Optimizing Conditions for Rat Pancreatic Islets Isolation

Amoli, Mahsa M.; Moosavizadeh, Roohollah; Larijani, Bagher

doi:10.1007/s10616-005-3586-5

Cited by 17 publications

(10 citation statements)

References 7 publications

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“…Pancreas digestion was carried out in a custom buffer of Hank’s Balanced Salt Solution (HBSS) containing 10 mM HEPES and 3.2 mM calcium chloride, adjusted to pH 7.6-7.8 at room temperature [ 10 – 12 ]. This digestion buffer was also used to prepare collagenase solutions.…”

Section: Methodsmentioning

confidence: 99%

Improved yield of canine islet isolation from deceased donors

Harrington

Williams

Otte³

et al. 2017

BMC Vet Res

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BackgroundCanine diabetes is a strikingly prevalent and growing disease, and yet the standard treatment of a twice-daily insulin injection is both cumbersome to pet owners and only moderately effective. Islet transplantation has been performed with repeated success in canine research models, but has unfortunately not been made available to companion animals. Standard protocols for islet isolation, developed primarily for human islet transplantation, include beating-heart organ donation, vascular perfusion of preservation solutions, specialized equipment. Unfortunately, these processes are prohibitively complex and expensive for veterinary use. The aim of the study was to develop a simplified approach for isolating canine islets that is compatible with the financial and logistical restrictions inherent to veterinary medicine for the purpose of translating islet transplantation to a clinical treatment for canine diabetes.ResultsHere, we describe simplified strategies for isolating quality islets from deceased canine donors without vascular preservation and with up to 90 min of cold ischemia time. An average of more than 1500 islet equivalents per kg of donor bodyweight was obtained with a purity of 70% (N = 6 animals). Islets were 95% viable and responsive to glucose stimulation for a week. We found that processing only the body and tail of the pancreas increased isolation efficiency without sacrificing islet total yield. Islet yield per gram of tissue increased from 773 to 1868 islet equivalents when the head of the pancreas was discarded (N = 3/group).ConclusionsIn summary, this study resulted in the development of an efficient and readily accessible method for obtaining viable and functional canine islets from deceased donors. These strategies provide an ethical means for obtaining donor islets.

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Section: Methodsmentioning

confidence: 99%

Improved yield of canine islet isolation from deceased donors

Harrington

Williams

Otte³

et al. 2017

BMC Vet Res

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“…The obtained solution was cultured for 24 h in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin–streptomycin (Sigma), and incubated at 37°C and in humidified atmosphere containing 5% CO 2 . [ 15 ]…”

Section: Methodsmentioning

confidence: 99%

The effect of time and temperature on viability and performance of Langerhans islets separated from Balb/c mouse after death

et al. 2015

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Background:Tissue transplantation plays a pivotal role in the treatment of diseases. Pancreatic beta cell transplantation is the best way to obtain normal blood glucose in patients with diabetes type 1. However, it is not clear how long endocrine pancreas cells can be used for transplantation after the donor's death. The present study was conducted to analyze the performance and viability of pancreatic islet cells after death.Materials and Methods:Pancreas was separated from Balb/c mice at different times (0, 1, 4, 6, 12, and 24 h after death) at temperatures of 4°C and 23°C, and was cultured in Roswell_Park_Memorial_Institute (RPMI) 1640. Insulin shock, MTT assay, aldehyde fuchsin staining, dithizone staining, and florescence microscopy methods were applied to analyze the performance of beta cells, cell viability, islets’ diagnosis, islet cells’ diagnosis, and viable and necrotic cells diagnosis, respectively.Results:Islets of Langerhans and beta cells were diagnosed. By increasing the temperature and time, the viability and performance of beta cells decreased significantly (P < 0.05).Conclusion:The best condition for keeping the islets of Langerhans in terms of viability and performance is 4 h after death at temperature of 4°C.

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“…Then cells were suspended in RPMI 1640 medium. The purity of the islets was examined by staining with Dithoizon [19].…”

Section: Pancreatic β-Cell Isolationmentioning

confidence: 99%

Effect of Maternal Diabetes on Pre- and Post-Natal Redox Status of F1 Rat Offspring

Kamel

Helmy²,

Hanafi³

et al. 2014

OJEMD

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Background: The oxidative stress and disturbed redox signaling during gestation my play an important role in the fetal programming of adult diabetes. Objective: The study aimed to investigate the effects of maternal diabetes on pre-and post-natal pancreatic and peripheral tissues redox and oxidative status in order to clarify their role in the diabetogenic programming of F1 offspring. Methods: Two groups of female Wistar rats were used (diabetic and control); diabetes was neonatally induced by STZ injection to 5-day old rats. 10 pregnancies of each group were terminated at GD 17 to obtain placentas and fetal pancreas, liver, muscle and adipose tissues for prenatal measurements. The rest of pregnancies were completed to term and the offspring were weaned to control diet or high-caloric (HCD) diet and followed up for 30 weeks. Every 5 weeks 10 male rats were sacrificed and serum and tissues were obtained for assessment of fasting blood glucose, tissues content of 8-oxo-dG, TBARS, GSH, GSSG, antioxidant enzymes and caspase-3. Results: The results indicated that, prenatally redox status of the foetuses of diabetic mothers is shifted toward more oxidizing environment which results from elevated oxidative stress and impaired antioxidants as indicated by elevated fetal tissues content of 8-oxo-dG, TBARS and GSSG and decreased GSH and GSH/GSSG ratio. All of these induce the apoptotic pathway in fetal pancreas. Postnatally, impaired glucose tolerance in the offspring of diabetic mothers is detected at 15 th week of age and no hyperglycemia was detected until age of 30 weeks in the offspring under CD while some of offspring under HCD at age 25 weeks and most of them at 30 weeks have developed hyperglycemia. The pancreas of the offspring of diabetic mothers suffers from oxidative stress from the 5 th week of age as indicated by elevated levels of nuclear and mitochondrial 8-oxo-dG contents and TBARS. Also, GSH level showed depletion with age and the activity of glutathione reductase was lower in the β-cell and hepatic tissues of the offspring of diabetic mothers. The prenatal shift of redox status persists postnatal and exaggerates with age which may explain the significant induction of the apoptotic pathway in the pancreatic β-cell. Conclusion: Maternal diabetes can crucially affect the redox status in fetal tissues prenatally and these effects can persist postnatal which may play an important role in the programming of the metabolic state of the offspring. Postnatal diet play im-M. A. Kamel et al. 112 portant role in accelerating development of metabolic derangements and aggravates oxidative stress in the tissues of F1 offspring.

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Optimizing Conditions for Rat Pancreatic Islets Isolation

Cited by 17 publications

References 7 publications

Improved yield of canine islet isolation from deceased donors

Improved yield of canine islet isolation from deceased donors

The effect of time and temperature on viability and performance of Langerhans islets separated from Balb/c mouse after death

Effect of Maternal Diabetes on Pre- and Post-Natal Redox Status of F1 Rat Offspring

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