Cryopreservation of isolated primary rat hepatocytes: enhanced survival and long-term hepatospecific function

OBJECTIVE: To investigate the long-term effect of cryopreservation on hepatocyte function, as well as attempt to improve cell viability and function through the utilization of the hypothermic preservation solution, HypoThermosol (HTS), as the carrier solution. SUMMARY BACKGROUND DATA: Advances in the field of bioartificial liver support have led to an increasing demand for successful, efficient means of cryopreservation of hepatocytes. METHODS: Fresh rat hepatocytes were cryopreserved in suspension in culture media (Media-cryo group) or HTS (HTS-cryo group), both supplemented with 10% DMSO. Following storage up to 2 months in liquid nitrogen, cells were thawed and maintained in a double collagen gel culture for 14 days. Hepatocyte yield and viability were assessed up to 14 days postthaw. Serial measurements of albumin secretion, urea synthesis, deethylation of ethoxyresorufin (CYT P450 activity), and responsiveness to stimulation with interleukin-6 (IL-6) were performed. RESULTS: Immediate postthaw viability was 60% in Media-cryo and 79% in HTS-cryo, in comparison with control (90%). Albumin secretion, urea synthesis and CYT P450 activity yielded 33%, 55%, and 59% in Media-cryo and 71%, 80%, and 88% in HTS-cryo, respectively, compared with control (100%). Assessment of cellular response to IL-6 following cryopreservation revealed a similar pattern of up-regulation in fibrinogen production and suppression of albumin secretion compared with nonfrozen controls. CONCLUSIONS: This study demonstrates that isolated rat hepatocytes cryopreserved using HTS showed high viability, long-term hepatospecific function, and response to cytokine challenge. These results may represent an important step forward to the utilization of cryopreserved isolated hepatocytes in bioartificial liver devices.     

Culture of C3A cells in alginate beads for fluidized bed bioartificial liver

Extracorporeal bioartificial liver has been designed to sustain the detoxification and synthetic function of the failed liver in patients suffering from acute liver failure until the time of liver allotransplantation or regeneration of their own. A fluidized bed, bioartificial liver improves the mass transfer velocity between the medium and the hepatocytes. Detoxification functions of the liver could be replaced by completely artificial systems, but the synthetic functions of hepatocytes may be obtained only by metabolically active cells. The aim of our study was to investigate the influence of C3A cell culture in alginate beads on synthetic function in a fluidized bed, bioartificial liver. Cells in alginate beads were prepared using an electrostatic droplet generator of our own design using low-viscosity alginate. Beads were cultured for 24 hours then 7 days in static conditions and then 24 hours of fluidization in the bioreactor to assess albumin production. We observed significantly increased albumin production by C3A cells entrapped in alginate beads during static culture. Fluidization increased albumin production compared with static culture. Fluidization performed after 7 days of static culture resulted in a significant increase in albumin synthesis. In conclusion, static culture of alginate beads hosting hepatic cells facilitates restoration of cell function.     

Improvement of the cold storage of isolated human hepatocytes

Increasing amounts of human hepatocytes are needed for clinical applications and different fields of research, such as cell transplantation, bioartificial liver support, and pharmacological testing. This demand calls for adequate storage options for isolated human liver cells. As cryopreservation results in severe cryoinjury, short-term storage is currently performed at 2-8°C in preservation solutions developed for the storage of solid organs. However, besides slowing down cell metabolism, cold also induces cell injury, which is, in many cell types, iron dependent and not counteracted by current storage solutions. In this study, we aimed to characterize storage injury to human hepatocytes and develop a customized solution for cold storage of these cells. Human hepatocytes were isolated from material obtained from partial liver resections, seeded in monolayer cultures, and, after a preculture period, stored in the cold in classical and new solutions followed by rewarming in cell culture medium. Human hepatocytes displayed cold-induced injury, resulting in >80% cell death (LDH release) after 1 week of cold storage in University of Wisconsin solution or cell culture medium and 3 h of rewarming. Cold-induced injury could be significantly reduced by the addition of the iron chelators deferoxamine and LK 614. Experiments with modified solutions based on the new organ preservation solution Custodiol-N showed that ion-rich variants were better than ion-poor variants, chloride-rich solutions better than chloride-poor solutions, potassium as main cation superior to sodium, and pH 7.0 superior to pH 7.4. LDH release after 2 weeks of cold storage in the thus optimized solution was below 20%, greatly improving cold storage of human hepatocytes. The results were confirmed by the assessment of hepatocellular mitochondrial membrane potential and functional parameters (resazurin reduction, glucagon-stimulated glucose liberation) and thus suggest the use of a customized hepatocyte storage solution for the cold storage of these cells.     

