Variation in human islet viability based on different membrane integrity stains.
Membrane integrity fluorescent staining is used routinely to evaluate islet viability. Results are used as one of the determining factors in islet product release criteria, and are used to assess the efficacy of different culture conditions. Recently, it has been observed that there is variation in the viability staining of freshly isolated islets based on which viability assay is used. This investigation compares three membrane integrity stains for the viability assessment of isolated human islets. Fluorescein diacetate/propidium iodide (FDA/ PI), the current standard method for assessing islet viability, demonstrates intense extracellular fluorescence, reducing the differential staining of intact islets. We further evaluated SYTO-13/ethidium bromide (SYTO/ EB) and calcein AM/ethidium homodimer (C/EthD) as alternative viability assays, and found considerable variation between FDA/PI and either SYTO/EB or C/EthD staining. Preparations of human islets were obtained from cadaveric pancreata after collagenase digestion, mechanical separation, and purification by continuous Ficoll gradient centrifugation. For each preparation, two replicate samples of 50 islets were counted for each stain, and the percent viability calculated. The results for SYTO/EB and C/EthD were nearly identical [57.6 +/- 7.3% and 57.9 +/- 7.2%, respectively (mean +/- SEM), N = 11]. FDA/PI-stained islets, however, showed consistently elevated values when compared to SYTO/EB. Accurate assessment of islet viability remains a critical determinant of islet product release. The discrepancies found between FDA/PI scoring and visual quality, compared with alternative stains, suggests that the FDA/PI stain may not be the optimal approach to assess islet viability.
Barnett
M J
MJ
Clinical Islet Program, University of Alberta, Edmonton, Canada.
McGhee-Wilson
D
D
Shapiro
A M J
AM
Lakey
J R T
JR
D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D017690:Cell Transplantation / Q000379:methods*; D017364:Collagenases / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D004996:Ethidium / Q000031:analogs & derivatives* / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation / Q000379:methods; D009336:Necrosis; D009930:Organic Chemicals; D011419:Propidium / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013997:Time Factors
D004396:Coloring Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C461159:SYTO 13; C085925:calcein AM; D011419:Propidium; C018533:ethidium homodimer; D017364:Collagenases; D004996:Ethidium; C018506:diacetylfluorescein