Introduction and Aims:We employ a hydrogel scaffold to improve the overall hepatocyte engraftment after transplantation, and to improve efficacy.The scaffold is comprised of a fibrinogen backbone cross-linked with polyethylene glycol diacrylate (PEG-DA) side chains that form a hydrogel when mixed with cells and photo-polymerized.This biocompatible and biodegradable hydrogel provides a mechanical support as well as immunological protection, while maintaining cell function for the critical period early after cell transplantation.To assess and optimize the short-term (3 day) viability and function of a hepatocyte cell line after cell encapsulation within the hydrogel construct.Methods: Cell viability (Huh7) was estimated by propidium iodide (PI) labeling and fluorescein diacetate (FDA) staining.Viability was quantified using an MTT assay.Albumin and the urea production (in the medium) was used to assess hepatocyte function.Results were compared with normal cell cultures without a scaffold. Results:The number of polymer encapsulated cells without additional PEG-DA as measured by the MTT assay increased by three-fold within three days, (P < 0.05).The cell number increased by two-fold when adding 2% additional PEG-DA.The viability of the encapsulated cells with 0% PEG-DA as measured by PI/FDA staining was comparable to cell cultures without the polymer (95%±1.1% after 3 days) but lower when adding 2% additional PEG-DA (85%±1% after 3 days).Albumin concentration was higher at day 3 in encapsulated cells: 12.8 mg/l (P < 0.05) with 0% PEG-DA, and 15.4 mg/l (P < 0.05) with 2% PEG-DA, compared to 9.5 mg/l in cell cultures without the polymer.The urea concentration also increased to 3.0 mg/dl and 2.7 mg/dl, with and without additional 2% PEG-DA, respectively (P < 0.05), compared to 3.3 mg/dl in cell cultures without the polymer.The cell/polymer constructs were injected to rat liver, spleen and subcutis; the constructs were stable for 3 days post transplantation, allowing for new vessels to develop.Conclusions: The in-vitro Huh7 viability and function after polymerization in PEGylated protein hydrogel constructs was comparable to cells without the polymer.This is an important step for the development of injectable tissue engineered liver analogs for transplantation.

POSTERStogether with the evidence of continued viability and minimal cell damage, suggests that this system can be scaled-up to form the biological component of a bioartificial liver.

BACKGROUND:University of Wisconsin (UW) solution (Viaspan) is currently used to preserve organs from nonheartbeating donors. Histidine-tryptophan-ketoglutarate (HTK) solution (Custodiol) is of proven efficacy in experimental pancreas preservation, but its efficacy in combined warm ischemia (WI) and cold ischemia (CI) is unknown. The viability of HTK-preserved porcine pancreatic grafts was assessed after various periods of WI and compared with grafts flushed and preserved with UW solution. METHODS:A total of 14 pigs were used: G1 (n=4, UW) and G2 (n=4, HTK) with 15-min WI and 16-hr cold storage; G3 (n=3, UW) and G4 (n=3, HTK) with 30-min WI and 16-hr cold storage. RESULTS:All animals in G1 and G2 were normoglycemic, whereas only 66% of pancreases were functioning in G3 and G4. HTK perfusion was associated with increased wet weight. Transient hyperinsulinemia was noted in all the groups on postoperative day 1 (mean range: 8.9-12.4 microU/L). Postoperative serum amylase and lipase were more pronounced in G3 and G4. However, HTK-stored grafts exhibited less evidence of biochemical pancreatitis as compared with UW-stored grafts on the first postoperative day in the group with 15-min WI. Mean K values of intravenous glucose tolerance tests on postoperative day 14 were similar in both groups. Vascular congestion was uniformly observed and was considered a typical feature of WI. CONCLUSIONS:Porcine pancreatic grafts are viable after 16-hr CI following 15-min WI in this experimental nonheartbeating donor model. HTK solution seems to provide reliable graft function in this setting and to be equivalent to UW.