Mild hypothermic preservation for transport purposes of the AMC bioartificial liver charged with porcine hepatocytes

BACKGROUND: Preservation conditions play a crucial role during transport of a bioartificial liver (BAL) from the laboratory to the hospital. We assessed the possibility to preserve the AMC-BAL loaded with freshly isolated porcine hepatocytes at mild hypothermic temperatures. METHODS: Two laboratory-scale AMC-bioreactors were loaded with 1 billion freshly isolated porcine hepatocytes per experiment (n=6). Bioreactors in the control group were kept for three days at 37 degrees C. Bioreactors in the transport group were kept at 37 degrees C during day 1, at 15 degrees C during day 2, and again at 37 degrees C during day 3. In addition, long-term mild hypothermic preservation periods of 45 and 110 hr at 15 degrees C and 26 degrees C, respectively, were assessed. The effect of mild hypothermic preservation on hepatocytes inside the bioreactors was tested by determination of cell damage parameters, as well as metabolic and hepatocyte-specific functions. RESULTS: A 24-hour period of mild hypothermic preservation did not reduce any hepatocyte-specific function. LDH release was significantly higher only at day 2. Albumin production at day 2 and lidocaine elimination at day 3 were significantly higher with glucose consumption and lactate production being significantly lower at both test days. Long-term mild hypothermic preservation had a drastic negative effect on cellular viability and hepatocyte-specific function. CONCLUSIONS: Mild hypothermic preservation at temperatures as low as 15 degrees C and for a duration of 24 hr is a feasible method to preserve BAL systems loaded with freshly isolated porcine liver cells and will simplify the logistics of BAL transport from the laboratory to the hospital.     

Induction of necrosis and DNA fragmentation during hypothermic preservation of hepatocytes in UW,HTK, and Celsior solutions

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.     

Protective effects of a hibernation-inducer on hepatocyte injury induced by hypothermic preservation

Background/Purpose For hepatocyte-based cell therapy to be realistic, the method chosen for cryopreservation or hypothermic preservation is critical. The aim of the present study was to clarify whether D-Ala2-Leu5-enkephalin (DADLE), a hibernation inducer, has protective effects on hepatocytes with regard to hypothermic preservation injury. Methods A suspension of rat hepatocytes was stored at 4 °C for 24 h with or without DADLE. Their viability was measured by the trypan blue dye exclusion method, and alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels in the preservation solution were measured. After 24 h of cold storage, viable hepatocytes were cultured at 37 °C for another 24 h. Then albumin production and lidocaine clearance were measured. Results DADLE significantly improved the survival rate of hepatocytes. The levels of ALT and LDH in the preservation solution with DADLE were significantly lower than those in the preservation solution without DADLE. The treated viable hepatocytes maintained both albumin synthesis and lidocaine clearance. Conclusions DADLE appears to have protective effects on hepatocytes with regard to hypothermic preservation injury in vitro. This hibernation-inducer is useful in prolonged hypothermic preservation for hepatocyte-based therapy.     

The immediate function of the kidney after 24-to 72-hr preservation. Hypothermic storage versus mechanical perfusion.

Seventy-two dog kidneys were stored under hypothermia as described by Collins and Sacks between 24 and 72 hr and then transplanted. The immediate function of the kidneys was measured by p-aminohippuric acid and inulin clearances. Twenty-four hr proved to be the maximum safe preservation time with both methods. The immediate function of the kidneys stored under hypothermia could not be improved by the addition of furosemide to the flushing solution. These results were compared with those gained by mechanical perfusion of the organ. Kidney function after 72 hr of hypothermic mechanical perfusion was significantly better than after 24 hr of hypothermic storage.     

