MINIMIZATION OF MICROBIAL-CONTAMINATION FOR POTENTIAL ISLET XENOGRAFTS USING SPECIFIC PATHOGEN-FREE PIGS AND A PROTECTED ENVIRONMENT DURINGTISSUE-PREPARATION

One major risk of islet xenotransplantation is transmission of infecti ons. We thus compared microbial contamination during preparation of is lets from 4 pigs conventionally breeded and slaughtered or 8 specific pathogen free (SPF) pigs, and different environmental conditions durin g pancreas excision. Pancreas harvested in a slaughterhouse (for conve ntional pigs) or in a protected autopsy room (for SPF pigs) were soake d in betadine solution and submitted to enzymatic digestion with colla genase. Islets were purified on histopaque gradient with a COBE 2991 p rocessor. For each step of the process, a 10 mi aliquot was harvested and microbial contamination was analysed. For ail animals, contaminati on of livers, which were not soaked in betadine solution, was also exa mined. Analysis of livers from the 4 conventional pigs showed polymicr obial contaminations(1 122 +/- 841 CFU/mg) with several species of Sta phylococcus, Streptococcus, Bacillus and Enterobacteriaceae. For these conventional pigs, soaking of pancreas in betadine solution and prese nce of antibiotics in all media decreased the pancreatic contamination compared to hepatic contamination, but were unable to suppress it, as transport solution and crude suspension obtained after the digestion step with collagenase showed persistent contamination (9.7 +/- 2.4 and 10.5 +/- 4 CFU/ml, respectively). After islet purification by histopa que gradient, no medium remained contaminated. During analysis of the 8 SPF pigs, no liver exhibited contamination. Analysis of medium from each preparation step showed complete absence of contamination for 7 p ancreases. Only one contamination with Staphylococcus simulans was obs erved for one pancreas in transport solution (6 CFU/ml), and persisted in digestion medium (16 CFU/ml). Finally, all purified suspensions we re completely sterile. In conclusion, breeding conditions of pig islet donors, and contoled environment for pancreas excision, considerably influence the risk of microbial contamination. In order to limit the r isk, SPF pigs are a suitable and compulsory source of islets.