Cryogenic preservation of isolated rat Islets of Langerhans: Effect of cooling and warming rates

Bank, Harvey L.; Davis, R.; Emerson, David L.

doi:10.1007/bf01219798

Cited by 45 publications

(17 citation statements)

References 18 publications

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“…Long Evans Hooded (LEH) male rats (220-250 g) were fast-H. L. Bank: Fluorescent islet cell viability assay ed for 12 to 18 h before the isolation of the islets. Pancreatic islets of Langerhans were isolated using the collagenase isolation procedure described previously [13,14]. A single step, 11% Ficoll (LSM, Bionetics Lab, Litton, Kensington, Md, USA) separation was used to separate islets from the tissue digest [14].…”

Section: All National Institute Of Health and Institutional Animal Rementioning

confidence: 99%

“…Pancreatic islets of Langerhans were isolated using the collagenase isolation procedure described previously [13,14]. A single step, 11% Ficoll (LSM, Bionetics Lab, Litton, Kensington, Md, USA) separation was used to separate islets from the tissue digest [14]. The islets were collected at the buffer/ficoll interface, and washed with phosphate buffered saline (PBS).…”

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confidence: 99%

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Assessment of islet cell viability using fluorescent dyes

Bank

1987

Diabetologia

100

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Summary.A rapid fluorometfic method has been developed to evaluate the viability of isolated islet cells. The assay differentiates between viable and nonviable cells by the simultaneous use of the inclusion and exclusion dyes acridine orange and propidium iodide. When viewed by fluorescent microscopy, viable cells fluoresce green, while nonviable cells fluoresce bright red. Although the acridine orange and propidium iodide assay measures membrane integrity, the results of this assay correlate with other measures of cell viability. Compared to trypan blue exclusion, this assay is easier to read, more stable, and has fewer staining artifacts. The assay enables the rapid estimation of the viability of a population of islet cells prior to time-consuming experiments rather than retrospectively. This assay can also be used with intact islets. Stained islets can be divided into three distinct groups: green fluorescing islets contain insulin, red fluorescing islets contain little or no insulin and a third class of islets containing some non-viable cells fluoresce red, green, and yellow. The yellow colour is due to the superimposition of red and green fluorescing cells. Key words:Islet viability, fluorochromes, B cells, acridine orange, propidium iodide, trypan blue exclusion, membrane integrity.A reliable method for rapidly determining the viability of isolated islet cells would be useful in studies on transplantation, cryopreservation, insulin release, and biosynthesis. Functional assays to determine insulin content or release and morphological assays including histochemistry or electron microscopy are time-consuming and expensive for routine viability assays.An alternative approach uses inclusion and/or exclusion dyes to test the integrity of the plasma membrane. Several fluorometric dyes were tested as indicators of membrane integrity. Fluorescein diacetate (FDA) has been used as a fluorometric assay of cell viability. FDA is a nonpolar ester which passes through plasma membranes and is hydrolysed by intracellular esterases to produce free fluorescein. The polar fluorescein is confined within cells with an intact plasma membrane and can be observed under appropriate excitation conditions [1][2][3][4]. However, staining of islet cells with FDA results in high background fluorescence. This fluorescence is presumably due to extracellular hydrolysis of the FDA or to fluorescein that leaked through damaged membranes.Acridine orange (AO) is a membrane-permeable, monovalent, cationic dye [5,6] which binds to nucleic acids. A low concentration of AO causes a green fluorescence, while a high concentration causes a red fluorescence. Propidium iodide (PI) is impermeable to intact plasma membranes, but it easily penetrates the plasma membrane of dead or dying cells [7] and intercalates with DNA or RNA forming a bright red fluorescent complex [8][9][10][11]. Both intracellular and extracellular background fluorescence is minimal after staining with optimal concentrations of these dyes.A combination of low concentrations of AO and...

