Isolation of Viable Porcine Islets by Selective Osmotic Shock Without Enzymatic Digestion

Atwater, I; Guajardo, Margarita H.; Caviedes, Pablo; Jeffs, S.; Parrau, D.; Valencia, M.; Roméro, Christine; Arriagada, Christian; Caamaño, E.; Salas, Anna; Olguin, Francesca; Atlagich, Miljenko; Maas, Ruth; Mears, David; Rojas, Eduardo

doi:10.1016/j.transproceed.2009.11.030

Cited by 9 publications

(21 citation statements)

References 49 publications

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“…Porcine islets were isolated using the selective osmotic shock method 34 . In this method, young adult pigs (20kg) were intravenously heparinized (400U/kg) then euthanized according to IACUC guidelines.…”

Section: ) Materials and Methodsmentioning

confidence: 99%

Applications of particulate oxygen-generating substances (POGS) in the bioartificial pancreas

et al. 2017

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Type-1 Diabetes (T1D) is a devastating autoimmune disorder which results in the destruction of beta cells within the pancreas. A promising treatment strategy for T1D is the replacement of the lost beta cell mass through implantation of immune-isolated microencapsulated islets referred to as the bioartificial pancreas. The goal of this approach is to restore blood glucose regulation and prevent the long-term comorbidities of T1D without the need for immunosuppressants. A major requirement in the quest to achieve this goal is to address the oxygen needs of islet cells. Islets are highly metabolically active and require a significant amount of oxygen for normal function. During the process of isolation, microencapsulation, and processing prior to transplantation, the islets’ oxygen supply is disrupted, and a large amount of islet cells are therefore lost due to extended hypoxia, thus creating a major barrier to clinical success with this treatment. In this work, we have investigated the oxygen generating compounds, sodium percarbonate (SPO) and calcium peroxide (CPO) as potential supplemental oxygen sources for islets during isolation and encapsulation before and immediately after transplantation. First, SPO particles were used as an oxygen source for islets during isolation. Secondly, silicone films containing SPO were used to provide supplemental oxygen to islets for up to 4 days in culture. Finally, CPO was used as an oxygen source for encapsulated cells by co-encapsulating CPO particles with islets in permselective alginate microspheres. These studies provide an important proof of concept for the utilization of these oxygen generating materials to prevent beta cell death caused by hypoxia.

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Section: ) Materials and Methodsmentioning

confidence: 99%

Applications of particulate oxygen-generating substances (POGS) in the bioartificial pancreas

et al. 2017

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“…To mimic hyperglycemic conditions, we set up the dedifferentiation process by comparing isolated islet properties from islets that were cultured for 24 h or Day 1 and Day 4 ( Figure 1 A) under high glucose conditions (16.7mM glucose or the equivalent of 3 times physiological blood glucose levels). The process of islet purification involved non-enzymatic method [ 23 , 24 ] from non-diabetic donors to minimize mechanical and cellular stresses from enzyme digestion and high temperature treatment. Figure 1 B represents a healthy isolated islet.…”

Section: Resultsmentioning

confidence: 99%

“…Ethical approval for the usage of human islets was granted by the Human Research Ethics Board at the Ramathibodi Hospital Mahidol University. Islet isolation was performed using non-enzymatic method as described in [ 23 ] Pancreatic tissues were cleaned by standard practice, then mixed and incubated with approximately 35–40 ml of RPMI 1640 medium supplemented with 600 mM glucose in 20 min at room temperature. The minced pancreatic tissue was washed and switched to zero-glucose RPMI medium.…”

Section: Methodsmentioning

confidence: 99%

Direct suppression of human islet dedifferentiation, progenitor genes, but not epithelial to mesenchymal transition by liraglutide

