical significance although the trend was for less enzyme release in livers stored in UW solution. This technique permits rapid assessment and refinement of new storage methods and new solutions for liver preservation prior to testing in a large animal transplant model. The results suggest that UW solution is superior to other preservation solutions and would permit successful 24-hr storage of livers.     

供体胰腺保存方法的改进

目的 探索在胰腺保存液中添加胰蛋白酶抑制剂保护受损供体胰腺的方法方法按照获取的供体胰腺分为对照组、完整组和破损组.在完整组和破损组的UW 液中添加乌司他丁,于胰腺灌注前取保存胰腺的UW 液送测淀粉酶浓度,并比较各组的胰岛得率、纯度、活率和胰岛素释放指数.结果 对照组的UW 液中的淀粉酶含量为(31 ± 21)U,完整组...     

Effect of sugars in the preservation solution on liver storage in rats.

We have performed 128 rat liver transplants in order to examine the effect of sugars in preservation solutions on cold storage of rat livers. Glucose (Mw. 180), sucrose (Mw. 348), and raffinose (Mw. 594) were tested. Rat livers were preserved at 4 degrees C for 12, 16, 18, and 24 h in standard Eurocollins solution (EC solution) (solution A) or in one of three modified EC solutions in which 194 mM/liter glucose in standard EC solution was replaced by 140 mM/liter of glucose (solution B), sucrose (solution C), or raffinose (solution D). The osmolarity of the modified solutions (solution B-D) was 320 mOsm/liter. Using standard EC solution (solution A), the 1-week survival rate of rats receiving livers preserved for 12, 16, 18, or 24 h was 6/8, 4/8, 1/8, and 0/4, respectively. With solution B, in which 194 mM/liter glucose was replaced by 140 mM/liter glucose, 1 week survivors following transplantation of livers preserved for 12, 16, 18 or 24 h were 4/8, 3/8, 2/8 and 0/4, respectively. Solution C, which was identical to solution A except for the replacement of 194 mM/liter glucose by 140 mM/liter sucrose, gave the following 1-week survival rates: 5/8 for 12 h, 5/8 for 16 h, 2/8 for 18 h, and 0/4 for 24 hours preservation, respectively. Using solution D, which differed from A in the replacement of glucose by 140 mM/liter raffinose, the 1-week survival rates of rats grafted with livers preserved for 12, 16, 18, and 24 h were 6/8, 5/8, 3/8 and 0/4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 mM 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 mM 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 degrees 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-aminotransferase into the perfusate was higher in livers preserved with Eurocollins, with or without modification, than in the livers preserved with UW solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Natural biopolymer for preservation of microorganisms during sampling and storage

Stability of microbial cultures during sampling and storage is a vital issue in various fields of medicine, biotechnology, food science, and forensics. We have developed a unique bacterial preservation process involving a non-toxic, water-soluble acacia gum polymer that eliminates the need for refrigerated storage of samples.The main goal of this study is to characterize the efficacy of acacia gum polymer for preservation of pathogenic bacteria (Bacillus anthracis and methicillin-resistant Staphylococcus aureus—MRSA) on different materials, used for swabbing and filtration: cotton, wool, polyester, rayon, charcoal cloth, and Whatman paper.Acacia gum polymer used for preservation of two pathogens has been shown to significantly protect bacteria during dehydration and storage in all tested samples at the range of temperatures (5-45 °C for MRSA and 40-90 °C for B. anthracis). Our results showed higher recovery as well as higher viability during the storage of both bacteria in all materials with acacia gum. Addition of acacia gum polymer to swabbing materials or filters will increase efficacy of sample collection and identification of pathogenic bacteria from locations such as hospitals or the environment. Proposed approach can also be used for long-term storage of culture collections, since acacia gum contributes to viability and stability of bacterial cultures.     

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.     

Optimizing the concentration of hydroxyethylstarch in a novel intestinal-specific preservation solution

Introduction

Our lab has developed an effective nutrient-rich solution that facilitates energy production and control of oxidative stress during static cold storage of the intestine; however, the requirement for oncotic agents, such as hydroxyethylstarch (HES), has not been evaluated. This study investigated the effectiveness and requirement for HES in an intraluminal preservation solution during a clinically relevant period of cold storage.

