J P Kitzmann1, T Karatzas2, K R Mueller1, E S Avgoustiniatos3, A C Gruessner4, A N Balamurugan3, M D Bellin3, B J Hering3, K K Papas5. 1. Department of Surgery, University of Arizona, Tucson, Arizona; Schulze Diabetes Institute, University of Minnesota, Minneapolis, Minnesota. 2. Department of Surgery, University of Arizona, Tucson, Arizona; Second Department of Propedeutic Surgery, School of Medicine, University of Athens, Athens, Greece. 3. Department of Surgery, University of Arizona, Tucson, Arizona. 4. Schulze Diabetes Institute, University of Minnesota, Minneapolis, Minnesota. 5. Department of Surgery, University of Arizona, Tucson, Arizona; Schulze Diabetes Institute, University of Minnesota, Minneapolis, Minnesota. Electronic address: kkpapas@surgery.arizona.edu.
Abstract
BACKGROUND: Replacement of β-cells with the use of isolated islet allotransplantation (IT) is an emerging therapy for type 1 diabetics with hypoglycemia unawareness. The current standard protocol calls for a 36-72-hour culture period before IT. We examined 13 clinical islet preparations with ≥2 purity fractions to determine the effect of culture on viability. METHODS: After standard islet isolation and purification, pure islet fractions were placed at 37°C with 5% CO2 for 12-24 hours and subsequently moved to 22°C, whereas less pure fractions were cultured at 22°C for the entire duration. Culture density was targeted at a range of 100-200 islet equivalents (IEQ)/cm(2) adjusted for purity. Islets were assessed for purity (dithizone staining), quantity (pellet volume and DNA), and viability (oxygen consumption rate normalized to DNA content [OCR/DNA] and membrane integrity). RESULTS: Results indicated that purity was overestimated, especially in less pure fractions. This was evidenced by significantly larger observed pellet sizes than expected and tissue amount as quantified with the use of a dsDNA assay when available. Less pure fractions showed significantly lower OCR/DNA and membrane integrity compared with pure. The difference in viability between the 2 purity fractions may be due to a variety of reasons, including hypoxia, nutrient deficiency, toxic metabolite accumulation, and/or proteolytic enzymes released by acinar tissue impurities that are not neutralized by human serum albumin in the culture media. CONCLUSIONS: Current clinical islet culture protocols should be examined further, especially for less pure fractions, to ensure the maintenance of viability before transplantation. Even though relatively small, the difference in viability is important because the amount of dead or dying tissue introduced into recipients may be dramatically increased, especially with less pure preparations.
BACKGROUND: Replacement of β-cells with the use of isolated islet allotransplantation (IT) is an emerging therapy for type 1 diabetics with hypoglycemia unawareness. The current standard protocol calls for a 36-72-hour culture period before IT. We examined 13 clinical islet preparations with ≥2 purity fractions to determine the effect of culture on viability. METHODS: After standard islet isolation and purification, pure islet fractions were placed at 37°C with 5% CO2 for 12-24 hours and subsequently moved to 22°C, whereas less pure fractions were cultured at 22°C for the entire duration. Culture density was targeted at a range of 100-200 islet equivalents (IEQ)/cm(2) adjusted for purity. Islets were assessed for purity (dithizone staining), quantity (pellet volume and DNA), and viability (oxygen consumption rate normalized to DNA content [OCR/DNA] and membrane integrity). RESULTS: Results indicated that purity was overestimated, especially in less pure fractions. This was evidenced by significantly larger observed pellet sizes than expected and tissue amount as quantified with the use of a dsDNA assay when available. Less pure fractions showed significantly lower OCR/DNA and membrane integrity compared with pure. The difference in viability between the 2 purity fractions may be due to a variety of reasons, including hypoxia, nutrient deficiency, toxic metabolite accumulation, and/or proteolytic enzymes released by acinar tissue impurities that are not neutralized by human serum albumin in the culture media. CONCLUSIONS: Current clinical islet culture protocols should be examined further, especially for less pure fractions, to ensure the maintenance of viability before transplantation. Even though relatively small, the difference in viability is important because the amount of dead or dying tissue introduced into recipients may be dramatically increased, especially with less pure preparations.
Authors: C Ricordi; D W Gray; B J Hering; D B Kaufman; G L Warnock; N M Kneteman; S P Lake; N J London; C Socci; R Alejandro Journal: Acta Diabetol Lat Date: 1990 Jul-Sep
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Authors: Efstathios S Avgoustiniatos; William E Scott; Thomas M Suszynski; Henk-Jan Schuurman; Rebecca A Nelson; Phillip R Rozak; Kate R Mueller; A N Balamurugan; Jeffrey D Ansite; Daniel W Fraga; Andrew S Friberg; Gina M Wildey; Tomohiro Tanaka; Connor A Lyons; David E R Sutherland; Bernhard J Hering; Klearchos K Papas Journal: Cell Transplant Date: 2012-08-02 Impact factor: 4.064
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