Rattanaamnuaychai

Roshorm

Wilasrusmee

et al. 2020

Heliyon

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β-cell dedifferentiation has been accounted as one of the major mechanisms for β-cell failure; thus, is a cause to diabetes. We study direct impacts of liraglutide treatment on ex vivo human dedifferentiated islets, and its effects on genes important in endocrine function, progenitor states, and epithelial mesenchymal transition (EMT). Human islets from non-diabetic donors, were purified and incubated until day 1 and day 4, and were determined insulin contents, numbers of insulin (INS + ) and glucagon (GCG + ) cells. The islets from day 3 to day 7 were treated with diabetic drugs, the long acting GLP-1 receptor agonist, liraglutide. As observed in pancreatic islets of type 2 diabetic patients, ex vivo dedifferentiated islets showed more than 50% reduced insulin contents while number of glucagon increased from 10% to about 20%. β-cell specific genes: PDX1 , MAFA , as well as β-cell functional markers: GLUT1 and SUR1, were significantly depleted more than 40%. Notably, we found increased levels of glucagon regulator, ARX and pre-glucagon transcripts, and remarkably upregulated progenitor expressions: NEUROG3 and ALDH1A identified as β-cell dysfunction markers in diabetic models. Hyperglucagonemia was often observed in type 2 patients that could lead to over production of gluconeogenesis by the liver. Liraglutide treatments resulted in decreased number of GCG + cells, increased numbers of GLP-1 positive cells but did not alter elevated levels of EMT marker genes: ACTA2, CDH-2, SNAIL2, and VIM . These effects of liraglutide were blunted when FOXO1 transcripts were depleted. This work illustrates that ex vivo human isolated islets can be used as a tool to study different aspects of β-cell dedifferentiation. Our novel finding suggests a role of GLP-1 pathway in beta-cell maintenance in FOXO1-dependent manner. Importantly, dedifferentiated islets ex vivo is a useful model that can be utilized to verify the actions of potential drugs to diabetic β-cell failure.

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“…Similarly, ECM-based materials have been shown to regulate survival, proliferation, and insulin secretion in purified β-cell cultures, while incompletely isolated islets that retain some of their native ECM have been shown to have markedly reduced apoptosis rates and significantly greater function with respect to insulin response in vitro than completely isolated and purified islets. This nonenzymatic method, first described by Atwater et al, uses rapid osmosis to isolate islet cells from the rest of the pancreatic cells [4]. This is possible because the beta cells of islets contain glucose transporters (Glut 2).…”

Section: Introductionmentioning

confidence: 99%

Selective Osmotic Shock (SOS)-Based Islet Isolation for Microencapsulation

Enck

McQuilling

Orlando

et al. 2016

Cell Microencapsulation

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Islet transplantation (IT) has recently been shown to be a promising alternative to pancreas transplantation for reversing diabetes. IT requires the isolation of the islets from the pancreas, and these islets can be used to fabricate a bio-artificial pancreas. Enzymatic digestion is the current gold standard procedure for islet isolation but has lingering concerns. One such concern is that it has been shown to damage the islets due to nonselective tissue digestion. This chapter provides a detailed description of a nonenzymatic method that we are exploring in our lab as an alternative to current enzymatic digestion procedures for islet isolation from human and nonhuman pancreatic tissues. This method is based on selective destruction and protection of specific cell types and has been shown to leave the extracellular matrix (ECM) of islets intact, which may thus enhance islet viability and functionality. We also show that these SOS-isolated islets can be microencapsulated for transplantation.

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Isolation of Viable Porcine Islets by Selective Osmotic Shock Without Enzymatic Digestion

Cited by 9 publications

References 49 publications

Applications of particulate oxygen-generating substances (POGS) in the bioartificial pancreas

Applications of particulate oxygen-generating substances (POGS) in the bioartificial pancreas

Direct suppression of human islet dedifferentiation, progenitor genes, but not epithelial to mesenchymal transition by liraglutide

Selective Osmotic Shock (SOS)-Based Islet Isolation for Microencapsulation

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