Hypothermic storage and cryopreservation of cartilage. An experimental study

Osteochondral autografts of femoral condyles in dogs were frozen at different cooling velocities after exposure to either glycerol or dimethyl sulfoxide to determine the freezing regimen best suited for preservation of intact cartilage. Autografts were also subjected to hypothermic storage in tissue culture media for ascertainment of how long they can be stored under these conditions. Autografts maintained in vitro under various conditions of storage were examined after transplantation. Autografts were chosen for this portion of the investigation in order to study the effects of storage uncomplicated by immunologic interactions. No differences were found between the cryoprotective actions of glycerol and dimethyl sulfoxide. The freezing rate that produced the least damage in the cartilage was 2 degrees/min. Cartilage survived ten days of hypothermic storage in tissue culture medium. Allografts frozen by the standard method of glycerolization and cooling at 1 degree/min were also studied. These showed eventual loss of chondrocytes and conversion of hyaline cartilage to fibrocartilage.     

Chemoimmunoprophylaxis of an experimental bladder cancer with retinoids and Bacillus Calmette Guérin

The function of kidneys stored for 48 to 72 hours in hypertonic crystalloid, intracellular solution, Euro-Collins (360 mOsm./l.), was compared with those stored in a new hyperosmolar (520 mOsm./l.) colloid solution designated as TP-II. The TP-II solution contained less K2HPO4 (1.05 gm./l.) and KH2PO4 (317 gm./l.) and more glucose (40 gm./l.) than the Euro-Collins, and also had an albumin concentration of 4.3 gm./dl. Kidneys obtained from beagle dogs were flushed with either Euro-Collins or TP-II solution (4C). Hypothermic storage followed for either 48 or 72 hours, prior to autotransplantation into the iliac fossa and contralateral nephrectomy. Four experimental groups were transplanted as follows: group 1 (n = 8) and group 2 (n = 7) received kidneys that were flushed with 250 ml. of Euro-Collins or TP-II solution, respectively, prior to 48 hours hypothermic storage. Group 3 (n = 5) and group 4 (n = 5) received kidneys that were flushed in the same way as those in groups 1 and 2, respectively, but were stored for a 72-hour period. TP-II appears to be superior to Euro-Collins for hypothermic storage of kidneys for periods as long as 48 hours. When hypothermic storage times are extended to 72 hours, the number of kidneys with normal function after transplantation is reduced for both solutions, however, TP-II solution has a slight advantage over Euro-Collins solution. Further studies will hopefully clarify this issue and lead to the application of TP-II in the clinical setting.     

HYPOTHERMIC ISCHAEMIA OF THE LIVER: A RE-PERFUSION PHENOMENON

Background : The effects of hypothermic injury to the liver were investigated on an isolated perfusion circuit by comparing porcine livers with varying degrees of preservation injury. Methods : A group of unstored livers (n= 5) were compared to livers stored in University of Wisconsin (UW) solution for 18 h (n= 5), and a group of livers stored in Hartmann's solution for 18 h (n= 5). Results : We observed that the degree of platelet sequestration was directly related to the severity of the preservation injury. After 2 h of isolated liver perfusion, the perfusate platelet count fell from 148 ± 14 × 10⁹/L to 84 ± 13 × 10⁹/L for control livers. In comparison for livers stored in UW solution, the platelet count fell from 173 ± 43 × 10⁹/L to 61 ± 14 × 10⁹/L, representing a 64.8% fall, while for those stored in Hartmann's solution, an even more profound fall from 152 ± 36 × 10⁹/L to 19 ± 9 × 10⁹/L (87.5% fall) was observed. The difference between the UW-stored and Hartmann's-stored livers was significant (P < 0.05). However, using this model, the degree of leukocyte sequestration did not differentiate the groups. Both histological and ultrastructural examination of liver biopsies taken immediately following revascularization demonstrated that for mild degrees of preservation injury following hypothermic storage, changes occur to the sinusoidal lining cells well before changes to the parenchymal elements. Conclusions : These findings substantiate the hypothesis that the primary injury associated with hypothermia involves the sinusoidal lining cells (non-parenchymal elements), that it is predominantly a reperfusion phenomenon and that efforts at improving preservation should therefore be targeted primarily at these cells and not the hepatocytes.     