Delayed graft function (DEGF) remains an obscure phenomenon in organ transplantation. For the optimal washing of the compounds of the different organ flush solutions, adequate temperature and equilibrium of electrolytes have to be provided. A total of 29 landrace pigs weighing 37.3–5.4 kg were included in this study. According to the model, the left hemipancreas was perfused with Histidine-Tryptophan-Ketoglutarate (HTK)-solution and autotransplanted after 24 h (G1, n = 13) and 48 h (G2, n = 4) of cold storage (CS). Results were compared with grafts perfused with UW-solution and autotransplanted after 24 h (G3, n = 8) and 48 h (G4, n = 4) CS respectively. Daily measurements of glycemia, glucosuria, amylase and lipase were carried out. HTK perfusion resulted in an increase in wet weight of the grafts after 24 h and 48 h CS (P = 0.031 vs UW). Postoperative glycemia levels in pancreases flushed with HTK-solution were higher after 48 h than after 24 h CS until the 6th postoperative day, when the glycemia returned to normal range (P = 0.02), suggesting a delayed endocrine graft function. The mean IVGTT values attained after full function were comparable in G1 and in G3 (–1.22 ± 0.23 vs. –1.5 ± 0.65). The rises in serum amylase and lipase levels were more pronounced after 48 h CS in both HTK and UW groups, (P = n.s.). Appearance of interstitial and intracellular edema after CS and reperfusion did not influence the function.Conclusion: HTK-solution is suitable for 24 h pancreatic preservation in vivo; the perfusion requires at least 4 min for electrolyte equilibration. Long preservation time (48 h) resulted in a transitory DEGF.

438 Delayed graft function appears to be a rare but realistic complication in pancreas transplantation. Clinical studies have suggested that prolonged cold ischemia time, donor age and cardiocerebrovascular non traumatic causes are responsible for DGF. In the following study the function of pancreas autotransplants in pigs were analyzed after 24 h and 48 h cold storage of the graft with HTK solution with the aim to study the influence of prolonged cold ischemia on the immediate and the delayed graft function with a model mimicking the clinical setting. Methods: A total of 17 landrace pigs weighing 36.5 ± 6.1 Kg were included in this study. According to the model, the left hemipancreas was perfused with Histidine-Tryptophan-Ketoglutarate (HTK)-solution. During the flushing, samples of the perfusate were collected during 10 min to look at the electrolyte equilibration which is required for adequate function of the preservation buffer system. Segmental grafts were autotransplanted after 24 (n=13) and 48 (n=4) hours cold storage. Thereafter, daily measurement of glycemia, glucosuria, amylase and lipase was done. Results: HTK perfusion resulted in an increase in wet weight of the grafts after 24 and 48 h CS, with a mean ± SD of 2.32 ± 2.6 and 1.5 ± 2.5 respectively (p = n.s. for 24 h versus 48 h and for weight of all the grafts before and after CS). Electrolyte equilibration process was almost accomplished within 10 min. of flushing. All the animals were normoglycemic after autotransplantation. Postoperative glycemia levels were higher after 48 h than after 24 h CS until the 6th postoperative day when the glycemia returned to normal range (p = 0.02), suggesting a delayed endocrine graft function (DEGF) for pancreata stored for 48 h. The mean ± SD K-values were −1.18 ± 0.23 for grafts preserved 24 h and −1.22 ± 0.23 after 48 h CS (p = 0.6). The mean ± SD rise in serum amylase and lipase levels was 2890 ± 1063 and 827 ± 1308 U/L after 24 h CS and 3339 ± 2382 and 1587 ± 1492 after 48 h CS (p = 0.2). Appearance of interstitial and intracellular edema after CS and reperfusion did not influence the function. Conclusion: DGF appears to be correlated with the length of cold ischemia time (48 vs 24 hrs) with adequate electrolyte equilibration. The degree of edema is not influencing the immediate graft function.