Assessment of islet cell viability using fluorescent dyes (original) (raw)
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Clinical & Biomedical Research, 2021
Introduction: The success of islet transplantation for patients with unstable type 1 diabetes mellitus depends, in part, on the number of isolated islets and their quality, which is assessed by functional and viability tests. The test currently employed to evaluate islet viability, used by the Collaborative Islet Transplant Registry to release products for transplantation, is fluorescein diacetate/propidium iodide (FDA/PI) staining. However, the efficacy of this method relies on researcher experience; in this context, a quantitative method may be useful. The aim of this study was to compare islet viability as assessed by flow cytometry and the FDA/PI assay.
A Practical Guide to Rodent Islet Isolation and Assessment
Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes.
Acta Diabetologica, 2004
The aim of the present study was to evaluate, by use of fluorescence microscopy and immunofluorescence stainings, the use of a fluorescent membrane potential sensitive probe as a means to identify and monitor changes in membrane potential of individual cell types in whole islets of Langerhans over time. Our work supports the use of the fluorescent probe bis-(1,3 dibutylbarbituric acid) trimethine oxonol (diBAC 4(3)), in identification of single α and β cells in the periphery of mouse pancreatic islets cultured on extracellular matrix. At a low extracel-lular glucose concentration (3 mM), heterogeneous staining of the islets was observed. Approximately 97% of the peripheral cells that stained brightly with diBAC4 were glucagon positive. Additional diBAC4 studies, demonstrated that an increase in glucose concentration from 3 to 10 mM is paralleled by repolarization of α cells and depolarization of β cells. This suggests that reciprocity of glucagon and insulin release upon glucose stimulation is coupled to divergent changes in membrane potential of these cell types and supports the use of diBAC 4(3) as a means to detect changes in secretion in both cell types.
A Simplified Approach to Human Islet Quality Assessment
Transplantation, 2010
Background-The successful treatment of patients with type 1 diabetes by islet transplantation is affected by a multitude of factors, of which infusion of the highest quality tissue is essential. The current standard pre-transplant quality assessments lack sensitivity, accuracy, and objectivity in the determination of islet viability and potency. We hypothesized that a multi-parametric approach focused on islet cell metabolic state, mitochondrial integrity, and in vitro glucose stimulated insulin secretion could provide data predictive of in vivo function. The objective of this study was to validate a novel set of islet quality assays and develop a simplified islet quality scoring system for both basic research and clinical applications.
Isolated islets in diabetes research
The Indian journal of medical research, 2007
This review highlights some recent developments and diversified applications of islets in diabetes research as they are rapidly emerging as a model system in biomedical and biotechnological research. Isolated islets have formed an effective in vitro model in antidiabetic drug development programme, screening of potential hypoglycaemic agents and for investigating their mechanisms of action. Yet another application of isolated islets could be to understand the mechanisms of beta cell death in vitro and to identify the sites of intervention for possible cytoprotection. Advances in immunoisolation and immunomodulation protocols have made xeno-transplantation feasible without immunosuppression thus increasing the availability of islets. Research in the areas of pancreatic and non pancreatic stem cells has given new hope to diabetic subjects to renew their islet cell mass for the possible cure of diabetes. Investigations of the factors leading to differentiation of pancreatic stem/progen...
