Minimisation of microbial contamination for potential islet xenografts using specific pathogen-free pigs and a protected environment during tissue preparation.
One major risk of islet xenotransplantation is transmission of infections. We thus compared microbial contamination during preparation of islets from 4 pigs conventionally breeded and slaughtered or 8 specific pathogen free (SPF) pigs, and different environmental conditions during pancreas excision. Pancreas harvested in a slaughterhouse (for conventional pigs) or in a protected autopsy room (for SPF pigs) were soaked in betadine solution and submitted to enzymatic digestion with collagenase. Islets were purified on histopaque gradient with a COBE 2991 processor. For each step of the process, a 10 ml aliquot was harvested and microbial contamination was analysed. For all animals, contamination of livers, which were not soaked in betadine solution, was also examined. Analysis of livers from the 4 conventional pigs showed polymicrobial contaminations (1,122 +/- 841 CFU/mg) with several species of Staphylococcus, Streptococcus, Bacillus and Enterobacteriaceae. For these conventional pigs, soaking of pancreas in betadine solution and presence 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 histopaque 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 pancreases. Only one contamination with Staphylococcus simulans was observed for one pancreas in transport solution (6 CFU/ml), and persisted in digestion medium (16 CFU/ml). Finally, all purified suspensions were completely sterile. In conclusion, breeding conditions of pig islet donors, and controlled environment for pancreas excision, considerably influence the risk of microbial contamination. In order to limit the risk, SPF pigs are a suitable and compulsory source of islets.
Gouin
E
E
University/INRA/ENVN, Nantes, France.
Rivereau
A S
AS
Darquy
S
S
Cariolet
R
R
Jestin
A
A
Reach
G
G
Sai
P
P
D000818:Animals; D001407:Bacillus / Q000302:isolation & purification; D017364:Collagenases; D004755:Enterobacteriaceae / Q000302:isolation & purification; D007515:Islets of Langerhans / Q000382:microbiology; D016381:Islets of Langerhans Transplantation / Q000379:methods* / Q000592:standards; D008099:Liver / Q000382:microbiology; D008834:Microclimate; D009926:Organ Preservation / Q000379:methods; D010180:Pancreatectomy / Q000379:methods / Q000592:standards; D011206:Povidone-Iodine; D013047:Specific Pathogen-Free Organisms; D013210:Staphylococcus / Q000302:isolation & purification; D013291:Streptococcus / Q000302:isolation & purification; D013552:Swine; D014183:Transplantation, Heterologous / Q000379:methods* / Q000592:standards
D011206:Povidone-Iodine; D017364:Collagenases