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Section: All National Institute Of Health and Institutional Animal Rementioning

confidence: 99%

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confidence: 99%

Assessment of islet cell viability using fluorescent dyes

Bank

1987

Diabetologia

100

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“…Dimethyl sulfoxide (DMSO) was chosen as the cryoprotectant in this study (22), and two methods for exposing the tissue to DMSO (2 M) before freezing were compared. The first, based on the method of Rajotte et al (27), involves a much longer prefreeze exposure to the cryoprotectant solutions (45 min) than does the second (6 min), which is based on the method of Bank et al (18). The first method probably allows time for full permeation of the cryoprotectant into the tissue, whereas the second does not (9).…”

Section: Methodsmentioning

confidence: 99%

Interaction of Cooling Rate, Warming Rate, and Extent of Permeation of Cryoprotectant in Determining Survival of Isolated Rat Islets of Langerhans During Cryopreservation

Taylor

Benton

1987

Diabetes

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Cryopreservation of islets of Langerhans offers a number of important benefits for attempts to cure diabetes by transplantation. In the published literature, a variety of cooling rates, ranging from 0.25 to 75 degrees C/min, in conjunction with warming rates of 4-200 degrees C/min have been proposed to give optimal preservation of islets. In view of the general importance of rates of temperature change in determining survival and because of the possibility of modulating tissue immunogenicity by freezing and thawing, we have studied the interaction of cooling rate and warming rate for isolated rat islets that had been either fully or partially equilibrated with 2 M dimethyl sulfoxide (DMSO). Batches of islets were stored at -196 degrees C after cooling at 0.3, 3.0, 10, 30, 60, 150, or greater than 1000 degrees C/min and then warmed at either 10 or 50 degrees C/min. Survival was assessed by measuring the secretion of insulin during static incubation in alternating nonstimulatory and stimulatory media. Cooling rates extending over three orders of magnitude proved not to be a major determinant of survival when the islets were equilibrated with 2 M DMSO: greater than 50% survival was achieved at all cooling rates studied when the warming rate was at 50 degrees C/min. Peak survival (83%) was attained at a cooling rate of 0.3 degrees C/min, but only slightly lower recoveries were obtained at 60 and greater than 1000 degrees C/min. However, in islets only partially equilibrated with cryoprotectants, functional recovery was highly dependent on the cooling and warming rates, with peak survivals after slow cooling and rapid warming. Full permeation of the tissue with cryoprotectant offered maximal recovery of function.

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“…Banks et al [272] found that freezing in 1 mol/1 DMSO at 75 ~ gave optimal viability by in vitro parameters. Rajotte et al [275] found different conditions to be optimal: stepwise suspension in 2 mol/1DMSO, seeding with ice at -7.5 ~ freezing to -75 ~ at 0.25 ~ min, storage at -196 ~ and warming at 7.5 ~ Bretzel et al [276] have also normalized blood glucose in diabetic rats with isologous islets frozen in 1 mol/1 DMSO at 2 ~ and stored for 4 weeks at -196 ~ before rapid thawing and intraportal transplantation.…”

Section: Cryopreservation Of Islet Tissue a Variety Of Cells Ormentioning

confidence: 99%

“…Several investigators have used in vitro tests to determine the optimal conditions for freezing and thawing of adult islets [271,272] or fetal rat pancreas [273]. Critical factors include the cooling and warming rates, the concentration and penetration of agents added to protect against intracellular ice crystal formation, and the osmatic changes that occur during removal of the cryoprotectant [270].…”

Section: Cryopreservation Of Islet Tissue a Variety Of Cells Ormentioning

confidence: 99%

Pancreas and islet transplantation

Sutherland

1981

Diabetologia

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Cryogenic preservation of isolated rat Islets of Langerhans: Effect of cooling and warming rates

Cited by 45 publications

References 18 publications

Assessment of islet cell viability using fluorescent dyes

Assessment of islet cell viability using fluorescent dyes

Interaction of Cooling Rate, Warming Rate, and Extent of Permeation of Cryoprotectant in Determining Survival of Isolated Rat Islets of Langerhans During Cryopreservation

Pancreas and islet transplantation

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