A Good Breath of Oxygen for Beta-Like Cells Obtained From Porcine Exocrine Pancreatic Tissue M.C. Gioviale, G. Damiano, F. Cacciabaudo, V.D. Palumbo, M. Bellavia, G. Cassata, G. Spinelli, G. Buscemi, and A.I. Lo Monte ABSTRACT Ischemia is the most important factor that affects organ survival during harvesting. The two-layer method (TLM) is one of several cold storage solutions that seeks to preserve organs and cells avoiding in vivo and in vitro ischemia. We compared the retrieval of beta-like elements from exocrine pancreatic cells using TLM versus University of Wisconsin (UW) solutions. For this purpose pancreata laparoscopically harvested from 20 female pigs were preserved in UW solution or TLM before digestion. The resulting exocrine cells were divided into 2 groups: the first was cultured in a designed medium to allow differentiation into beta-like cells and the second was cryopreserved before the differentiation process at -196°C for 8 weeks before culture in the same medium. The results revealed that TLM was better than UW as a preservation solution in terms of beta-cell viability and insulin secretion. We suggest that the use of TLM solution allows one to obtain less damaged cells for research purposes. D URING their removal organs readily encounter pre- cocious degenerative phenomena caused by ischemia, which damages tissues, leading to failure of cell metabolism. During multiorgan removal perfusion solutions at low temperature limit these phenomena by reducing cell swell- ing and lowering cellular energy needs. 1,2 The two-layer method (TLM) for organ preservation provides oxygen- ation of organs during periods of cold ischemia, 3–10 by means of a double layer of University of Wisconsin (UW) solution and perfluorochemical (PFC). The usefulness of the TLM method has been validated in clinical fields. 2 Furthermore, it may be useful for in vitro studies to improve the biological phenomena of resistance to cellular ischemic stress, transdifferentiation, growth, renewal, and senescence. For this purpose, we sought to test the effects of TLM compared with UW solution preservation of pan- creata removed from non– heart-beating porcine donors seeking to obtain beta-like cells from exocrine tissue. 11 MATERIALS AND METHODS Whole pancreata were collected from 20 female pigs (large white/ Landrace) of ages ranging from 12 to 18 months and a mean weight of 120  40 kg. The operative procedure approved by the our Ethics Committee was performed in accordance with International Guidelines for Animal Experiments. 12 The anesthesia was induced with premedication including ketamine (Ketavet, Farmaceutici Gellini, Peschiera Borromeo, Italy; 10 mg/kg), atropine (Atropina Solfato, Monico Spa, Venezia Mestre, Italy; 0.03 mg/kg), and diazepam (Valium, Roche, Monza, Italy; 0.4 mg/kg), followed by a mixture of isoflurane/oxygen supplied by orotracheal intubation. General anesthesia was maintained with 1%–1.7% isoflurane (Rhodia Chem SpA, Ospiate di Bollate, Italy) in 50% oxygen supplemented by a continuous infusion of fentanyl (Fentanest, Pfizer, Latina, Italy; 8 g/kg/h) and midazolam (Ipnovel, Roche, Monza, Italy; 0.5 mg/kg/h). A midline xyphopubic incision was performed to access the abdomen. After exsanguination through a vena caval incision and cardiac arrest, we isolated the duodenal “C” loop and through robust ties obtained an “en bloc” duodenum- pancreasspleen specimen that was immediately immersed in a basin containing melted ice mixed with UW solution (ViaSpan, Bristol-Myers Squibb, Italy) containing added dexamethasone, insulin, and glutathione. The operative time was defined as that elapsed between the start of the operation and the removal of the pancreas. Warm ischemic time (WIT) was defined as the time between cessation of the heart beat and placement of the pancreas into the preservation solution. From the Dipartimento di Discipline Chirurgiche ed Onco- logiche (M.C.G., G.D., F.C., V.D.P., M.B., G.S., G.B., A.I.L.M.), Università degli Studi di Palermo, and Istituto Zooprofilattico Sperimentale della Sicilia (G.C.) “A. Mirri,” Palermo, Italy. Address reprint requests to Attilio Ignazio Lo Monte, Diparti- mento di Discipline Chirurgiche ed Oncologiche, Università degli Studi di Palermo, Via del Vespro, 129 90127 Palermo, Italy. E-mail: ailomonte@unipa.it © 2011 by Elsevier Inc. All rights reserved. 0041-1345/–see front matter 360 Park Avenue South, New York, NY 10010-1710 doi:10.1016/j.transproceed.2011.02.039 Transplantation Proceedings, 43, 1173–1177 (2011) 1173