Role of peroxynitrite anion in renal hypothermic preservation injury

BACKGROUND: Peroxynitrite anions may play a role in normothermic renal ischemia and reperfusion. The purpose of this study was to determine if endogenous peroxynitrite anion is involved in renal preservation injury. METHODS: Experiments were conducted in isolated canine renal tubules and in a canine autotransplant model of hypothermic preservation injury. RESULTS: Isolated renal tubules demonstrated progressive loss of membrane transport function after reperfusion with increasing cold storage times in UW solution as assessed by tetraethylammonium cation transport (TEA). This transport defect was not altered by reperfusion in the presence of WW85, a peroxynitrite decomposition catalyst. Likewise, tubule LDH release was not altered by WW85. Renal tubules did not demonstrate any evidence of peroxynitrite formation after cold storage (0-120 h) or after subsequent reperfusion in vitro as measured by nitrotyrosine adduct formation. Addition of exogenous peroxynitrite (1 mM) directly to freshly isolated renal tubules produced strong nitrotyrosine signals but failed to alter membrane function (TEA uptake). Conversely, SIN-1, a peroxynitrite generator molecule, failed to produce a nitrotyrosine signal in extracted renal tubule proteins but significantly impaired transport function. Finally, function of cold stored canine autografts was not affected by the scavenging of peroxynitrite anions (WW85) before kidney harvest and immediately at reperfusion. Tissue biopsies from cold stored kidney autografts also failed to show evidence of peroxynitrite synthesis either after cold storage (72 h) or after kidney transplantation (60 min reperfusion). CONCLUSIONS: This study concludes that peroxynitrite anions are not formed and are not involved in renal preservation injury.     

Hypothermic preservation of endothelialized gas-exchange membranes

Endothelialization of the blood contacting surfaces of blood-contacting medical devices, such as cardiovascular prostheses or biohybrid oxygenators, represents a plausible strategy for increasing their hemocompatibility. Nevertheless, isolation and expansion of autologous endothelial cells (ECs) usually requires multiple processing steps and time to obtain sufficient cell numbers. This excludes endothelialization from application in acute situations. Off-the-shelf availability of cell-seeded biohybrid devices could be potentially facilitated by hypothermic storage. In this study, the survival of cord-blood-derived endothelial colony forming cells (ECFCs) that were seeded onto polymethylpentene (PMP) gas-exchange membranes and stored for up to 2 weeks in different commercially available and commonly used preservation media was measured. While storage at 4°C in normal growth medium (EGM-2) for 3 days resulted in massive disruption of the ECFC monolayer and a significant decline in viability, ECFC monolayers preserved in Chillprotec could recover after up to 14 days with negligible effects on their integrity and viability. ECFC monolayers preserved in Celsior, HTS-FRS, or Rokepie medium showed a significant decrease in viability after 7 days or longer periods. These results demonstrated the feasibility of hypothermic preservation of ECFC monolayers on gas-exchange membranes for up to 2 weeks, with potential application on the preservation of pre-endothelialized oxygenators and further biohybrid cardiovascular devices.     

Twelve-hour and twenty four-hour preservation of small bowel allografts by simple hypothermia. Survival utilizing cyclosporine

Canine small bowel was harvested and stored by simple hypothermic technique. After 12- and 24-hr storage, respectively, the small bowel graft was allotransplanted into recipients. All animals receiving 12-hr stored grafts (n = 9) survived beyond 5 days. In the 24-hr storage group, 67% of the animals (n = 9) survived beyond 5 days. Successful storage for such extended periods by simple hypothermia has not been achieved previously. Donor pretreatment with antibiotics as well as extensive intraluminal irrigation of the harvested small bowel are considered to be important technical features in this successful preservation.     

