Selective destruction of leucocytes by freezing as a potential means of modulating tissue immunogenicity: Membrane integrity of lymphocytes and macrophages

Taylor, Michael J.; Bank, Harvey L.; Benton, Michael J.

doi:10.1016/0011-2240(87)90011-3

Cited by 30 publications

(18 citation statements)

References 30 publications

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“…However, these dyes have a limited usefulness for evaluating islet cells [12]. Our results agree with "previous reports that the percentage of cells stained with trypan blue increases with time [22][23][24][25]. The constraints of a 5-10 min window of stability for trypan blue limits the number of samples that can be stained at a single time.…”

Section: Discussionsupporting

confidence: 77%

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Assessment of islet cell viability using fluorescent dyes

Bank

1987

Diabetologia

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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: Discussionsupporting

confidence: 77%

“…An additional application of the AO/PI assay is testing the viability of mononuclear cells. For example, this laboratory recently reported excellent agreement between the AO/PI assay results and the blastogenic potential of lymphocytes as well as the ability of macrophages to secrete lysozyme [22].…”

Section: Discussionmentioning

confidence: 92%

Assessment of islet cell viability using fluorescent dyes

Bank

1987

Diabetologia

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100

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“…Transplantation of large quantities of L islets will lead to the transplantation of L islets with various antigens and the possibility of rejection. Taylor's report9 have shown, in cases of frozen storage, that immunologically competent cells in the L islets, particularly lymphocytes, are extirpated at a freezing rate of at least 25°C per min. These interesting problems should be investigated in the future.…”

Section: Discussionmentioning

confidence: 99%

Transplantation of islets of Langerhans

Nakagawara

1994

J Hep Bil Pancr Surg

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The present study attempts to evaluate the possibility of using cryopreserved pancreatic islets in rats. Insulin‐releasing activity was studied for the purpose of clarifying the function of cryopreserved islets. Cryopreserved pancreatic islets were also transplanted into the portal veins of recipient rats. Rats recovered from the diabetic state, and the normalized condition was maintained for up to 20 weeks, although 4 weeks were needed before blood and urine glucose reached normal levels after transplantation. Intact B cells were found in the transplanted islet cell masses in the liver of the recipients, but B cells of the recipient's pancreases (streptozotocin‐treated rats) showed a marked decrease, as well as degenerative changes.

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“…Regardless of the possible reduction of rejection provided by better HLA matching when using islet cryopreservation and banking, one interesting aspect of cryopreserved islets is that they might be less prone to rejection than noncryopreserved ones (6). In spite of the potential practical importance of this phenomenon, its mechanism remains controversial (7)(8)(9).…”

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confidence: 99%

Influence of Cryopreservation on the Sensitivity of Human Islets to Tumor Necrosis Factor

Vara

Arias-Dı́az²,

Garcı́a³

et al. 1996

Pancreas

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Tumor necrosis factor ( T N F a ) has been shown to inhibit insulin release and it has been postulated to be an important effector in islet rejection. We studied the effect of cryopreservation on glucose oxidation rate (GOR), lipid synthesis, hormone secretion (insulin, glucagon, somatostatin, thyrotropin-releasing hormone), and cyclic guanosine 3'3'-monophosphate (cGMP) content of human islets, in the presence or absence of TNFa, looking for changes that could explain a different susceptibility to rejection for cryopreserved islets. Islets were isolated from multiple organ donor pancreata by collagenase digestion. The islets were then cultured for 7 days, cryopreserved (-0.25"C/min), and stored in liquid N,.After 24 h of culture, thawed islets were cultured for another 24 h in the presence or absence of TNFa. Islets were then washed to remove the cytokine and incubated in Krebs-Ringer bicarbonate (5 or 20 mM glucose), and both the cGMP content of the islets and the hormone concentration in the medium were determined by radioimmunoassay. GOR was measured as the production of 14C0, from 5 or 20 mM ~-[U-'~C]glucose, and de novo lipid synthesis was determined as ~-[U-'~C]glucose incorporation into different lipidic fractions. Cryopreservation did not significantly modify the hormone response to glucose but it partially reversed the TNFa-induced inhibitory effect on insulin release in the presence of 20 mM glucose. In addition, the inhibitory effect of TNFa on phosphatidylcholine labeling was attenuated in cryopreserved islets compared with noncryopreserved islets. TNFa significantly stimulated islet nitrite production and cGMP accumulation, both effects being of a similar magnitude in cryopreserved and noncryopreserved islets. Our results suggest that cryopreservation can modify the metabolic and hormone response of human islets to TNFa. This effect is not mediated by changes in the TNFainduced islet nitric oxide production or cGMP accumulation.

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Selective destruction of leucocytes by freezing as a potential means of modulating tissue immunogenicity: Membrane integrity of lymphocytes and macrophages

Cited by 30 publications

References 30 publications

Assessment of islet cell viability using fluorescent dyes

Assessment of islet cell viability using fluorescent dyes

Transplantation of islets of Langerhans

Influence of Cryopreservation on the Sensitivity of Human Islets to Tumor Necrosis Factor

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