Establishment of fluorescein diacetate and ethidium bromide (FDAEB) assay for quality assessment of isolated islets.
One of the most important factors in clinical islet transplantation is isolation of a great number of islets with good viability. According to viability assessments of isolated islets, the static incubation test and the perifusion test of islets, which are used retrospectively, take much time and need various apparatus. But viability assessments of isolated islets for clinical islet transplantation require a simple, rapid, sensitive, and prospective method. We have developed a microfluorometric viability assay for isolated human, porcine, and dog islets of pancreata using fluorescein diacetate and ethidium bromide (FDAEB). Fluorescein diacetate (FDA) causes live cells to fluoresce green under blue light excitation (490 nm) and ethidium bromide (EB) causes dead cells to fluoresce red. In this study, we investigated the applicability of FDAEB staining to quality assessment of isolated islets for clinical use by correlation with the counting method with insulin secretion of islets. Discrimination of living from dead islets by insulin secretion correlated well with viability as determined by FDAEB staining. The proportion of living islets within isolated canine islets, as measured by microfluorometric counting, was found to correlate highly significantly on low-temperature (24 degrees C) culture (R = 0.831, p < 0.001) and on 37 degrees C culture (R = 0.553, p < 0.05) with the insulin contents of the same islets. Therefore, it is possible to differentiate degrees of viability, and a scoring system is described for this purpose. The FDAEB assay prospectively and easily provides a rapid, accurate, and objective measurement of the proportion of living cells and dead cells in isolated islets for clinical islet transplantation.
Miyamoto
M
M
Department of Organ Reconstruction, Field of Clinical Application, Institute for Frontier Medical Sciences, Kyoto University, Japan.
Morimoto
Y
Y
Nozawa
Y
Y
Balamurugan
A N
AN
Xu
B
B
Inoue
K
K
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D003592:Cytophotometry / Q000379:methods*; D004285:Dogs; D004996:Ethidium / Q000737:chemistry*; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism; D016381:Islets of Langerhans Transplantation; D013696:Temperature
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007328:Insulin; D004996:Ethidium; C018506:diacetylfluorescein