Ultrastructural analysis of human endothelial cells after hypothermic storage in organ preservation solutions

BACKGROUND. Protection of vascular endothelium is a critical factor in organ preservation for transplantation. This study aims at a morphological assessment of endothelial cell injury in a comparison of storage solutions, using a cell culture model of cold preservation and rewarming. MATERIALS AND METHODS. Human umbilical vein endothelial cells (HUVEC) were cultured in monolayer and exposed to hypothermic storage in University of Wisconsin (UW), histidine-tryptophane-ketoglutarate (HTK), and EuroCollins solutions for 6 h and subsequent rewarming for 30 min or 6 h. Alterations of subcellular structures and cell-cell contacts were analyzed by transmission electron microscopy (TEM) and light microscopic assessment after actin and nuclear staining. RESULTS. Structural alterations of mitochondria, endoplasmic reticulum, nuclei, and cytoskeletal fibers as well as disruption of intercellular contacts were found after cold storage in HTK and EuroCollins solutions. In contrast, storage in UW solution resulted in minimum changes of stress fibers only. A rapid rearrangement of structural alterations was achieved during rewarming in cell culture medium in all experimental groups. CONCLUSIONS. Preservation of endothelial cell structure is best achieved by UW solution. Ultrastructural cell damage is a direct consequence of hypothermic storage and is fully reversible during rewarming after short storage times.     

Cold-preserved rat liver viability testing by proton nuclear magnetic resonance relaxometry.

The results of a series of experiments in the cold-preserved rat liver, applying a newly developed method of pretransplant viability testing, are described. The livers were stored either under simple hypothermic conditions (KHB) or in EC, HTK, or UW preservation solution. Livers were stored up to 48 hr and reperfused after a period of hypothermic storage of 1, 7, 14, or 21 hr. In a parallel series of experiments, with livers stored under identical conditions, repeated proton relaxometry measurements (0, 1, 7, 14, 24, 32, 48 hr) were undertaken; and ATP, ADP, AMP (Atkinson's energy charge), and water content of livers, as well as pH of storage solution, were estimated. Based on a strong correlation between proton spin-spin relaxation time T2 and tissue water content (edema), this new method may be useful to estimate the amount of cell swelling during hypothermic storage from a surgical biopsy of about 200 mg within a few minutes. There was, however, no significant correlation found between energy charge and/or pH and water content, T2, or bile flow. Our method could be useful as a rapid test method in experimental cold liver storage models and may be of interest in human liver transplantation as a viability indicator in combination with other parameters.     

Large-Scale Production and Cultivation of Hepatocytes on Biosilon Microcarriers

A method for large-scale production of hepatocytes on microcarriers have been developed for the purpose of bioartificial liver support system. Hepatocytes obtained by collagenase treatment of rat liver were efficiently attached and spread on a microcarrier surface in the presence of O2-saturated perfluorodecalin. In order to compare the metabolic activities of hepatocytes under long-term cultivation on microcarriers with those of cells under conventional monolayer culture, some liver-specific functions were investigated. Microcarrier-attached hepatocytes cultured in the absence of serum for 8 days synthesized and secreted albumin and fibronectin. Moreover, hepatocytes on microcarriers retained the ability to conjugate bilirubin for 4-5 days. With respect to these specific metabolic properties, microcarrier-attached hepatocytes were comparable to those from routine dish culture. These results suggest that this method developed for large-scale production of hepatocytes on microcarriers will allow one to obtain metabolically active cells suitable for extracorporeal liver support systems.     

