title: A closed system for islet isolation and purification using the COBE2991 cell processor may reduce the need of clean room facilities.
abstract: During the isolation of human islets of Langerhans the digest has repeated direct contact with the ambient atmosphere. In order to fulfill GMP requirements in clinical applications, the entire cell preparation must be performed in clean room facilities. We hypothesized that the use of a closed system, which avoids the direct exposure of tissue to the atmosphere, would significantly ease the preparation procedure. To avoid the direct atmosphere exposure we tested a modification of the isolation and purification process by performing all islet preparation steps in a closed system. In this study we compared the isolation outcome of the traditional open preparation technique with the new closed system. Pancreata from 6-month-old hybrid pigs were procured in the local slaughterhouse. After digestion/filtration the digest was cooled, collected, and concentrated in centrifugation containers and purified thereafter in the COBE2991 by top loading (control). In the control group 502 +/- 253 IEQ per gram pancreas were purified. The total preparation time amounted to 12 h. In the closed system the digest was cooled and directly pumped into the COBE2991 for centrifugation followed by supernatant expelling. Bag filling, centrifugation, and expelling were repeated several times. Islets in pellet form were then purified by adding a gradient (bottom loading). Using this closed system 1098 +/- 489 IEQ per gram pancreas were purified with a total cell viability of 67 +/- 10% and a beta-cell viability of 41 +/- 13%. The total preparation time reduced to 6 h. After 24 h of cell culture the viability of beta-cells was still 56 +/- 10% and was only reduced after the addition of proapoptotic IL-1 and TNF-alpha to 40 +/- 4%, indicating that freshly isolated islets are not apoptotic. In conclusion, the closed system preparation is much faster, more effective, and less expensive than the traditional islet preparation. The closed system may be applicable for human islets preparations to restrict the need of clean room facilities for islet preparations to a minimum and may open the way for islet preparations without clean room demand.
authors.affiliation: Department of General, Visceral, and Transplantation Surgery, University of Tübingen, 72076 Tübingen, Germany.
authors.forename: R A
authors.initials: RA
authors.lastname: Klaffschenkel
authors.forename: A
authors.initials: A
authors.lastname: Biesemeier
authors.forename: M
authors.initials: M
authors.lastname: Waidmann
authors.forename: H
authors.initials: H
authors.lastname: Northoff
authors.forename: W
authors.initials: W
authors.lastname: Steurer
authors.forename: A
authors.initials: A
authors.lastname: Königsrainer
authors.forename: N
authors.initials: N
authors.lastname: Lembert
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods* / Q000592:standards; D002470:Cell Survival / Q000502:physiology; D002499:Centrifugation, Density Gradient / Q000379:methods*; D017364:Collagenases / Q000008:administration & dosage; D004780:Environment, Controlled; D006652:Histological Techniques / Q000379:methods / Q000592:standards; D006801:Humans; D017053:Infection Control / Q000295:instrumentation* / Q000379:methods / Q000592:standards; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism; D016381:Islets of Langerhans Transplantation / Q000379:methods* / Q000592:standards; D010179:Pancreas / Q000166:cytology; D013552:Swine; D013820:Thermolysin / Q000008:administration & dosage; D016896:Treatment Outcome
chemical_list: D017364:Collagenases; C106635:Liberase; D013820:Thermolysin