Methods

Rat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a ‘back table’ intraluminal flush with a nutrient-rich preservation solution containing varying amounts of HES (n = 6 per group): Group 1, 0%; Group 2, 2.5%; Group 3, 5%; Group 4, 10%. Energetics, oxidative stress, and morphology were assessed over a 24 h time-course of cold storage.

Results

Overall, the 5% HES solution, Group 3, demonstrated superior energetic status (ATP and total adenylates) compared to all groups, P < 0.05. Malondialdehyde levels indicated a reduction in oxidative stress in Groups 3 and 4 (P < 0.05). After 12 h, median modified Parks’ grades for Groups 2 and 3 were significantly lower than Groups 1 and 4, P < 0.05.

Conclusion

Our data suggests that when employing an intraluminal preservation solution for static organ storage, oncotic support is a fundamental requirement; 5% HES is optimal.     

Cold storage of rat hepatocyte suspensions for one week in a customized cold storage solution--preservation of cell attachment and metabolism

Background & Aims

Primary hepatocytes are of great importance for basic research as well as cell transplantation. However, their stability, especially in suspension, is very low. This feature severely compromises storage and shipment. Based on previous studies with adherent cells, we here assessed cold storage injury in rat hepatocyte suspensions and aimed to find a cold storage solution that preserves viability, attachment ability and functionality of these cells.

Methods

Rat hepatocyte suspensions were stored in cell culture medium, organ preservation solutions and modified TiProtec solutions at 4°C for one week. Viability and cell volume were determined by flow cytometry. Thereafter, cells were seeded and density and metabolic capacity (reductive metabolism, forskolin-induced glucose release, urea production) of adherent cells were assessed.

Results

Cold storage injury in hepatocyte suspensions became evident as cell death occurring during cold storage or rewarming or as loss of attachment ability. Cell death during cold storage was not dependent on cell swelling and was almost completely inhibited in the presence of glycine and L-alanine. Cell attachment could be greatly improved by use of chloride-poor solutions and addition of iron chelators. Using a chloride-poor, potassium-rich storage solution containing glycine, alanine and iron chelators, cultures with 75% of the density of control cultures and with practically normal cell metabolism could be obtained after one week of cold storage.

Conclusion

In the solution presented here, cold storage injury of hepatocyte suspensions, differing from that of adherent hepatocytes, was effectively inhibited. The components which acted on the different injurious processes were identified.     

Development of a closed-loop artificial pancreas.

A closed-loop glucose controlled insulin infusion system was developed, consisting of elements for continuous blood glucose analysis, a computer control system, and infusion systems. Improvements include decreased size, cost reduction and better performance. The algorithm used was a piecewise linear representation of the sigmoidal curve commonly employed. The apparatus has been applied to simulation of the healthy beta cell and glucose clamp studies.     

Hepatic uptake of solutes from the preservation solution during hypothermic storage: a (1)H NMR study in rat liver.

The hepatic uptake of histidine and carnosine (histidyl-alanine), used as buffer agents in four preservation solutions, was studied during 24-h hypothermic storage of rat livers by use of (1)H nuclear magnetic resonance (NMR) spectroscopy. Results demonstrated that there was a progressive, concentration-linked passive diffusion of histidine into liver tissues throughout the storage period. A similar inward diffusion of carnosine was also noted. Of the carbohydrate osmotic buffers in the preservation solutions, mannitol permeated the liver tissues to a greater degree and more rapidly than raffinose after the flushing with equivalent concentrations and storage at hypothermia. In general, many solutes from preservation solutions will increasingly penetrate the hepatic inter- and intracellular spaces during extended hypothermic preservation and (1)H NMR spectroscopy is one technique that can assist in the identification of these changes.     

Development of a modified vitrification strategy suitable for subsequent scale-up for hepatocyte preservation

This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VS_basic (40% (v/v) ethylene glycol 0.6 M sucrose, i.e. 7.17 M ethylene glycol 0.6 M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VS_basic and increase the solution’s total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5 days.     

MiMiR: a comprehensive solution for storage,annotation and exchange of microarray data

Background  

The generation of large amounts of microarray data presents challenges for data collection, annotation, exchange and analysis. Although there are now widely accepted formats, minimum standards for data content and ontologies for microarray data, only a few groups are using them together to build and populate large-scale databases. Structured environments for data management are crucial for making full use of these data.