Survival of isolated human islets of Langerhans maintained in tissue culture
Journal of Clinical Investigation, 1976
of human pancreatic islets to diabetic patients may require that donor islets be kept viable in vitro for extended time periods before transfer to the recipient. We have maintained isolated pancreatic islets obtained from the human cadaveric pancreas in tissue culture for 1-3 wk, after which we studied the structure and function of the islets. Electron micrographs of the cultured islets showed a satisfactory preservation of both s-cells and a2-cells. After culture for 1 wk, the islet oxygen uptake proceeded at a constant rate at a low glucose concentration (3.3 mM) and was significantly enhanced by raising the glucose concentration to 16.7 mM. Likewise, after culture for 1 wk, the islets responded with an increased insulin release when exposed to 16.7 mM glucose with or without added theophylline (10 mM). Islets cultured for 1-3 wk were able to incorporate ['H]leucine into proinsulin, as judged by gel filtration of acid-alcohol extracts. Glucagon release from the cultured islets was reduced significantly by 16.7 mM glucose alone, but stimulated by glucose (16.7 mM) plus theophylline (10 mM). It is concluded that viable pancreatic islets can be isolated from the pancreas of adult human donors and maintained in tissue culture for at least 1 wk without loss of the specific functions of the a2-and A-cells. It remains to be established whether such islets will survive and remain functionally competent after transplantation to human recipients. This work was presented in part at the meeting of the Scandinavian Society for the Study of Diabetes, Bergen,
Validation of methodologies for quantifying isolated human islets: an islet cell resources study
Clinical transplantation, 2009
Background-Quantification of islet mass is a crucial criterion for defining the quality of the islet product ensuring a potent islet transplant when used as a therapeutic intervention for select patients with type I diabetes. Methods-This multi-center study involved all 8 member institutions of the National Institutes of Health-supported Islet Cell Resources (ICR) consortium. The study was designed to validate the standard counting procedure for quantifying isolated, dithizone-stained human islets as a reliable methodology by ascertaining the accuracy, repeatability (intra-observer variability), and intermediate precision (inter-observer variability). The secondary aim of the study was to evaluate a new software-assisted digital image analysis method as a supplement for islet quantification. Results-The study demonstrated the accuracy, repeatability and intermediate precision of the standard counting procedure for isolated human islets. This study also demonstrated that softwareassisted digital image analysis as a supplemental method for islet quantification was more accurate and consistent than the standard manual counting method. Conclusions-Standard counting procedures for enumerating isolated stained human islets is a valid methodology, but computer-assisted digital image analysis assessment of islet mass has the added benefit of providing a permanent record of the isolated islet product being evaluated that improves quality assurance operations of current good manufacturing practice (cGMP).
Cell Transplantation, 2009
The ability to consistently and reliably assess the total number and the size distribution of isolated pancreatic islet cells from a small sample is of crucial relevance for the adequate characterization of islet cell preparations used for research or transplantation purposes. Here, data from a large number of isolations were used to establish a continuous probability density function describing the size distribution of human pancreatic islets. This function was then used to generate a polymeric microsphere mixture with a composition resembling those of isolated islets, which, in turn, was used to quantitatively assess the accuracy, reliability, and operator-dependent variability of the currently utilized manual standard procedure of quantification of islet cell preparation. Furthermore, on the basis of the best fit probability density function, which corresponds to a Weibull distribution, a slightly modified scale of islet equivalent number (IEQ) conversion factors is proposed that...
Cell and Tissue Banking, 2003
Once human islets are isolated, they are typically transplanted into type 1 diabetic recipients within 2 h of isolation. This time restriction makes it difficult for patients to travel from distant locations to receive an islet transplant and it also makes it difficult to complete pre-release quality control assessments (i.e., endotoxin and gram stain) before the expiration of the islet product. Therefore, there were two goals for this study. The first was to measure the stability of islets after a 24 h culture period using CMRL media 1066 (CMRL) supplemented with either fetal bovine serum (FBS); albumin or insulin transferrin and selenium (ITS). The second was to determine the impact of cell concentration and media depth on islet stability. The results of the study indicated that culture recoveries at 37 C with CMRL + ITS (also known as Memphis media) were higher (64.1 ± 8.3%) than with CMRL supplemented with FBS (38.7 ± 9.7%) or albumin (47.6 ± 8.2%) and that post-culture islet viabilities, post-culture purities and stimulation indexes (SIs) were comparable. In the second series of experiments, the results showed that islets recoveries and SIs in cultures with low islet concentrations (300 IE/ml) were significantly better than cultures at high islet concentrations (1500 IE/ml). Additionally, at a shallow media depth (1.4 vs. 7 mm of media) the SI of the islets improved, and this effect was independent of the additive (i.e., FBS, albumin and ITS). Abbreviations: AO/PI -Acridine orange and propidium iodide; BMI -Body mass index; CIT -Cold ischemic time; CMRL -CMRL media 1066; FBS -Fetal bovine serum; Fisher's PLSD -Fisher's protected least significant difference; HBSS -Hanks' balanced salt solution; ICPL -Islet and Cell Processing Laboratory; IE -Islet equivalent; ITS -Insulin transferrin selenium; SI -Stimulation index; UW -University of Wisconsin solution. ? From the Human Islet Transplants in Seattle