Hypothermic preservation of rat liver microorgans (lmos) in Viaspan® and BG35 (bes-gluconate-peg35) solutions. Study of ammonia metabolism during rewarming to evaluate their possible use as biological component of a bal system.

BERARDI FLORENCIA; PIZARRO M. DOLORES; SCANDIZZI ANGEL L.; MEDIAVILLA MARÍA G.; TIRIBELLI CLAUDIO; RODRIGUEZ JOAQUIN V.; MAMPRIN MARIA E

Rosario

Congreso; 49th Annual Meeting of the Society for Cryobiology,; 2012

Society for Cryobiology

Bioartificial Livers (BAL) were designed as a bridge to maintain patients with liver failure until they recover or receive a liver transplant. In these devices, the patient?s blood or plasma is circulated extra-corporeally through a bioreactor that houses a metabolically active component, as isolated hepatic cells or cultured cells. In this work, we tested LMOs from rat liver. LMOs are tissue slices that retain the microarchitecture of the liver lobe and its physiological characteristics. The use of LMOs is also attractive because it obviates the stages of cell isolation and cultivation. So, our group has set up a technique to manually obtain LMOs and constructed a flat-based BAL model suitable to use fresh LMOs. Moreover, we have developed a novel preservation solution, BG35, with similar efficacy than ViaSpan® to protect LMOs against cold preservation injury. This would allow having the biological component viable, functional and ready to be used when is needed for our BAL. Ammonia accumulates in the blood of patients with liver failure, causing metabolic and neurological disturbances, being crucial that any cell or tissue to be employed in a BAL can perform this task. Since the Urea Cycle is the major pathway of ammonia removal, the objective of this work was to study gene expression and activity of Carbamoyl Phosphate Synthetase I (CPSI) and Ornithine Transcarbamylase (OTC), two key enzymes, after LMOs preservation in BG35 and ViaSpan® (0ºC, 48 hr). We have assayed in vitro the LMOs ability to detoxify an overload of ammonia in order to evaluate them for their successive application into a BAL. LMOs were manually cut (338±27 μm thickness, n=25). 50 LMOs were stored at 0°C in 60 mL of BG35 or ViaSpan®. Freshly-cut LMOs were used as controls. After cold storage, LMOs were rewarmed (37°C, Krebs Henseleit Rewarming solution, 120 min) in a Dubnoff metabolic shaker under 95%O2:5% CO2 atmosphere. Samples were taken after 60 and 120 min. All groups showed a good maintenance of their viability. After 120 min, the amount of LDH released by LMOs cold preserved in BG35 (20.1 ± 5.6 %) and ViaSpan® (23.4 ± 5.5 %) was similar to Controls (16.0 ± 4.4 %, n=3). The preservation conditions did not affect ammonia metabolism: after 2 hs of rewarming, LMOs cold preserved in BG35 and ViaSpan® could detoxify 29.3 ± 7.9 % and 25.7 ± 4.9 % of the initial overload, similarly than Controls (29.7 ± 10.7 %, n=6). Though there were differences in relative mRNA levels between Controls and the two preserved groups, the values of CPSI and OTC enzymatic activity were comparable in all studied groups. In summary, we have shown that our preservation conditions did not affect ammonia metabolism and cold preserved LMOs maintain the physiological and biochemical liver functions tested, which allows their use as biological component of a BAL system.