Liver Preservation Techniques for Transplantation

The development of optimal methods for preservation is important for the advancement of liver transplantation. This study compares hypothermic storage (HS) and hypothermic pulsatile perfusion (HPP) with various solutions, using an isolated normothermic perfusion model (LIPM). Canine livers were removed from mongrel dogs without warm ischemia and flushed with either heparinized Ringer's lactate (control and HPP-preserved groups) or the solution used for hypothermic storage (TP-V or modified Collins). The type of preservation and solution for each of the experimental groups was as follows: group I (n = 7), no preservation, fresh; group II (n = 7), 24-h HS with TP-V (a hyperosmolar colloid solution containing sucrose, dextrose, and ATP-MgCl2); group III (n = 7), 24-h HS with modified Collins (C-2), an intracellular crystalloid solution; group IV (n = 5), 24-h HP with TP-V; group V (n = 6), 24-h HPP with Belzer solution, containing ATP-MgCl2; group VI (n = 3), 24-h HPP with albumin. After the preservation period, livers were placed on HPP at 37 degrees C with albumin-mannitol solution for 3-h testing in an LIPM. Perfusate samples were taken at 1-h intervals to assess liver function. LDH, SGOT, alkaline phosphatase, lactic acid, LAP, GGT, pO2, pCO2, pH, osmolarity, AMP, ADP, and ATP were studied. Histologic studies were performed, as were representative HIDA scans. Using the LIPM, livers preserved by HS and HPP with TP-V solution appeared to be superior to those preserved with modified Collins, Belzer, and albumin solutions. In these non-TP-V groups, the greatest cellular and organ damage was observed. TP-V HPP appeared to give the best overall liver functional response and histologic results and is recommended as the preferred method for 24-h liver preservation.     

Hypothermic maintenance of hepatocyte spheroids

Primary hepatocytes form spheroids under some culture conditions. These spheroids exhibit many tissue-like ultrastructures and retain many liver-specific functions over a long period of time. They are attractive for many applications employing liver cells. The ability to maintain their viability and functions at a reduced temperature to allow for transportation to the site of their application will facilitate their use. Furthermore, with their structural and functional similarity, they could possibly be used as a model system for studying various liver ischemias. The effect of hypothermic treatment was assessed by oxygen consumption rate, ATP, H2O2, and caspase 8 content, as well as albumin and urea synthesis, during and posttreatment. No single outcome variable gives a superlative quantification of hypothermic damage. Taken together, the hypothermic treatment can be seen as increasingly damaging as the temperature decreases from 21 degrees C to 15 degrees C and 4 degrees C. The addition of the chemical protectants glutathione, N-acetyl-L-cystein (NAC), and tauroursodeoxycholic acid (TUDCA) decreased the damaging effect of hypothermic treatment. This protection effect was even more profound when spheroids were preincubated with the protectant for 24 h, and was most prominent at 4 degrees C. The viability of the hypothermically treated hepatocyte spheroids was confirmed by laser scanning confocal microscopy. The method reported provides a means of maintaining spheroids' viability and may allow for their distribution to application sites at a distance.     

Prolonged vital cartilage graft preservation using tissue culture methods

Summary Cartilage grafting is one of the most commonly performed procedures in plastic surgery. Since storage of both autologous and allogenic cartilage is necessary, different preservation methods have been used with varying success. The use of chemical preservation procedures such as formaldehyde, Merthiolate or Cialit lead to a loss of viability of the graft. This work presents a study of the cell viability of cartilage grafts stored in different solutions (saline, RPMI 1640 and DMEM) for 100 days. Cartilage immersed in saline solution lost 50% of its viability after a storage period of 6 days, after 30 days no viable cells could be observed. In contrast to this, cartilage immersed in tissue culture media retained its viability (>80%) during the whole storage period. No differences were found between either culture medium. The results of these experiments suggest that viable cartilage tissue can be successfully stored over a long period of time using tissue culture procedures.     

Seventy-two-hour preservation of the canine liver by machine perfusion

The UW solution effectively preserves the dog liver for up to 48 hr by simple cold storage. This solution contains lactobionate as the primary impermeant. Another solution developed for machine perfusion of the kidney is similar to the UW solution but contains gluconate in place of lactobionate. In this study the UW gluconate solution was used for the continuous hypothermic machine perfusion of dog livers for 72 hr. Dog livers were continuously perfused at 5 degrees C through the portal vein at a pressure of 16-18 mm Hg and transplanted. Seven of 8 dogs survived for 7 or more days following orthotopic transplantation. The livers functioned as well as those preserved for 48 hr by cold storage in the UW solution as indicated by various liver-function tests. Successful machine perfusion was only achieved when the perfusate contained a high concentration of potassium (125 mM) but not with a high concentration of sodium (125 mM). This study demonstrates the feasibility of machine-perfusion preservation of the liver that yields longer preservation of equal quality compared to simple cold storage. For the development of truly long-term preservation (5 or more days) and better quality short-term preservation, machine perfusion may be the method of choice.