Islet preparation purity is overestimated, and less pure fractions have lower post-culture viability before clinical allotransplantation

J. P. Kitzmann, T. Karatzas, K. R. Mueller, E. S. Avgoustiniatos, Angelika C Gruessner, A. N. Balamurugan, M. D. Bellin, B. J. Hering, Klearchos K Papas

Research output: Contribution to journalArticle

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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)/cm2 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.

Original languageEnglish (US)
Pages (from-to)1953-1955
Number of pages3
JournalTransplantation Proceedings
Volume46
Issue number6
DOIs
StatePublished - 2014

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DNA
Dithizone
Membranes
Poisons
Hypoglycemia
Oxygen Consumption
Serum Albumin
Culture Media
Peptide Hydrolases
Transplantation
Maintenance
Staining and Labeling
Food
Therapeutics
Hypoxia

ASJC Scopus subject areas

  • Surgery
  • Transplantation

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Islet preparation purity is overestimated, and less pure fractions have lower post-culture viability before clinical allotransplantation. / Kitzmann, J. P.; Karatzas, T.; Mueller, K. R.; Avgoustiniatos, E. S.; Gruessner, Angelika C; Balamurugan, A. N.; Bellin, M. D.; Hering, B. J.; Papas, Klearchos K.

In: Transplantation Proceedings, Vol. 46, No. 6, 2014, p. 1953-1955.

Research output: Contribution to journalArticle

Kitzmann, J. P. ; Karatzas, T. ; Mueller, K. R. ; Avgoustiniatos, E. S. ; Gruessner, Angelika C ; Balamurugan, A. N. ; Bellin, M. D. ; Hering, B. J. ; Papas, Klearchos K. / Islet preparation purity is overestimated, and less pure fractions have lower post-culture viability before clinical allotransplantation. In: Transplantation Proceedings. 2014 ; Vol. 46, No. 6. pp. 1953-1955.
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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)/cm2 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.",
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T1 - Islet preparation purity is overestimated, and less pure fractions have lower post-culture viability before clinical allotransplantation

AU - Kitzmann, J. P.

AU - Karatzas, T.

AU - Mueller, K. R.

AU - Avgoustiniatos, E. S.

AU - Gruessner, Angelika C

AU - Balamurugan, A. N.

AU - Bellin, M. D.

AU - Hering, B. J.

AU - Papas, Klearchos K

PY - 2014

Y1 - 2014

N2 - 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)/cm2 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.

AB - 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)/cm2 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.

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